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WO2024182860A1 - Methods and compositions for in vitro haematopoiesis and lymphopoiesis - Google Patents

Methods and compositions for in vitro haematopoiesis and lymphopoiesis Download PDF

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WO2024182860A1
WO2024182860A1 PCT/AU2024/050206 AU2024050206W WO2024182860A1 WO 2024182860 A1 WO2024182860 A1 WO 2024182860A1 AU 2024050206 W AU2024050206 W AU 2024050206W WO 2024182860 A1 WO2024182860 A1 WO 2024182860A1
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cells
population
generating
defined medium
psc
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Edouard Stanley
Ali MOTAZEDIAN
Shicheng SUN
Elizabeth Ng
Andrew Elefanty
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Murdoch Childrens Research Institute
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Murdoch Childrens Research Institute
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Priority to AU2024233447A priority Critical patent/AU2024233447A1/en
Publication of WO2024182860A1 publication Critical patent/WO2024182860A1/en
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Definitions

  • the present application relates to methods for the generation of human pluripotent stem cell-derived cells involved in haematopoiesis and lymphopoiesis, such as arterial endothelial cells, innate lymphoid cells, natural killer (NK)-like cells, and T cells.
  • the present application also relates to haemogenic arterial endothelial cells, innate lymphoid cells, natural killer (NK)- like cells, T cells and erythroid cells and myeloid cells, and progenitors of such cells, produced by such methods.
  • lymphoid commitment in vitro is an additional challenge, an event that is highly dependent on NOTCH ligands.
  • In vitro lymphopoiesis from either primary human haematopoietic progenitors or PSCs, requires exogenous supply of NOTCH ligands provided with immobilized recombinant proteins, or ectopic expression by mouse stromal cells, such as OP9 and MS5.
  • the relative opacity of many in vitro PSC differentiation platforms not only impacts the reproducibility of these methods, but also affects the exact type of cells that are generated. In turn, this lack of clarity also represents concerns for manufacturing lymphoid cells from PSCs for clinical applications, which may result in variable treatment outcomes.
  • NK cells from PSCs which display effective cell killing activity and show promise for biomedical applications.
  • PSCs PSCs which display effective cell killing activity and show promise for biomedical applications.
  • NK cells it is likely such cells arise from a population that resembles yolk-sac derived erythroid-myeloid progenitors.
  • the inventors have surprisingly identified novel culture methods that robustly generated NOTCH-ligand expressing haemogenic endothelial cells, whose gene profile resembles that of arterial haemogenic endothelial cells found the in AGM, and which permit sufficient priming of haematopoietic progenitors to the lymphoid lineage enabling the generation of PSC-derived innate lymphoid cells, natural killer (NK)-like cells, and T cells, erythroid cells and myeloid cells.
  • NK natural killer
  • T cells erythroid cells and myeloid cells.
  • the inventors provide a simplified culture system that enables the generation of the aforementioned cell types without the provision of exogenous NOTCH ligands or co-culture with exogenous stromal cells.
  • the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • AECs arterial endothelial cells
  • a fourth defined medium comprising at least one of SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells, and optionally cry opreserving the population of DLL4-expressing AECs.
  • the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • AECs arterial endothelial cells
  • a fourth defined medium comprising SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells, and optionally cryopreserving the population of DLL4-expressing AECs.
  • the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • AECs arterial endothelial cells
  • the present invention provides method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of: 2a. generating a monolayer of DEE4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
  • the present invention provides method for generating a mixed population of PSC-derived innate lymphoid cells (IFCs) and PSC-derived NK-like cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • the present invention provides a method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • 3c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • the present invention provides a method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
  • 3a generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3, 3b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
  • AECs arterial endothelial cells
  • 3c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • the present invention provides a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • the present invention provides a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • 4a generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3, 4b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
  • the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
  • the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • the present application provides method for generating a cell population enriched in PSC-derived erythroid cells, wherein the PSC-derived erythroid cells are CD235a+CD14- cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • FGF2, and IL3, and Flt3L for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells
  • the present invention provides a method for generating a cell population enriched in PSC-derived myeloid cells, wherein the PSC-derived erythroid cells are CD235a-CD14+ cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • FGF2, and IL3, and Flt3L for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells
  • a ninth defined medium comprising one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), and IL34, and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a CD14+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
  • MCSF Macrophage Colony-Stimulating Factor
  • GM-CSF Granulocyte Macrophage Colony-Stimulating Factor
  • IL34 optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7
  • the present invention provides a population of DLL4- expressing arterial endothelial cells (AECs) obtained from the method of any one of the first to the third aspects, wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
  • AECs DLL4-expressing arterial endothelial cells
  • the present invention provides a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells obtained from the method of any one of the fourth to the fifth aspects.
  • ILCs PSC-derived innate lymphoid cells
  • the present invention provides a cell population enriched in PSC-derived ILCs obtained from the method of any one of the sixth to the seventh aspects.
  • the present invention provides a cell population enriched in PSC-derived T cells obtained from the method of any one of the eighth to the ninth aspects.
  • the present invention provides a cell population enriched in PSC-derived NK-like cells obtained from the method of any one of the tenth to the twelfth aspects.
  • the present invention provides a population of PSC- derived erythroid cells obtained from the method of the thirteenth aspect.
  • the present invention provides a population of PSC- derived myeloid cells obtained from the method of the fourteenth aspect.
  • the present invention provides a population of PSC-derived DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
  • AECs PSC-derived DLL4-expressing arterial endothelial cells
  • the present invention provides a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells.
  • ILCs PSC-derived innate lymphoid cells
  • the present invention provides a cell population enriched in PSC-derived ILCs.
  • the present invention provides a cell population enriched in PSC-derived T cells.
  • the present invention provides a cell population enriched in PSC-derived NK-like cells.
  • a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • a method for generating a population of DEE4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells comprising the sequential steps of: la.
  • a third defined medium comprising a TGF-beta pathway inhibitor, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal- endothelial cells, ld.
  • a fourth defined medium comprising SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells.
  • a method for generating a population of DEE4-expressing arterial endothelial cells (AECs), wherein the DEE4-expressing AECs are CD34+ cells comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • a method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells comprising the sequential steps of:
  • AECs arterial endothelial cells
  • a method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells comprising the sequential steps of: 2a. generating a monolayer of DEE4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
  • AECs arterial endothelial cells
  • 3c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IE7 and at least one of Flt3E, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mE, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • AECs arterial endothelial cells
  • 3c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells comprising the sequential steps of:
  • AECs arterial endothelial cells
  • a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells comprising the sequential steps of:
  • AECs arterial endothelial cells
  • a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells comprising the sequential steps of:
  • a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells comprising the sequential steps of: 5a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
  • AECs arterial endothelial cells
  • a method for generating a cell population enriched in PSC-derived erythroid cells, wherein the PSC-derived erythroid cells are CD235a+CD14- cells comprising the sequential steps of:
  • AECs arterial endothelial cells
  • a method for generating a cell population enriched in PSC-derived myeloid cells, wherein the PSC-derived erythroid cells are CD235a-CD14+ cells comprising the sequential steps of:
  • AECs arterial endothelial cells
  • 8a generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3, 8b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
  • the first defined medium further comprises Y-27263, preferably the concentration of Y-27263 is about 1 to 50 pM, preferably about 8 to 12 pM, preferably about 10 pM.
  • iPSCs induced pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs are selected from the group consisting of iPSC RM3.5 (male) 77407 GFP , iPSC PB0-01 (male), iPSC PB0-04 (female), iPSC PB0-05 (female), iPSC PB0-06 (male), iPSC PB0-10 (male), and iPSC CRL2429 (ATCC).
  • ESCs are selected from the group consisting of ESC Hl (male), ESC H9 (female) 77407 :GFP , ESC ⁇ soxi7:tdTOMATO;Ruxic:GFP and ESC HES3(female) M7XL7;GF7> .
  • the concentration of Activin A in the first defined medium is about 10 to about 50 ng/mL, preferably about 30 ng/mL
  • the concentration of CHIR99021 in the first defined medium is about 1 to about 10 pM, preferably about 4 pM
  • the concentration of FGF2 in the first defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL
  • the concentration of PIK90 in the first defined medium is about 10 to about 300 nM, preferably about 100 nM.
  • the concentration of A83-O1 in the third defined medium is about 0.1 pM to about 10 pM, preferably about IpM
  • the concentration of VEGF in the third defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL
  • the concentration of BMP4 in the third defined medium is about 10 to about 100 ng/mL, preferably about 30 ng/mL
  • the concentration of FGF2 in the third defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL.
  • the concentration of SCF in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL
  • the concentration of VEGF in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL
  • the concentration of BMP4 in the fourth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL
  • the concentration of FGF2 in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL.
  • step la The method of any one of statements 1 to 24, wherein the time sufficient for generating the population of MIXL1+ cells at the end of step la is about 2 to about 72 hours, preferably about 24 hours.
  • step lb The method of any one of statements 1 to 25, wherein the time sufficient for generating a population of CD13+ early mesoderm cells at the end of step lb is about 2 to about 72 hours, preferably about 24 hours.
  • the concentration of SCF in the fifth defined medium is about 50 to about 500 ng/mL, preferably about 100 ng/mL
  • the concentration of VEGF in the fifth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL
  • the concentration of FGF2 in the fifth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL
  • the concentration of IL3 in the fifth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL
  • the concentration of Flt3L in the fifth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL.
  • the concentration of Flt3L in the sixth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL
  • the concentration of VEGF in the sixth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL
  • the concentration of FGF2 in the sixth defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL
  • the concentration of IL7 in the sixth defined medium is about 0.1 to about 10 ng/mL, preferably about 1 ng/mL.
  • the concentration of Flt3L in the seventh defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL
  • the concentration of VEGF in the seventh defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL
  • the concentration of FGF2in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL
  • the concentration of IL7 in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL
  • IL 15 in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL.
  • step 2e The method of any one of statements 4, 5, and 16 to 35, wherein the time sufficient for generating the mixed population of CD7+CD161+RAG1- cells and CD161+CD56+ cells at the end of step 2e is about 48 to about 192 hours, preferably about 96 hours.
  • PSC-derived innate lymphoid cells are CD161+CD7+ cells and/or CD161+RAG1- cells.
  • the concentration of Flt3L in the sixth defined medium is about 1 to about 100 ng/mL, preferably about 10 ng/mL
  • the concentration of VEGF in the sixth defined medium is about 5 to about 500 ng/mL, preferably about 50 ng/mL
  • the concentration of FGF2 in the sixth defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL.
  • step 3c The method of any one of statements 6, 7, and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+RAG1- cells at the end of step 3c is about 7 to about 21 days, preferably about 11 days.
  • step 3c The method of any one of statements 6, 7, and 16 to 35, wherein the cell population enriched in CD161+RAG1- cells at the end of step 3c is at least 50% of total cells.
  • step 4c The method of any one of statements 8, 9, and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD4+CD8a+ cells at the end of step 4c is about 7 to about 21 days, preferably about 11 days.
  • step 5c The method of any one of statements 12, and 16 to 35, wherein the time sufficient for generating the cell suspension comprising the population of CD161+RAG1+ cells at the end of step 5c is about 3 to about 14 days, preferably about 7 days.
  • step 60 The method of any one of statements 10, 11, 12 and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+CD56+ cells at the end of step 5e is about 48 to about 192 hours, preferably about 96 hours.
  • step 5e The method of any one of statements 10, 11, 12 and 16 to 35, wherein the cell population enriched in CD161+CD56+ cells at the end of step 5e is at least 70% of total cells.
  • step 6d The method of any one of statements 13 and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+CD56+ cells at the end of step 6d is about 48 to about 240 hours, preferably about 120 hours.
  • a population of DLL4-expressing arterial endothelial cells obtained from the method of any one of statements 1 to 3 and 16 to 35, wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
  • a cell population enriched in PSC-derived ILCs obtained from the method of any one of statements 6, 7, 16 to 36, 39, 43, and 52 to 54.
  • a cell population enriched in PSC-derived T cells obtained from the method of any one of statements 8, 9, and 16 to 36, 39, 43, 52, 55, and 56.
  • a cell population enriched in PSC-derived NK-like cells obtained from the method of any one of statements 10 tol3, 16 to 37, 39, 40, 43, 44, 52, and 57 to 63.
  • a population of CD34+CD7+ lymphoid haematopoietic progenitor cells obtained from step 2c of any one of statements 4, 5, 16 to 37, 39 and 40.
  • a population of CD161+RAG1+ cells obtained from step 5c or 5d of any one of statements 12, 16 to 37, 39, 58 and 59.
  • AECs PSC-derived DLL4-expressing arterial endothelial cells
  • AECs DLL4-expressing arterial endothelial cells
  • a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells consisting of the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • a fourth defined medium comprising at least one of SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells.
  • a method for generating a population of DEE4-expressing arterial endothelial cells (AECs), wherein the DEE4-expressing AECs are CD34+ cells consisting of the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • a second defined medium comprising a TGF-beta pathway inhibitor, a Wnt pathway activator, a BMP pathway inhibitor, FGF and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc.
  • incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal- endothelial cells, ld.
  • a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells consisting of the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • a method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells consisting of the sequential steps of: 2a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
  • a method for generating a mixed population of PSC-derived innate lymphoid cells (IFCs) and PSC-derived NK-like cells consisting of the sequential steps of:
  • AECs arterial endothelial cells
  • AECs arterial endothelial cells
  • 3c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • 3a generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3, 3b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
  • AECs arterial endothelial cells
  • 3c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • AECs arterial endothelial cells
  • AECs arterial endothelial cells
  • a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • AECs arterial endothelial cells
  • 5c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating a cell suspension comprising a population of CD161+RAG1+ cells, 5d. separating the cell suspension from the monolayer and sorting the cell suspension for a cell population enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells;
  • a seventh defined medium comprising IL15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IL 15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • AECs arterial endothelial cells
  • AECs arterial endothelial cells
  • AECs arterial endothelial cells
  • Figures 1A to II illustrate differentiation of human pluripotent stem cells to DLL4+ AECs and lymphoid haematopoietic cells. Specifically, each figure provides the following:
  • Figure 1A is a schematic representation showing sequential stages of PSC differentiation towards DLL4+ AECs, haemogenic endothelium, haematopoietic progenitors, lymphoid commitment, and the generation of ILCs. The approximate corresponding days are indicated.
  • Figure IB is a bright field image of a monolayer of cells at differentiation day 6, comprising DLL4+ AECs. Scale bar, 100 [im.
  • Figure 1C is a set of flow cytometric analysis of day 6 cultures showing the coexpression of the AEC markers DLL4, CXCR4, CDH5 (VECAD) on CD34+ cells. Red dots indicate CD34+DLL4+ double positive populations in each plot. Frequency of cell fractions are indicated in the relevant quadrants.
  • Figure ID is a bar graph summarising the flow cytometry analysis of CD34 and DLL4 expression in day 6 samples derived from multiple independent differentiation experiments using 9 distinct parental PSC lines (and 2 additional subclones of RM3.5 iPSCs and H9 ESCs). Cell line details are provided in Methods section. The number of experiments for each line is shown.
  • Figure IE is a set of flow cytometry analysis tracing the progressive differentiation of haematopoietic populations, including CD43+CD34+ HSPCs at day 12, CD34+CD7+ lymphoid haematopoietic progenitor cells at day 15, CD34-CD7+ and CD1+RAG1+ lymphoid cells at day 19. Green dots indicate RAG1 :GFP+ cells. Frequencies of cell fractions are indicated. [00041]
  • Figure 1G is a set of bright field and fluorescence images showing the emergence and accumulation of RAG1:GFP cells at day 15 and day 19. Inset shows a higher magnification of the indicated rectangle.
  • BF Bright Field. Scale bar, 100 pm.
  • Figure 1H is a set of flow cytometric analysis of CD45+ cells at day 23 of differentiation showing the expression of lymphoid associated markers CD7, CD 161 and RAG1, and the NK cell marker CD56. Frequencies of cell fractions are indicated.
  • Figure II is a set of bar graphs depicting quantification of frequency of the CD7+CD161+ innate lymphoid cells and CD161+CD56+ NK-like cells in multiple independent experiments incorporating 3 distinct parental PSC lines.
  • FIGS. 2A to 2F illustrate that scRNA-seq reveals AEC and haematopoietic differentiation resembles human AGM and fetal liver haematopoiesis. Specifically, each figure provides the following:
  • Figure 2A is a graph of UMAP projection showing a comparison between cells isolated from the AGM and fetal liver tissues (left) and cells generated by PSC differentiation in vitro (right).
  • Stromal cell strom; Endothelial cell, Endo; haematopoietic stem/progenitor-like cell, HSPC; lymphoid progenitor expressing RAG genes, RAG+ Lymph; innate lymphoid cells, ILC; monocyte, mono; erythroid cells, Eryth; granulocyte, Granu; megakaryocyte, Mega; eosinophils, Eosino; epithelial cells, Epi.
  • Figure 2B is pair of dotplots showing differentially expressed genes of haemogenic and haematopoietic cells from PSC (right) and from fetal tissues (left).
  • Figure 2C is a set of UMAP projections showing the expression of AEC associated genes CD34, SOX17, CDH5, GJA4, CXCR4, and GJA5). and NOTCH ligand genes (DLL4, DLK1, JAG1, and JAG2) on PSC-derived cells in vitro.
  • Figure 2D is a pie chart showing transcriptomics based ACTINN predication showing in vitro PSC-derived endothelial cells within the endothelial cell cluster projected to the arterial endothelial cells collected from the AGM of CS 14/15 embryos (AE-AGM CS 14/15).
  • Figure 2E is a set of UMAP projections showing the expression of key genes related to endothelial-to-haematopoietic transition and haemogenic endothelial cells in the fraction of day 12 and day 15 CD34+CDH5+RUNX1+ cells.
  • Figure 2F is set of UMAP projections (upper panel) and flow cytometric validation (lower panel) showing the persistent expression of DLL4 on CD34+ endothelial cells on day 12, day 15 and day 19.
  • FIGS 3A to 3F illustrate lymphoid cell development in the PSC-derived arterial haematopoietic culture. Specifically, each figure provides the following:
  • Figure 3A is a UMAP projection showing haematopoietic cell types in the PSC-based arterial haematopoietic culture. Cells represent hematopoietic cell clusters in Figure 2A.
  • Haemogenic endothelial cells HE; haematopoietic stem/progenitor-like cells, HSPC; common myeloid progenitor-like cells, CMP; erythroid progenitors, Eryth_Pro; myeloid progenitors, Mye_Pro; three lymphoid progenitor populations (Lymp_Prol, Lymph_Pro2, and Lymph_Pro3), T cell progenitor (T_Pro), T cell progenitor in cycling (T_Pro_cyc), innate lymphoid cells (ILC), and innate lymphoid cells in cycling (ILC_cyc). Colours indicate cell types.
  • Figure 3B is a set of UMAP projections showing the development of haematopoietic cell types in real-time (Day 6, 12, 15, 19 and 25) and in pseudo-time. Colours indicate cell types.
  • Figure 3C is a dot plot showing differentially expressed genes distinguish cell types generated in the PSC-based arterial haematopoietic culture.
  • Figure 3E is a UMAP projection showing three sub-clusters within the ILCs: NK-ILC1 like cells a (NK/ILCla), NK-ILC1 like cells b (NK/ILClb), and ILC2 like cells (ILC2-like).
  • Figure 3F is a heatmap representation of the expression levels of selected ILC associated genes in cells assigned to clusters representing NK-ILC1 like cells a (NK/ILCla), NK-ILC1 like cells b (NK/ILClb), and ILC2 like cells (ILC2-like).
  • FIG. 4A to 4F illustrate identification of IL7 as a determinant factor of fate choices between the T and the ILC lineages. Specifically, each figure provides the following:
  • Figure 4D is a set of fluorescent and bright field (BF) images showing the expression of RAGLGFP and cell growth of sorted GFP+ cells on the day of sorting and replating (day 0, sort) and after four days in culture (day 4) under the indicated conditions supplemented with IL7 (20ng/ml), IL15 (20ng/ml), or IL7 + IL15 (both 20ng/ml).
  • IL7 20ng/ml
  • IL15 20ng/ml
  • IL7 + IL15 both 20ng/ml
  • Figure 4E is a set of flow cytometry plots showing that IL15 (20 ng/ml) and IL7+IL15 (both 20ng/ml) support cell growth and the generation of CD161+CD56+ NK-like cells but IL7 (20ng/ml) does not.
  • Figure 4F is a bar graph showing quantification of output cell numbers per 5,000 input RAG1:GFP+ cells (as normalized to 1) showing IL7+IL15 robustly supported the generation of CD161+CD56+ NK-like cells from RAG1+ lymphoid progenitors. Bar graph is a representation of 2 independent experiments, data points represent technical replicates, data is shown in mean +SEM.
  • FIG. 5A to 5F illustrate differentiation of PSCs to arterial endothelial cells. Specifically, each figure provides the following:
  • Figure 5A is a set of bright field images showing early days (day 1, 2 and 3) of PSC differentiation to arterial endothelial cells.
  • Figure 5B is a flow cytometry plot showing efficient generation of MIXL1 :GFP+ primitive streak cells on day 1 of PSC differentiation. Frequency of MIXL1+ cells is indicated.
  • Figure 5C is a set of flow cytometry plots tracking expressions of the mesodermal marker CD 13, and the endothelial cell marker KDR(VEGFR2). Differentiating cells are CD13+KDR- representing mesodermal progenitors, followed by upregulation of KDR on day 3.
  • Figure 5D is a set of flow cytometry plots showing reproducible generation of arterial endothelial cells from 10 PSC lines in one differentiation experiments.
  • Figure 5E is a bar graph showing quantification of the number of CD34+DLL4+ cells per input PSC on day 6 of differentiation. Data is shown with six technical replicates for four PSC lines of biologically independent backgrounds: PB005, iPSC; RM3.5, iPSC; H9, ESC; CRL-2429, iPSC.
  • Figure 5F is a set of flow cytometry analysis showing differentiation from cryopreserved PSC-derived arterial cell cultures at day 7 (24 hours after thaw), day 14 (CD45+CD34+, haematopoiesis), and day 20 (CD7+RAG1+, CD161+RAG1-, and CD161- RAG1+ lymphopoiesis). Frequencies of cells of each fraction are indicated.
  • FIG. 6A to 6J illustrate scRNA-seq analysis and characterization of the PSC- derived arterial haematopoietic cultures. Specifically, each figure provides the following:
  • Figure 6A is a set of UMAP projections showing samples representing cells collected at different timepoints of PSC differentiation (PSC Day 6, 12, 15, 19 and 25), and samples representing cells collected from the AGM and foetal liver tissues.
  • Figure 6B is a set of UMAP projections showing the expression cell type specific genes: stromal cell (COL3A F). arterial endothelial cell ⁇ CD34 and GJA4); haematopoietic stem/progenitor-like cell (CD34. SPINK2), lymphoid haematopoietic cell (CD7), lymphoid progenitor expressing RAG genes (RAG I). innate lymphoid cell (KLRB1), monocyte (CSF/R). erythroid cell (HBA2). granulocyte (S100A9), megakaryocyte (PF4). eosinophils (CP A3).
  • stromal cell ⁇ CD34 and GJA4
  • Figure 6C is a set of immunofluorescence images for human DLK1 and CDH5 of arterial-haematopoietic culture on day 8. Red, DLK1. Green, CDH5; Blue, DAPI. Scale bar, 100 pm.
  • Figure 6D is a set of UMAP projections (upper panel) and flow cytometric validation (right panel) showing expression of Jagged 1 (JAG1) on CD34+ endothelial cells on day 6.
  • JAG1 Jagged 1
  • CD34- stromal cells also expressed JAG1 with persistent expression to day 15 of PSC differentiation. Frequencies of cell fractions are indicated.
  • Figure 6E is a set of UMAP projections showing the expression of JAG1, CXCR4, and DLL4 in fetal tissues across developmental time, including AGM (week 4.5, 5, 5.5, and 6) and fetal liver (week 5.5, 6, 8, 11, 15).
  • Figure 6F is a bar graph showing quantification of JAG1+ cells in the arterial endothelial cell cluster of fetal tissues across development time.
  • Figure 6G is a CellChat analysis of NOTCH ligand-receptor pair commination between of PSC-derived cell types in the arterial haematopoietic cultures on day 12, 15 and 19: stromal cells, haematopoietic stem/progenitor-like cells (HSPC), endothelial cells, innate lymphoid cells (ILCs) and lymphoid progenitor expressing RAG genes.
  • stromal cells haematopoietic stem/progenitor-like cells (HSPC), endothelial cells, innate lymphoid cells (ILCs) and lymphoid progenitor expressing RAG genes.
  • HSPC haematopoietic stem/progenitor-like cells
  • ISCs innate lymphoid cells
  • RAG genes lymphoid progenitor expressing RAG genes.
  • Figure 6H is a set of flow cytometry analysis, bright field images, and cytospin analysis demonstrating erythroid and myeloid differentiation from day 12 AECs following addition of the indicated growth factors.
  • the percentage of cells in pertinent gates is indicated. Images are of single wells of a 96 well tray containing cells differentiated in the presence of indicated growth factors, noting the overt haemoglobinisation apparent in cell populations treated with Erythropoietin (EPO) for two weeks.
  • EPO Erythropoietin
  • cytospin analysis shows cells with a distinctive macrophage morphology arising from cultures supplemented with M-CSF.
  • Figure 61 is a set of flow cytometry analysis demonstrating T cell (TCR+CD3+) & B lymphoid (RAG1+ CD19+) differentiation from day 12 AECs following co-culture with stromal cells. For the flow cytometry plots, the percentage of cells in pertinent gates is indicated.
  • Figure 6J is a set of is a set of brightfield and immunofluorescence images showing the tube forming ability of differentiation day 6 RM-tTom endothelial cells following disaggregation and re-seeding on Matrigel. Scale bar, 200 pm.
  • FIGS 7A to 7E illustrate scRNA-seq analysis of the lymphoid components in PSC- derived arterial haematopoietic cultures. Specifically, each figure provides the following:
  • Figure 7A is a set of UMAP projections showing samples representing haematopoietic cells collected at different timepoints of PSC differentiation (PSC Day 6, 12, 15, 19 and 25). This figure is corelated to Figure 3A. Colours indicate differentiation day.
  • Figure 7B is a set of UMAP projections showing rare cells expressing B cell development genes MME/CD10, CD19, MS4A1/CD20.
  • Figure 7C is a heatmap showing top 2000 differentially expressed genes across the four cell populations of haematopoietic stem/progenitor like cells (HSPC), three lymphoid progenitors (Lymph_Prol, Lymph_Pro2, and Lymph_Pro3).
  • HSPC haematopoietic stem/progenitor like cells
  • Lymph_Prol Lymph_Pro2
  • Lymph_Pro3 lymphoid progenitors
  • Figure 7D is set of gene ontology analysis the cell type specific gene modules shown in the Figure 7C.
  • Figure 7E is a set of UMAP projections showing the expression of ILC genes and NK genes in the PSC-derived ILC sub-populations. This figure is related to Figure 3E.
  • FIG. 8A to 8J illustrates IL7 concentrations determine cell fate choices between the T and the ILC lineages and RAG1 gene expression. Specifically, each figure provides the following:
  • Figure 8A is set of UMAP projections showing the expressions of IL7R, KLRB1 CD161), RAG1 and CD4 expression on PSC-derived haematopoietic cells.
  • Figure 8B is a UMAPC projection showing undetected IL7 mRNA in cells from the entire PSC-derived arterial haematopoietic culture.
  • Figure 8D is a set of violin plots of scRNA-seq showing the expression of ILC-related genes (KLRB1, NKG7, NCAMI) expressed in /MG/-low cells, while /MG /-high cells show higher levels of expressions of T cell development related genes ⁇ CD4.
  • the mean of RAG1 average expression across all clusters was calculated by the AverageExpression function in R; cells that were above the mean average expression of 2.259 (4sf) were labelled as “7MG7-high” and those lower as “TMGJ-low”.
  • Figure 8E is a set of violin plots of scRNA-seq showing day 19 culture contains predominantly /?AG7-low cells and day 25 culture contains both RAG 1 -high and RAG 1 -low cells.
  • Figure 8F is a set of fluorescent and bright field (BF) images showing the expression of RAG1 :GFP and cell growth of sorted GFP+ cells on the day 1, 2 and 3 after replating and cultured under the indicated conditions supplemented with IL7 (20ng/ml), IL15 (20ng/ml), or IL7 + IL 15 (both 20ng/ml). This figure is related to Figure 4D.
  • Figure 8H is a set of flow cytometry analysis of CD45+ cells isolated from cultures at day 30 in which IL 15 had been added from day 15.
  • transitional phrase “consisting essentially of” is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.
  • the term “about” as used herein contemplates a range of values for a given number of ⁇ 25% the magnitude of that number. In other embodiments, the term “about” contemplates a range of values for a given number of ⁇ 30%, ⁇ 20%, ⁇ 15%, ⁇ 10%, or ⁇ 5% the magnitude of that number. For example, in one embodiment, “about 3 pM” indicates a value of 2.7 to 3.3 pM (i.e.
  • differentiation processes include ordered, sequential events
  • the timing of the events may be varied by at least 25%.
  • a particular step may be disclosed in one embodiment as lasting one day, the event may last for more or less than one day.
  • “one day” may include a period of about 18 to about 30 hours.
  • periods of time may vary by ⁇ 20%, ⁇ 15%, ⁇ 10%, or ⁇ 5% of that period of time.
  • Periods of time indicated that are multiple day periods may be multiples of “one day,” such as, for example, about two days may span a period of about 36 to about 60 hours, and the like.
  • time variation may be lessened, for example, where 1 day is 24 ⁇ 3 hours; 3 days is 72 ⁇ 3 hours; 4 days is 96 ⁇ 3 hours; 5 days is 120 ⁇ 3 hours; 6 days is 144 ⁇ 3 hours; 7 days is 168 ⁇ 3 hours; 11 days is 264 ⁇ 3.
  • about 3 days may be 2.5, 3 or 3.5 days, about
  • 4 days may be 3.5, 4 or 4.5 days, about 5 days may be 4.5, 5 or 5.5 days, about 6 days may be
  • about 21 days may be 20, 20.5, 21, 21.5, or 22 days.
  • pluripotent stem cell and “PSC” refer to cells that display pluripotency.
  • human pluripotent stem cell and “hPSC” refer to cells derived, obtainable or originating from human tissue that display pluripotency.
  • the hPSC may be a human embryonic stem cell or a human induced pluripotent stem cell.
  • Human pluripotent stem cells may be derived from inner cell mass or reprogrammed using Yamanaka factors from many fetal or adult somatic cell types.
  • the generation of hPSCs may be possible using somatic cell nuclear transfer.
  • human embryonic stem cell refers to cells derived, obtainable or originating from human embryos or blastocysts, which are self-renewing and pluri- or toti-potent, having the ability to yield all of the cell types present in a mature animal.
  • Human embryonic stem cells can be isolated, for example, from human blastocysts obtained from human preimplantation embryos, in vitro fertilized embryos, or onecell human embryos expanded to the blastocyst stage.
  • induced pluripotent stem cell and “iPSC” and “hiPSC” (human iPSC) refer to cells derivable, obtainable or originating from adult somatic cells of any type reprogrammed to a pluripotent state through the expression of exogenous genes, such as transcription factors, including but not limited to a preferred combination of OCT4, SOX2, KLF4 and c-MYC.
  • hiPSC show levels of pluripotency equivalent to hESC but can be derived from an individual for autologous therapy with or without concurrent gene correction prior to differentiation and cell delivery.
  • the method disclosed herein could be applied to any pluripotent stem cell derived from any individual or a hPSC subsequently modified to generate a mutant model using gene-editing or a mutant hPSC corrected using gene-editing.
  • Gene-editing could be by way of CRISPR, TALEN or ZF nuclease technologies.
  • cell culture refers to any in vitro culture of cells.
  • the term “culturing” refers to the process of growing and/or maintaining and/or manipulating a cell Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.
  • the terms “primary cell culture,” and “primary culture,” refer to cell cultures that have been directly obtained from cells in vivo, such as from a tissue specimen or biopsy from an animal or human. These cultures may be derived from adults as well as fetal tissue.
  • a "progenitor cell” is a cell which is capable of differentiating along one or a plurality of developmental pathways, with or without self-renewal. Typically, progenitor cells are unipotent or oligopotent and are capable of at least limited self- renewal.
  • differentiate relate to progression of a cell from an earlier or initial stage of a developmental pathway to a later or more mature stage of the developmental pathway.
  • undifferentiated in this context, relate to a cell from an earlier or initial stage of a developmental pathway or a cell that has not yet developed into a specialized cell type. It will be appreciated that in this context “differentiated' does not mean or imply that the cell is fully differentiated and has lost pluripotency or capacity to further progress along the developmental pathway or along other developmental pathways. Differentiation may be accompanied by cell division.
  • the stage or state of differentiation of a cell may be characterized by the expression and/or non-expression of one or more specific markers.
  • the expression of “signature” or “milestone” markers may be used in determining or defining the stage or state of differentiation instead of using the period of time defined in days and/or hours.
  • markers is meant nucleic acids or proteins that are encoded by the genome of a cell, cell population, lineage, compartment or subset, whose expression or pattern of expression changes throughout development. Nucleic acid marker expression may be detected or measured by any technique known in the art including nucleic acid sequence amplification (e.g. polymerase chain reaction) and nucleic acid hybridization (e.g.
  • Protein marker expression may be detected or measured by any technique known in the art including flow cytometry, immunohistochemistry, immunoblotting, protein arrays, protein profiling (e.g. 2D gel electrophoresis), although without limitation thereto.
  • Such terms are commonplace and well-understood by the skilled person when characterizing cell phenotypes.
  • a skilled person would conclude the presence or evidence of a distinct signal for the marker when carrying out a measurement capable of detecting or quantifying the marker in or on the cell.
  • the presence or evidence of the distinct signal for the marker would be concluded based on a comparison of the measurement result obtained for the cell to a result of the same measurement carried out for a negative control (for example, a cell known to not express the marker) and/or a positive control (for example, a cell known to express the marker).
  • a positive cell may generate a signal for the marker that is at least 1.5-fold higher than a signal generated for the marker by a reference cell (e.g. negative control cell) or than an average signal generated for the marker by a population of reference or negative control cells, e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher, at least 100-fold higher, or even higher.
  • a reference cell e.g. negative control cell
  • an average signal generated for the marker by a population of reference or negative control cells e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher, at least 100-fold higher, or even higher.
  • a positive cell may generate a signal for the marker that is 3.0 or more standard deviations, e.g., 3.5 or more, 4.0 or more, 4.5 or more, or 5.0 or more standard deviations, higher than an average signal generated for the marker by a population of reference or negative control cells.
  • the terms “culture medium,” “cell culture medium,” “defined medium,” “first defined medium,” “second defined medium,” “third defined medium,” “fourth defined medium,” “fifth defined medium”, “sixth defined medium”, and “seventh defined medium” refer to media that are suitable to support the growth of cells in vitro (i.e., cell cultures, cell lines, etc.). It is not intended that the term be limited to any particular culture medium. For example, it is intended that the definition encompass maintenance media as well as other media for the differentiation or specialization of cells. Indeed, it is intended that the term encompass any culture medium suitable for the growth of the cell cultures and cells of interest.
  • the cell culture medium used in various steps includes a basal medium which is supplemented.
  • the basal medium is an STAPEL Medium (Ng E.S., et al. Nat. Biotechnol. 2016;34: 1168-1179).
  • ODM an STAPEL Medium
  • the STAPEL medium was prepared by mixing 0.5% OsrHSA, 0.5% BSA(bovostar), , 0.05% polyvinyl alcohol (Sigma- Aldrich), IxGlutaMAX, Ixascorbic acid-2-phosphate (Sigma- Aldrich), ITSE AF blood-free cell culture media supplement (50pgml-l; InVitria), linoleic and linolenic acid Soybean oil (125ngml-l; Sigma- Aldrich), synthetic cholesterol (4pgml-l; Sigma- Aldrich), and protein-free hybridoma mix II (5%) in IMDM/F12 media.
  • enriched is used to refer to a population of cells which contains a significant proportion of a specific subset or subtype of cell, wherein the set of cells may contain 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95% or 100% of the specific subset/subtype of cell.
  • Enriched as in an enriched population of cells, can be defined phenotypically based upon the increased number of a specific subset or subtype of cells having a particular marker, or combination of markers, or having one or more markers and lacking one or more other markers, in a fractionated, or expanded, set of cells as compared with the number of cells having the marker, combination of markers, or having one or more markers and lacking one or more other markers, in the unfractionated or unexpanded set of cells.
  • tissue means an aggregate of cells.
  • the cells in the tissue are cohered or fused.
  • “reduced,” “reduction,” “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “increases”, or “enhance” or “activate” or “to a greater extent” are all used herein to generally mean an increase of a property, level, or other parameter, including by a statistically significant amount; for the avoidance of any doubt, the terms “increased”, “increase”, “to a greater extent,” “enhance” or “activate” can refer to an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, at least about a 20
  • a “reference level” refers to the level of a marker or parameter in a normal, otherwise unaffected cell population or tissue (e.g., a cell, tissue, or biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, e.g., cell, tissue, or a biological sample obtained from a patient prior to being diagnosed with a disease, or a biological sample that has not been contacted with an agent or composition as disclosed herein).
  • a reference level can also refer to the level of a given marker or parameter in a subject, organ, tissue, or cell, prior to administration of a treatment, e.g., with an agent or via administration of a composition.
  • a “control” or an “appropriate control” refers to an untreated, otherwise identical cell, subject, organism, or population (e.g., a cell, tissue, or biological sample that was not contacted by an agent or composition described herein) relative to a cell, tissue, biological sample, or population contacted or treated with a given treatment.
  • an appropriate control can be a cell, tissue, organ or subject that has not been contacted with an agent or subjected to the same methods as described herein.
  • assessing the expression of various genes includes comparing the fold change.
  • the fold change is used to measure the change in the expression level of genes.
  • the expression of a gene can be expressed as relative expression comparative to a housekeeping gene.
  • agonist or “activator” may be used interchangeably and as used herein means an activator, for example, of a pathway or signalling molecule.
  • An agonist of a molecule can retain substantially the same, or a subset, of the biological activities of the molecule (e.g. FGF).
  • FGF biological activity of the molecule
  • an FGF agonist or FGF activator means a molecule that selectively activates FGF signalling.
  • inhibitor means a selective inhibitor, for example of a pathway or signalling molecule.
  • An inhibitor or antagonist of a molecule e.g. BMP4 inhibitor
  • BMP4 inhibitor can inhibit one or more of the activities of the naturally occurring form of the molecule.
  • a BMP4 inhibitor is a molecule that selectively inhibits BMP signalling mediated by BMP4.
  • cell population or “population of XXX cells” as used herein is meant as an in vitro or ex vivo collection of cells.
  • step la is performed before step lb
  • step lb is performed before step 1c, and so on.
  • the term “monolayer” as used herein refers to a 2D adherent cell culture.
  • the methods of the present invention comprise a TGF-beta pathway activator.
  • the TGF-beta pathway activator is selected from the group consisting of Activin A, TGF-betal, TGF-beta2, TGF-beta3, IDE1/2 (IDE1 (l-[2- [(2Carboxyphenyl)methylene]hydrazide]heptanoic acid), IDE2 (Heptanedioic acid-l-(249 cyclopentylidenehydrazide)), and Nodal.
  • the TGF-beta pathway activator is Activin A.
  • the concentration of Activin A in the medium used in the methods of the invention is about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29 ng/mL, about 30 ng/mL.
  • the methods of the present invention comprise a WNT pathway activator or a WNT agonist.
  • the WNT pathway activator or WNT agonist is selected from the group consisting of CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)5-(5- methyl- 1 H-imidazol-2-yl)-2-pyrimidinyl] amino] ethyl] amino] -3 -pyridinecarbonitrile), Wnt 1 , Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a/b, Wnt-7b, Wnt48 8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-lOa, Wnt-lOb, Wnt-11, Wnt-16b, RSPO co-agonists, lithium chlor
  • the Wnt pathway activator is CHIR99021.
  • the WNT agonist in the cell culture media is CHIR99021 ((CHIR) CAS 252917-06-9).
  • the concentration of CHIR99021 in the medium used in the methods of the invention is about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3 pM, about 3.1 pM, about 3.2 pM, about 3.3 pM, about 3.4 pM, about 3.5 pM, about 3.6
  • the methods of the present invention comprise FGF.
  • the FGF is selected from the group consisting of FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20 and FGF23.
  • the FGF is FGF2.
  • the concentration of FGF2, also known as basic fibroblast growth factor (bFGF), in the medium used in the methods of the invention is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28
  • the methods of the present invention comprise a PI3K pathway inhibitor.
  • the PI3K pathway inhibitor is selected from the group consisting of AS 252424 (5-[[5-(4-Fluoro-2-hydroxyphenyl)-2-furanyl]methylene]-2,4- thiazolidinedione), AS 605240 (5-(6-Quinoxalinylmethylene)-2,4-thiazolidine-2, 4-dione), AZD 6482 ((-)-2-[[(lR)l-[7-Methyl-2-(4-morpholinyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-9- yl]ethyl]amino]benzoic acid), BAG 956 (a,a,-Dimethyl-4-[2-methyl-8-[2-(3-pyridinyl)ethynyl]- lH-imidazo[4,5c]quinol
  • the PI3K pathway inhibitor is PIK90.
  • the concentration of PIK90, in the medium used in the methods of the invention is about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 82 nM, about 84 nM, about 86 nM, about 88 nM, about 90 nM, about 92 nM, about 94 nM, about 96 nM, about 98 nM, about 100 nM, 100 nM, about 102 nM, about 104 nM, about 106 nM, about 108 nM, about 110 nM, about 112 nM, about 114 nM, about
  • the methods of the present invention comprise a Rho kinase inhibitor (ROCKi).
  • the Rho kinase inhibitor is Thiazovivin or Y- 27263.
  • the concentration of Y-27263 in the medium used in the methods of the invention is about 1 pM, about 2 pM, about 5 pM, about 8 pM, about 8.2 pM, about 8.4 pM, about 8.6 pM, about 8.8 pM, about 9 pM, about 9.2 pM, about 9.4 pM, about 9.6 pM, about 9.8 pM, about 10 pM, about 10.2 pM, about 10.4 pM, about 10.6 pM, about 10.8 pM, about 11 pM, about 11.2 pM, about 11.4 pM, about 11.6 pM, about 11.8 pM, about 12 pM, about 15 pM, about 20
  • the methods of the present invention comprise a TGF-beta pathway inhibitor.
  • the TGF-beta pathway inhibitor is selected from the group consisting of A-83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-lH-pyrazole- Icarbothioamide), D4476 (4-[4-(2,3-Dihydro-l,4-benzodioxin-6-yl)-5-(2-pyridinyl)- lHimidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]-2-pyridinyl]- N(tetrahydro-2H-pyran-4-yl)-benzamide), LY 364947 (4-[3-(2-Pyridinyl)-lH-pyrazol-4- yl
  • the TGF-beta pathway inhibitor is A-83-01.
  • the concentration of A83-O1 in the medium used in the methods of the invention is about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.82 pM, about 0.84 pM, about 0.86 pM, about 0.88 pM, about 0.9 pM, about 0.92 pM, about 0.94 pM, about 0.96 pM, about 0.98 pM, about 1 pM, about 1.02 pM, about 1.04 pM, about 1.06 pM, about 1.08 pM, about 1.1 pM, about 1.12 pM, about 1.14 pM, about 1.16 pM, about 1.18 pM, about 1.2 pM, about 1.4 pM, about 1.6 pM, about 1.8 pM, about 2
  • the methods of the present invention comprise a BMP pathway inhibitor.
  • the BMP pathway inhibitor is selected from the group consisting of Chordin, soluble BMPRla, soluble BMPRlb, Noggin, LDN-193189, and Dorsomorphin.
  • the BMP pathway inhibitor is LDN-193189.
  • the concentration of LDN-193189 in the medium used in the methods of the invention is about 50 nM, about 80 nM, about 110 nM, about 140 nM, about 170 nM, about 200 nM, about 205 nM, about 210 nM, about 215 nM, about 220 nM, about 225 nM, about 230 nM, about 235 nM, about 240 nM, about 245 nM, about 250 nM, about 255 nM, about 260 nM, about 265 nM, about 270 nM, about 275 nM, about 280 nM, about 285 nM, about 290 nM, about 295 nM, about 300 nM, about 325 nM, about 350 nM, about 375 nM, about 400 nM, about 425 nM, about 450 nM, about 475 nM, about 500 nM, about 525
  • the methods of the present invention comprise vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the VEGF includes human VEGF family members such as VEGFA as well as no-human VEGF.
  • the concentration of VEGF in the medium used in the methods of the invention is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/
  • the methods of the present invention comprise a BMP pathway activator.
  • the BMP pathway activator is selected from the group consisting of BMP4, BMP2 and BMP7.
  • the BMP pathway activator is BMP4.
  • the concentration of bone morphogenetic protein 4 (BMP4) in the medium used in the methods of the invention is about 5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL.
  • BMP4 bone morphogenetic protein 4
  • the methods of the present invention comprise stem cell factor (SCF).
  • SCF stem cell factor
  • concentration of SCF in the medium used in the methods of the invention is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 160 ng/mL, about 170 ng/mL, about 180 ng/mL, about
  • the concentration of interleukin 3 (IL3) in the medium used in the methods of the invention is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 8.2 ng/mL, about 8.4 ng/mL, about 8.6 ng/mL, about 8.8 ng/mL, about 9 ng/mL, about 9.2 ng/mL, about 9.4 ng/mL, about 9.6 ng/mL, about 9.8 ng/mL, about 10 ng/mL, about 10.2 ng/mL, about 10.4 ng/mL, about
  • the concentration of fms-related tyrosine kinase 3 ligand (FLT3-L) in the medium used in the methods of the invention is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 8.2 ng/mL, about 8.4 ng/mL, about 8.6 ng/mL, about 8.8 ng/mL, about 9 ng/mL, about 9.2 ng/mL, about 9.4 ng/mL, about 9.6 ng/mL, about 9.8 ng/mL, about 10 ng/mL, about 10.2 ng/mL, about 10.4 ng/mL, about 10.6 ng/mL, about 10.8 ng/mL, about 11 ng/mL, about 11.2 ng/mL,
  • the concentration of Interleukin- 15 (IL15) in the medium used in the methods of the invention is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/m
  • the concentration of Interleukin-7 (IL7) in the medium used in the methods of the invention is about 0.1 ng/mL, about 0.2 ng/mL, about 0.3 ng/mL, about 0.4 ng/mL, about 0.5 ng/mL, about 0.6 ng/mL, about 0.7 ng/mL, about 0.8 ng/mL, about 0.9 ng/mL, 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL,
  • the concentration of Macrophage Colony-Stimulating Factor is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, or about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL, about 325
  • the concentration of Granulocyte Macrophage Colony- Stimulating Factor is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, or about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL
  • the concentration of interleukin- 34 is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, or about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL, about 325 ng/mL, about 350
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • AECs DLL4-expressing arterial endothelial cells
  • the inventors of the present application have identified a need to develop a new monolayer differentiation method that robustly generated NOTCH-ligand expressing haemogenic endothelial cells, whose gene profile resembles that of arterial haemogenic endothelial cells found the in AGM.
  • ALK1, ALK2, ALK3, and ALK6 activin receptorlike kinase (ALK) and addition of BMP4 allows for the generation of the population of DLL4- expressing arterial endothelial cells (AECs). This approach is in contrast with methods known in the art which typically require early activation of BMP4.
  • the present invention provides a method for generating a population of DLL4- expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • AECs arterial endothelial cells
  • a fourth defined medium comprising at least one of SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells, and optionally cryopreserving the population of DLL4-expressing AECs.
  • the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • AECs arterial endothelial cells
  • a second defined medium comprising a TGF-beta pathway inhibitor, a Wnt pathway activator, a BMP pathway inhibitor, FGF and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc.
  • incubating the population of CD 13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating a population of CD13+ and KDR+ mesodermal-endothelial cells, ld.
  • the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb.
  • AECs arterial endothelial cells
  • the population of substantially undifferentiated pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).
  • the iPSCs are selected from the group consisting of iPSC RM3.5 (ma e) RAGI:GFP , iPSC PB0-01 (male), iPSC PB0-04 (female), iPSC PB0-05 (female), iPSC PB0-06 (male), iPSC PB0-10 (male), iPSC CRL2429 (ATCC), and the like.
  • the ESCs are selected from the group consisting of ESC Hl (male), ESC H9 (female) FAG/;GF/> , ESC ⁇ soxn.-tdTOMATo.-Ruxi ⁇ GFP ⁇ ESC HES3(female) Mm7;GFF , and the like.
  • the culturing in step la is performed in a first defined medium capable of and for a time sufficient for generation of a population of MIXL1+ cells.
  • the first defined medium comprises Activin A, CHIR99021 , FGF2, and PIK90.
  • the first defined medium is free of or is essentially free of or does not comprise BMP4.
  • the concentration of Activin A in the first defined medium is about 30 ng/mL (preferably, from 10 to 50 ng/mL), the concentration of CHIR99021 in the first defined medium is about 4 pM (preferably, from 1 to 10 pM), the concentration of FGF2 in the first defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL), and the concentration of PIK90 in the first defined medium is about 100 nM (preferably, from 10 to 300 nM).
  • the time sufficient for generation of a population of MIXL1+ cells is about 24 hours (preferably, from 2 to 72 hours).
  • the population of MIXL1+ cells at the end of step la is at least 75% of total cells.
  • the culturing in step la is optionally performed in a first defined medium further comprising Y-27263.
  • concentration of Y-27263 is about 1 to 50 pM, preferably about 8 to 12 pM, more preferably about 10 pM
  • the culturing in step lb is performed in a second defined medium capable of and for a time sufficient for generation of a population of CD 13+ early mesoderm cells.
  • the second defined medium comprises A83-O1, CHIR99021, LDN-193189, and FGF2.
  • the second defined medium is free of or is essentially free of or does not comprise BMP4.
  • the concentration of A83-01 in the second defined medium is about 1 pM (preferably, from 0.1 to 10 pM), the concentration of CHIR99021 in the second defined medium is about 3 pM (preferably, from 1 to 10 pM), the concentration of LDN-193189 in the second defined medium is about 250 nM (preferably, from 50 to 750 nM), and the concentration of FGF2 in the second defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL).
  • the time sufficient for generation of a population of CD 13+ early mesoderm cells is about 24 hours (preferably, from 2 to 72 hours).
  • the population of CD13+ early mesoderm cells at the end of step lb is at least 85% of total cells.
  • the culturing in step 1c is performed in a third defined medium capable of and for a time sufficient for generation of a population of CD 13+ and KDR+ mesodermal-endothelial cells.
  • the third defined medium comprises A83-01, VEGF, BMP4, and FGF2.
  • the concentration of A83- 01 in the third defined medium is about 1 pM (preferably, from 0.1 to 10 pM), the concentration of VEGF in the third defined medium is about 50 ng/mL (preferably, from 10 to 100 ng/mL), the concentration of BMP4 in the third defined medium is about 30 ng/mL (preferably, from 10 to 100 ng/mL), and the concentration of FGF2 in the third defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL).
  • the time sufficient for generation of a population of CD13+ and KDR+ mesodermal-endothelial cells is about 24 hours (preferably, from 2 to 72 hours).
  • the population of CD13+ and KDR+ mesodermal-endothelial cells at the end of step lc is at least 20% of total cells.
  • the culturing in step Id is performed in a fourth defined medium capable of and for a time sufficient for generation of CD34+ cells.
  • the CD34+ cells are DLL4-expressing arterial endothelial cells (AECs).
  • the CD34+ cells uniformly co-express the AEC markers VE-cadherin, CXCR4, and DLL4.
  • the fourth defined medium comprises SCF, VEGF, BMP4, and FGF2.
  • the population of CD34+ cells at the end of step Id is at least 70% of total cells.
  • one input pluripotent stem cell gives rise to approximately 6 DLL4+ CD34+ AECs at the and of step Id.
  • a population of DLL4-expressing arterial endothelial cells obtained from the method disclosed herein, wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
  • the population of CD34+ cells co-expresses CXCR4.
  • the population of CD34+ cells co-expresses CDH5 (VE-cadherin).
  • the present invention provides a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
  • the population of CD34+ cells co-expresses CXCR4.
  • the population of CD34+ cells co-expresses CDH5 (VE-cadherin).
  • the population of DLL4-expressing AECs can be optionally cryopreserved and subsequently thawed and further cultured.
  • the method further comprises cryopreserving the population of DLL4-expressing AECs following step Id.
  • a cryopreserved population of DLL4-expressing AECs obtained according to the methods described herein. That is, in one embodiment, there is provided a cryopreserved population of DLL4-expressing AECs produced according to steps la to Id as described above.
  • AECs arterial endothelial cells
  • NOTCH signalling is increased in emerging haematopoietic progenitor cells thereby obviating a requirement for addition of exogenous NOTCH ligands, and leading to lymphoid commitment by the haematopoietic progenitors and to enhanced generation of ILCs.
  • the present invention provides a method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • the present invention provides a method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • step 2a is carried out for a period of time and under conditions suitable for generation of a monolayer of the DLL4-expressing arterial endothelial cells (AECs) produced according to the methods described herein.
  • the population of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id and utilised in step 2a were cryopreserved and thawed prior to the commencement of step 2a.
  • the incubating in step 2b is performed in a fifth defined medium capable of and for a time sufficient for generation of CD34+CD43+ haematopoietic progenitor cells.
  • the fifth defined medium comprises SCF, VEGF, FGF2, IL3, and Flt3L.
  • the concentration of SCF in the fifth defined medium is about 100 ng/mL (preferably, from 50 to 500 ng/mL), the concentration of VEGF in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of IL3 in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of Flt3L in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL).
  • the time sufficient for generation of a CD34+CD43+ haematopoietic progenitor is about 144 hours (preferably, from 72 to 288 hours).
  • the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 2b is at least 25% of total cells.
  • the incubating in step 2c is performed in a sixth defined medium capable of and for a time sufficient for generation of CD34+CD7+ lymphoid haematopoietic progenitor cells.
  • the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7.
  • the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL), and the concentration of IL7 in the sixth defined medium is about 1 ng/mL (preferably, from 0.1 to 10 ng/mL).
  • the time sufficient for generation of CD34+CD7+ lymphoid haematopoietic progenitor cells is about 72 hours (preferably, from 24 to 144 hours).
  • the population of CD34+CD7+ lymphoid haematopoietic progenitor cells at the end of step 2c is at least 40% of total cells.
  • the incubating in step 2d is performed in a sixth defined medium capable of and for a time sufficient for generation of CD34-CD7+ and CD7+RAG1+ lymphoid cells.
  • the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7.
  • the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL), and the concentration of IL7 in the sixth defined medium is about 1 ng/mL (preferably, from 0.1 to 10 ng/mL).
  • the time sufficient for generation of CD34-CD7+ and CD7+RAG1+ lymphoid cells is about 96 hours (preferably, from 48 to 192 hours).
  • the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells at the end of step 2d is at least 45% of total cells.
  • the incubating in step 2e is performed in a seventh defined medium capable of and for a time sufficient for generation of mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells.
  • the seventh defined medium comprises Flt3L, VEGF, FGF2, IL7, and IL15.
  • the concentration of Flt3L in the seventh defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL)
  • the concentration of VEGF in the seventh defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL)
  • the concentration of FGF2 in the seventh defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL)
  • the concentration of IL7 in the seventh defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL)
  • the concentration of IL 15 in the seventh defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL).
  • the time sufficient for generation of mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells is about 96 hours (preferably, from 48 to 192 hours).
  • the population of PSC-derived innate lymphoid cells (ILCs) at the end of step 2e is at least 75% of total cells and the population of PSC-derived NK-like cells at the end of step 2e is at least 50% of total cells.
  • the population of PSC-derived innate lymphoid cells (ILCs) at the end of step 2e is at least 75% of total cells or the population of PSC-derived NK-like cells at the end of step 2e is at least 50% of total cells.
  • the PSC-derived innate lymphoid cells are CD161+CD7+ cells and CD161+RAG1- cells. In yet another preferred embodiment, the PSC-derived innate lymphoid cells (ILCs) are CD161+CD7+ cells or CD161+RAG1- cells. In yet another preferred embodiment, the population of CD161+CD7+ cells is at least 70% of total cells. In yet another preferred embodiment, the population of CD161+RAG1- cells is at least 70% of total cells. [000179] In one embodiment, there is provided a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells obtained from the method disclosed herein.
  • the PSC-derived ILCs are CD161+CD7+ cells. In another preferred embodiment, the PSC-derived ILCs are CD161+RAG1- cells. In another preferred embodiment, the NK-like cells are CD161+CD56+ cells.
  • the present invention provides a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells.
  • the PSC-derived ILCs are CD161+CD7+ cells.
  • the PSC-derived ILCs are CD161+RAG1- cells.
  • the NK-like cells are CD161+CD56+ cells.
  • the present invention provides a method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • 3c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • the present invention provides a method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • 3c incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • the present invention provides a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • the present invention provides a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • Steps 3a and 4a are identical.
  • step 3a or step 4a is carried out for a period of time and under conditions suitable for generation of a monolayer of the DLL4-expressing arterial endothelial cells (AECs) produced according to the methods described herein.
  • the population of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id and utilised in step 3 a or 4a were cryopreserved and thawed prior to the commencement of step 3 a or 4a.
  • Steps 3b and 4b are identical.
  • the incubating in step 3b or 4b is performed in a fifth defined medium capable of and for a time sufficient for generation of CD34+CD43+ haematopoietic progenitor cells.
  • the fifth defined medium comprises SCF, VEGF, FGF2, IL3, and Flt3L.
  • the concentration of SCF in the fifth defined medium is about 100 ng/mL (preferably, from 50 to 500 ng/mL), the concentration of VEGF in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of IL3 in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of Flt3L in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL).
  • the time sufficient for generation of a CD34+CD43+ haematopoietic progenitor is about 144 hours (preferably, from 72 to 288 hours).
  • the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 3b or 4b is at least 25% of total cells.
  • Steps 3c and 4c are identical except for the concentration of IL7 and for the types of cells generated.
  • the incubating in step 3c is performed in a sixth defined medium capable of and for a time sufficient for generation of cell population enriched in CD161+RAG1- cells.
  • the PSC-derived innate lymphoid cells are CD161+RAG1- cells.
  • the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 20 ng/mL (preferably, from 10 to 50 ng/mL).
  • the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), and the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 10 to 20 ng/mL).
  • the time sufficient for generation of cell population enriched in CD161+RAG1- cells is about 11 days (preferably, from 7 to 21 days).
  • the cell population enriched in CD161+RAG1- cells at the end of step 3c is at least 50% of total cells.
  • the sixth defined medium employed in step 3c is supplemented with IL15 starting from about 72h to about 168h following commencement of step 3c until completion of step 3c.
  • the concentration of IL 15 is about 20 ng/mL (preferably, from 1 to 100 ng/mL).
  • one input pluripotent stem cell gives rise to from 5 to 76 CD161+ CD7+ cells at the and of step 3c.
  • the incubating in step 4c is performed in a sixth defined medium capable of and for a time sufficient for generation of cell population enriched in CD4+CD8a+ cells.
  • the PSC- derived T cells are CD4+CD8a+ cells.
  • the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 0.1 ng/mL (preferably, from 0.05 to 1 ng/mL).
  • the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL)
  • the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL)
  • the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL).
  • the time sufficient for generation of cell population enriched in CD4+CD8a+ cells is about 11 days (preferably, from 7 to 21 days).
  • the cell population enriched in CD4+CD8a+ cells at the end of step 4c is at least 12% of total cells.
  • a cell population enriched in PSC-derived ILCs obtained from the method disclosed herein.
  • the PSC-derived ILCs are CD161+RAG1- cells.
  • a cell population enriched in PSC-derived T cells obtained from the method disclosed herein.
  • the PSC-derived T cells are CD4+CD8a+ cells.
  • the present invention provides a cell population enriched in PSC-derived ILCs.
  • the PSC-derived ILCs are CD161+RAG1- cells.
  • the PSC-derived T cells are CD4+CD8a+ cells.
  • the inventors also surprisingly found an alternative method for efficiently generating NK-like cells from CD34+CD7+ lymphoid haematopoietic progenitor cells. When said alternative method is used, a cell sorting step is not required.
  • RAG1 expression levels presaged gene expression profiles indicative of further commitment to the adaptive lymphoid lineage (RAGl-high) or the innate lymphoid lineage (RAG 1 -low).
  • RAG1 expression may be indicative of fate decisions between different branches of the ILC lineages and that PSC-derived NK-like cells can be generated from a RAG1+ intermediate.
  • the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising:
  • the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
  • AECs arterial endothelial cells
  • a seventh defined medium comprising IL15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IL 15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when present the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL and, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
  • the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of: 6a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
  • AECs arterial endothelial cells
  • separating the cell suspension from the monolayer removing the sixth defined medium from the suspension, and adding a seventh defined medium comprising IL15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IL15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when present the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
  • Steps 5a and 6a are identical.
  • step 5a or step 6a is carried out for a period of time and under conditions suitable for generation of a monolayer of the DLL4-expressing arterial endothelial cells (AECs) produced according to the method described herein.
  • AECs arterial endothelial cells
  • the population of DLL4-expressing AECs produced according to the method as previously described in steps la to Id and utilised in step 5a or 6a were cryopreserved and thawed prior to the commencement of step 5a or 6a.
  • step 5a 1 is carried out for a period of time and under conditions suitable for generation of a population of cells enriched in CD161+RAG1+ cells.
  • the population of cells enriched in CD161+RAG1+ cells is prepared according to the methods described herein.
  • Steps 5b and 6b are identical.
  • the incubating in step 5b or 6b is performed in a fifth defined medium capable of and for a time sufficient for generation of CD34+CD43+ haematopoietic progenitor cells.
  • the fifth defined medium comprises SCF, VEGF, FGF2, IL3, and Flt3L.
  • the concentration of SCF in the fifth defined medium is about 100 ng/mL (preferably, from 50 to 500 ng/mL), the concentration of VEGF in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of IL3 in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of Flt3L in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL).
  • the time sufficient for generation of a CD34+CD43+ haematopoietic progenitor is about 144 hours (preferably, from 72 to 288 hours).
  • the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 5b or 6b is at least 25% of total cells.
  • step c The two methods for generation of a cell population enriched in PSC-derived NK-like cells diverges from step c, onwards. That is, steps 5c to 5e are different from steps 6c to 6d.
  • the incubating in step 5c is performed in a sixth defined medium capable of and for a time sufficient for generation of a cell suspension comprising a population of CD161+RAG1+ cells.
  • the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 20 ng/mL (preferably, from 10 to 50 ng/mL).
  • the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL)
  • the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL)
  • the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL).
  • the time sufficient for generation of the cell suspension comprising the population of CD161+RAG1+ cells at the end of step 5c is about 7 days (preferably, from 3 to 14 days).
  • a person skilled in the art is aware of the typical or standard procedures used for separating a cell suspension from a cell monolayer.
  • a person skilled in the art is also aware of the typical or standard procedures used for sorting a cell suspension for or to obtain a cell population enriched in a particular cell type (such as through the expression of one or more genes or proteins, typically one or more markers expressed on the cell surface).
  • antibodies or similar agents specific for a given marker, or set of markers can be used to separate and isolate the desired cells using fluorescent activated cell sorting (FACS), panning methods, magnetic particle selection, particle sorter selection and other methods known to persons skilled in the art.
  • the sorting at step 5d is fluorescence-activated cell sorting (FACS).
  • the incubating in step 5e is performed in a seventh defined medium capable of and for a time sufficient for generation of cell population enriched in CD161+CD56+ cells.
  • the PSC-derived NK-like cells are CD161+CD56+ cells.
  • the seventh defined medium comprises Flt3L, VEGF, FGF2, and IL15, wherein the concentration of IL15 is about 20 ng/mL (preferably, from 1 to 100 ng/mL).
  • the seventh defined medium comprises Flt3L, VEGF, FGF2, IL7, and IL15, wherein the concentration of IL7 is about 20 ng/mL (preferably, from 10 to 50 ng/mL) and the concentration of IL15 is about 20 ng/mL (preferably, from 1 to 100 ng/mL).
  • the concentration of Flt3L in the seventh defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the seventh defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), and the concentration of FGF2 in the seventh defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL).
  • the time sufficient for generation of cell population enriched in CD161+CD56+ cells is about 96 hours (preferably, from 48 to 192 hours).
  • the cell population enriched in CD161+CD56+ cells at the end of step 5e is at least 70% of total cells.
  • one input pluripotent stem cell gives rise to approximately 1 to 2.5 CD161+ CD56+ cells at the and of step 5e.
  • the incubating in step 6c is performed in a sixth defined medium capable of and for a time sufficient for generation of a cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells.
  • the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7.
  • the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL)
  • the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL)
  • the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL)
  • the concentration of IL7 in the sixth defined medium is about 1 ng/mL (preferably, from 0.1 to 10 ng/mL).
  • the time sufficient for generation of cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells is about 72 hours (preferably, from 24 to 144 hours).
  • the population of CD34+CD7+ lymphoid haematopoietic progenitor cells at the end of step 6c is at least 40% of total cells.
  • step 6d comprises three separate sub-steps namely the sub-step of separating a cell suspension from a monolayer, the sub-step of removing the sixth defined medium from the suspension thereby obtaining cells from the suspension that is free from or essentially free from the sixth defined medium, and the sub-step of adding a seventh defined medium to the cells obtained from the previous sub-step.
  • the final sub-step of step 6d is performed in a seventh defined medium capable of and for a time sufficient for generation of cell population enriched in CD161+CD56+ cells.
  • the PSC-derived NK-like cells are CD161+CD56+ cells.
  • the seventh defined medium comprises Flt3L, VEGF, FGF2, IL7, and IL15, wherein the concentration of IL7 is about 20 ng/mL (preferably, from 1 to 100 ng/mL) and the concentration of IL 15 is about 20 ng/mL (preferably, from 1 to 100 ng/mL).
  • the concentration of Flt3L in the seventh defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL)
  • the concentration of VEGF in the seventh defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL)
  • the concentration of FGF2 in the seventh defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL).
  • the time sufficient for generation of cell population enriched in CD161+CD56+ cells is about 120 hours (preferably, from 48 to 240 hours).
  • the cell population enriched in CD161+CD56+ cells at the end of step 6d is at least 80% of total cells.
  • the cells in step 6d are maintained in said seventh defined medium for a time sufficient for generation of cell population enriched in CD161+CD16+ cells, preferably wherein the time is from 10 to 17 days, even more preferably 15 days.
  • the cells obtained after 15 days display an enhanced cytotoxic function compared to cells obtained maintained in said medium for 5 days.
  • a cell population enriched in PSC-derived NK- like cells obtained from the method disclosed herein.
  • the PSC- derived NK-like cells are CD161+CD56+ cells.
  • the present invention provides a cell population enriched in PSC-derived NK-like cells.
  • the PSC-derived NK-like cells are CD161+CD56+ cells.
  • Methods for generation of a cell population enriched in PSC-derived erythroid and myeloid cells [000209] The inventors surprisingly found that the method disclosed herein can be used to efficiently generate cells representing a broad spectrum of linages include erythroid and myeloid lineages.
  • a method for generating a cell population enriched in PSC-derived erythroid cells, wherein the PSC-derived erythroid cells are CD235a+CD14- cells comprising the sequential steps of:
  • AECs arterial endothelial cells
  • a method for generating a cell population enriched in PSC-derived myeloid cells, wherein the PSC-derived erythroid cells are CD235a-CD14+ cells comprising the sequential steps of:
  • AECs arterial endothelial cells
  • a ninth defined medium comprising one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), and IL34, and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a-CD14+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
  • MCSF Macrophage Colony-Stimulating Factor
  • GM-CSF Granulocyte Macrophage Colony-Stimulating Factor
  • IL34 optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7
  • Steps 7a and 8a are identical.
  • step 7a or step 8a is carried out for a period of time and under conditions suitable for generation of a monolayer of the DLL4-expressing arterial endothelial cells (AECs) produced according to the method described herein.
  • AECs arterial endothelial cells
  • the population of DLL4-expressing AECs produced according to the method as previously described in steps la to Id and utilised in step 7a or 8a were cryopreserved and thawed prior to the commencement of step 7a or 8a.
  • Steps 7b and 8b are identical.
  • the incubating in step 7b or 8b is performed in a fifth defined medium capable of and for a time sufficient for generation of CD34+CD43+ haematopoietic progenitor cells.
  • the fifth defined medium comprises SCF, VEGF, FGF2, IL3, and Flt3L.
  • the concentration of SCF in the fifth defined medium is about 100 ng/mL (preferably, from 50 to 500 ng/mL), the concentration of VEGF in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of IL3 in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of Flt3L in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL).
  • the time sufficient for generation of a CD34+CD43+ haematopoietic progenitor is about 144 hours (preferably, from 72 to 288 hours).
  • the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 7b or 8b is at least 25% of total cells.
  • the incubating in step 7c is performed in an eighth defined medium capable of and for a time sufficient for generation of cell population enriched in CD235a+ erythroid cells.
  • the PSC-derived CD235a+ erythroid cells are CD235a+CD14- cells.
  • the eighth defined medium comprises EPO, optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, wherein the concentration of EPO is about 2 units/mL (preferably, from 1 to 5 units/mL).
  • the concentration of Flt3L in the eighth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL)
  • the concentration of VEGF in the eighth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL)
  • the concentration of FGF2 in the eighth defined medium is about 20 ng/mL (preferably, from 10 to 20 ng/mL).
  • the time sufficient for generation of cell population enriched in CD235a+ cells is about 14 days (preferably, from 7 to 21 days).
  • the cell population enriched in CD235a+ cells at the end of step 7c is at least 80% of total cells.
  • a cell population enriched in PSC-derived erythroid cells obtained from the method disclosed herein.
  • the PSC- derived erythroid cells are CD235a+CD14- cells.
  • the incubating in step 8c is performed in a ninth defined medium capable of and for a time sufficient for generation of cell population enriched in CD 14+ myeloid cells.
  • the PSC-derived CD 14+ erythroid cells are CD235a- CD14+ cells.
  • the ninth defined medium comprises one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), and IL34, and optionally further comprises one or more of Flt3L, VEGF, FGF2, and IL7.
  • MCSF Macrophage Colony-Stimulating Factor
  • GM-CSF Granulocyte Macrophage Colony-Stimulating Factor
  • IL34 optionally further comprises one or more of Flt3L, VEGF, FGF2, and IL7.
  • the concentration of MCSF is about 50ng /mL (preferably, from 10 to 500 ng/mL). In a preferred embodiment, the concentration of GM-CSF is about 50ng /mL (preferably, from 10 to 500 ng/mL). In a preferred embodiment, the concentration of IL-34 is about lOOng /mL (preferably, from 10 to 500 ng/mL).
  • the concentration of Flt3L in the ninth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL)
  • the concentration of VEGF in the ninth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL)
  • the concentration of FGF2 in the ninth defined medium is about 20 ng/mL (preferably, from 10 to 20 ng/mL).
  • the time sufficient for generation of cell population enriched in CD 14+ cells is about 42 days (preferably, from 35 to 49 days).
  • the cell population enriched in CD235a+ cells at the end of step 7c is at least 50% of total cells.
  • a cell population enriched in PSC-derived myeloid cells obtained from the method disclosed herein.
  • the PSC- derived myeloid cells are CD14+CD235a- cells.
  • Other Cell Populations are provided.
  • a cell population may be obtained following any of the individual steps recited in any of the methods described herein. That is, by pausing or arresting the method, or obtaining a sample of cells after a first or any subsequent step of one of the methods described herein a population of cells may be obtained. Accordingly in one embodiment, the cell population obtained may be AECs obtained from step Id, or mesodermal-endothelial cells obtained from step 1c, or early mesoderm cells obtained from step lb, or MIXL1+ cells obtained from step la .
  • the cell population obtained may be population of CD34+CD43+ haematopoietic progenitor cells obtained from step 2b, a population of CD34+CD7+ lymphoid haematopoietic progenitor cells obtained from step 2c, a population of CD34-CD7+ lymphoid cells obtained from step 2d, a population of CD7+RAG1+ lymphoid cells obtained from step 2d, or a population of CD161+RAG1+ cells obtained from step 5c or 5d.
  • Pluripotent stem cells used in study is all human origin. Work related to human pluripotent stem cell lines was conducted in accordance with RCH Human Research Ethics Committee approval 33OO1A. Human PSC lines, including ESCs and iPSCs, used in this study are summarized as the follows: ESC Hl (male) (see, Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-7 (1998).), iPSC RM3.5 (male) ffAG2;GFP , ESC H9 (female) R4G2;GF (see, Motazedian, A. et al.
  • Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell- derived haematopoietic organoids. Nat Cell Biol 22, (2020).), ⁇ OX17MTOMAT °; RVX1C:GFP (see, Ng, E. S. et al. Differentiation of human embryonic stem cells to HOXA + hemogenic vasculature that resembles the aorta-gonad-mesonephros. Nat Biotechnol 34, 1168-1179 (2016).), ESC HES3(female) M2X£2 GF (see, Davis, R. P. et al.
  • hESCs and hiPSCs were sometimes grown in the presence of inactivated mouse embryonic fibroblasts in PSC media consisting of DMEM-F12, 20% knock-out serum replacement, Ixnon-essential amino acids, IxGlutaMAX, 0.11 mM -mercaptoethanol and FGF2 (10 ng/ml) as previously described (see, Costa, M., Sourris, K., Hatzistavrou, T., Elefanty, A. G. & Stanley, E. G. Expansion of human embryonic stem cells in vitro. Curr Protoc Stem Cell Biol Chapter 1, Unit 1C.1.1-1C.1.7 (2008).).
  • Day 0 Activin A, 30ng/ml; CHIR99021, 4pM; fibroblast growth factor (FGF) 2, 20ng/ml; PIK90, lOOnM; Y-27263 10 pM (optional for cell lines with poor viability after dissociation).
  • Day 1 A83-01, 1 pM; CHIR99021, 3pM; LDN-193189, 250 nM; FGF2, 20ng/ml.
  • Day 2 A83-01, 1 pM; vascular endothelium growth factor (VEGF), 50ng/ml; bone morphogenic protein (BMP4), 30ng/ml; FGF2, 20ng/ml.
  • VEGF vascular endothelium growth factor
  • BMP4 bone morphogenic protein
  • SCF stem cell factor
  • VEGF vascular endothelial growth factor
  • BMP4 lOng/ml
  • FGF2 50ng/ml
  • SCF stem cell factor
  • VEGF vascular endothelial growth factor
  • FGF2 50ng/ml
  • SCF lOOng/ml
  • VEGF vascular endothelial growth factor
  • FGF2 50ng/ml
  • IL3 interleukin 3
  • FLT3 ligand (FLT3-L) 10 ng/ml.
  • Activin A (338-AC, R&D Systems), BMP4 (314-BP, R&D Systems ), FGF2 (100- 18B, Peprotech), FLT3-L (300-19, Peprotech), IL3 (200-03, Peprotech), IL7 (200-07, Peprotech), IL15 (200-15, Peprotech), VEGF (100-20, Peprotech), SCF (synthesized by CSIRO), Y-27632 (72304, Stem Cell Technologies), CHIR99021 (4423, Tocris), PIK-90 (SI 187, Selleckchem), A83-01 (2939, Tocris), LDN-193189 (TB6053, Tocris).
  • G7 GFP+ hematopoietic cells were sorted using an Influx FACS sorter (BD) on day 19 of PSC differentiation. 5000 sorted RAG1 + cells were plated per well of 96-well round bottom plates, which enabled cells to be positioned in the centre of wells to visualise RAG! :GFP fluorescence. Cells were cultured with IL7 20ng/ml, IL15 20ng/ml, or IL7 and IL15 both 20ng/ml. Medium was carefully changed 2 days after replating (as day 2) and flow cytometry characterization was performed on day 4 after replating. Fluorescence images were taken using a Zeiss Observer Z1 fluorescent microscope and processed using Fiji for Mac OS X.
  • BD Influx FACS sorter
  • Anti-human conjugated bodies used for flow cytometry are as the follows.
  • CD4-PE BioLegend, 300508, RPA-T4, 1:30
  • CD7-APC BD Pharmingen, 561604, MT-701, 1:50
  • CD8a-PE-Cy7 BioLegend, 344712, SKI
  • CD13-PE-Cy7 BioLegend, 301712, WM15, 1:100
  • CD34-BV421 BioLegend, 343610, 581, 1:50
  • CD34-PE-Cy7 BioLegend, 343516, 581, 1:100
  • CD43-PE BioLegend, 343204, 10G7, 1:50
  • CD45-BV421 BioLegend, 304032, HI30; 1:30
  • CD56-PE BD Pharmingen, 555516, B 159, 1:50
  • CD127/IL7R-PE BioLegend, 351304, A019D5, 1:50
  • Conjugated antibodies were diluted in FACS wash buffer (PBS supplemented with 5% fetal bovine serum) and incubated with cells for 20 minutes on ice. The cell suspension was washed twice with FACS wash solution to remove unbound antibodies and resuspended in FACS wash solution containing 1 pg/ml propidium iodide. Cell surface staining was examined by Becton Dickenson (BD) LSRFortessa Cell Analyzer. Flow cytometry data was analyzed using the FlowLogic program (7.2.1, DataNova). Alternatively, cell purification was performed using a BD FACSaria FUSION or Infux cell sorter based on cell surface staining or the expression of a fluorescent reporter. Cells were collected using a 5ml FACS tube containing 0.5ml cold fetal calf serum. [000235] 1.7 Immunofluorescence and antibodies
  • K562 target cells were labelled with 100 pCi Chromium-51 (51Cr, PerkinElmer) for one hour at 37°C and subsequently co-cultured with PSC-derived NK cells or NK cells freshly isolated from healthy donors’ peripheral blood mononuclear cells (PBMC) by NK Cell Isolation Kit (Miltenyi Biotec). NK cells were added in triplicate wells at effector: target ratios from 4:1 to 1:1. Wells with target cells alone (spontaneous release) and target cells with 10% Triton X 100 (maximum release) were included as controls. After 4-hour or 16-hour co-culture, cells were spun down and supernatants were collected.
  • PBMC peripheral blood mononuclear cells
  • the amount of 5 ICr released in the supernatants was detected using a gamma counter (Wallac Wizard 1470).
  • the %specific lysis was calculated by [(experimental release - spontaneous release)/(maximum release - spontaneous release)] * 100. [000239] 1.8.1 K562 killing assay using flow cytometry.
  • K562 cells were maintained in RPMI +10% FCS +1% Pen Strep (medium changed weekly). For killing experiments, cells were passaged the day before and then given fresh medium on the morning of the experiment. Approximately 1 million K562 target cells suspended in 1 ml of phosphate buffered saline (PBS) were labelled with 1 pM carboxyfluorescein succinimidyl ester (CFSE) for 10 minutes at 37°, in the dark. Cells were pelleted and resuspended in 1 ml of PBS + 10% Fetal calf serum - and incubated for a further 30 mins at 37°.
  • PBS phosphate buffered saline
  • CFSE carboxyfluorescein succinimidyl ester
  • the R platform along with its suite of single cell bioinformatic packages with R version 4.2.1 was used to for single cell data analysis (www.R- project.org).
  • Seurat (v4.1.1) was used for single cell data pre-processing and subsequent downstream analysis and visualizations. Cells that were not within the quality control boundaries (see in GitHub) were excluded.
  • the standard Seurat pipeline including log normalization at a scale factor of 10000, scale data to centre gene expression values and principal component analysis (PCA) to reduce dimensions.
  • the FindCluster function was used to identify clusters within each sample.
  • PSC derived cells were integrated with fetal human embryonic AGM and fetal liver data at developmental stages week 4.5 to 15 (see, Calvanese, V. et al. Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature 604, 534-540 (2022).).
  • the integration was performed with Seurat’s FindlntegrationAnchors and IntegrateData function based on a list of genes identified by the SelectlntegrationFeatures function.
  • the human embryonic data, as a reference along with canonical markers was used for the identification of cell identities of each cluster.
  • the FindAllMarkers function was used to produce a list of genes that were specific to each cluster which were then used in visualization plots such as heatmaps, violin plots, dot plots and feature plots. Differential gene per cluster was performed with the FindMarkers function.
  • Analysis of endothelial to hematopoietic transition in Figure 2E utilize PSC derived cells from day 12 and 15 based on expression of CD31 or CD34 and RUNX1 or CDH5.
  • the analysis of blood cells from HSPC to lymphoid cells utilize a subset of PSC derived cells based on the “HSPC”, “ILC” and “RAG+Lymph” clusters identified in Figure 2A.
  • cells from “ILC” and “ILC_cyc” clusters were pooled from the Figure 3A. Additionally cell cycling genes were regressed out to avoid influence by ILC cells’ cycling state.
  • Genes displayed on Figure 8 heatmap ware the specific genes of each cluster based on reclustered and pooled cells identified as the “HSPC”, Lymph_prol, pro2 and pro3 clusters in Figure 3A.
  • Cells in Figure 8D were based on pooling of RAG1+ cells from day 19 and 25 samples.
  • the AverageExpression function was used to calculate the mean of RAG1 average expression across all clusters. Cells that were above the mean average expression of 2.259 (4sf) were labelled as “RAG-high” and those lower “RAG- low”. All subsets described above were re-clustered with the standard Seurat pipeline.
  • RNA sequencing raw data is available in the public GEO data repository under the Geo accession number: GSE217705.
  • CD34+CD43+ haematopoietic progenitor cells represented a substantial fraction of the culture, as assessed by flow cytometry ( Figure IE).
  • CD45+ blood cells begin to upregulate expression of the lymphoid lineage marker CD7, in response to the introduction of IL7 into the culture medium ( Figure IE).
  • RAG1 :GFP reporter lines a small fraction of RAGI+ cells were detected within the CD34+CD7+ haematopoietic cell population ( Figures IE and IF). The frequency this RAG1+ population increased over the next four days, an increase that was accompanied by downregulation of CD34 ( Figures IE and IF).
  • Fluorescence images also showed an accumulation of RAG1 :GFP+ cells from day 15 to day 19, suggestive of ongoing lymphoid differentiation (Figure 1G).
  • the majority of CD7+ cells were negative for RAG1 but positive for CD 161 (KLRB1), a cell surface marker frequently associated with innate lymphoid cells ( Figures 1H and II). Indeed, approximately half of the CD161+ cells also expressed the natural killer cell marker CD56.
  • Example 3 PSC-derived arterial haematopoietic culture (AHC) models human embryonic haematopoiesis.
  • SPINK2 and MLLT3 SPINK2 and MLLT3
  • erythroid cells HBZ, HBA1, and HBA2
  • myeloid haematopoietic lineages a variety of myeloid haematopoietic lineages
  • lymphoid lineages marked by RAG genes or ILC-associated genes Figures 2A, 2B, and 6B.
  • RM-tTom endothelial cells were thawed into T cell medium (supplemented with day 6 growth factors, as described in 1.2 above) and seeded onto adherent tissue culture plates to enable recovery. The following day, cells were detached from the plates using TrypLETM Select (ThermoFisher), counted, and then 150,000 cells were resuspended in T cell media containing VEGF 50ng/ml, EGF lOng/ml, FGF2 lOng/ml, hydrocortisone at 10 ng/ml.
  • Resuspended cells were then gently layered over a film of Matrigel (Merk) (300 pl/each well of a 24 well plate) and incubated for 24 hours. Cultures were imaged using a LSM900 confocal microscope and analysed using ImageJ.
  • the inventors detected activation of specific arterial-haemogenic genes, such as MECOM (EVI1 ), KCNK17, and SPINK2 (Figure 2E). Additionally, these AECs expressed a group of NOTCH-ligand genes, including DLL4, DLK1, JAG1 and JAG2 ( Figure 2C). Flow cytometry and immunofluorescence validated protein expression of these genes ( Figures 2F, 6C, and 6D). The inventors found CD34+ cells persistently expressed DLL4 from day 6 to day 19, creating conditions favourable for lymphopoiesis ( Figure 2F). Interestingly, although JAG1 was expressed by both endothelial cells and stromal cells, its endothelial expression gradually diminished from day 6 to day 19 ( Figure 6D).
  • lymph_prol on day 15
  • lymph_pro2 and lymph_pro3 on day 19
  • ILC-like cells KLRB1, NKG7 and GNLY ILC-like cells
  • T cell progenitors RAG/, CD5 and TCF7
  • lymph_pro2 showed a high-level expression of IL7 receptor gene (IL7R) and an upregulation of the NOTCH-pathway genes, NOTCH1 and HES4.
  • lympho_pro3 showed a downregulation of IL7R but increased expression of KLRB1, which may suggest a cell fate potentially directed towards an ILC-like phenotype.
  • analysis of a cohort of T cell differentiation genes indicated the lymph_pro2 and lympho_pro3 clusters showed characteristics consistent with differentiation trajectories towards T cell lineage and the ILC lineage, respectively (Figure 3D).
  • the transcription factors BCL11B and TCF7 key drivers of T cell commitment, were enriched in lymph_pro2 and T cell progenitors, consistent with the expression patterns of RAG1, RAG2, and PTCRA. Additionally, the elevated expression of IL7R in lymph_pro2 and T progenitor clusters, but not in lymph_pro3 and ILC populations, suggests IL7 signalling might be important for ILC versus T cell lineage specification.
  • lymphoid differentiation within the AHC system disclosed herein is highly sensitive to IL7 concentrations and reaffirms that high levels of IL7 favours the development of innate lymphoid lineages.
  • the highly defined nature the AHC system provides myriad opportunities to dissect key events in lymphoid lineage commitment and differentiation.
  • lymphoid derived NK cells Given the expansion of lymphoid derived NK cells in response to IL7 and IL 15, the inventors next examined if these same conditions were able generate NK cells from haematopoietic progenitors that arise at earlier stages of the AHC system disclosed herein, without a requirement for prior enrichment of progenitors using FACS.
  • suspension cells were separated from the AHC on this day and these cells were cultured in media supplemented with only IL7 and IL15.
  • flow cytometry analysis showed the efficient generation of CD161+CD56+ cells (Figure 8G). This was accompanied by a reduced number of RAG1+ cells.
  • PSCs were cultured according to the methods described in Examples 1 and 2 so as to give rise to a population of CD34+CD45+ haematopoietic progenitor cells by day 12 of culturing.
  • erythroid lineage cells cultures were grown in a medium comprising EPO (2 units/ml).
  • myeloid lineage cells cultures were grown in a medium comprising MCSF (50 ng/ml).
  • T lineage cells day 12 haematopoietic cells were transferred to monolayers of MS 5 cells expressing high levels of human DLL4 (MS5-hDLL4) or OP9-DLL4 cells; or day 15 CD34+CD45+ haematopoietic progenitor cells were co-cultured with MS5-hDLL4 cells using an air-liquid interface culture to generate artificial thymic organoids.
  • OP9 DLL4 Monolayer differentiations, one day prior to initiation of T-cell cultures, a tissue-culture treated 12-well plate was plated with 3 - 5 x 10 4 OP9 cells expressing high levels of human DLL4 (henceforth DLL4hi) per well in a-MEM with 10% FCS, lx GlutaMAX and lx Penicillin/Streptomycin.
  • Cell passage was performed by harvesting and mechanically-dissociating the whole cells within each well and passing the cell mixture through a 40 pm membrane to exclude cell clumps.
  • the flowthrough fraction was collected by centrifugation and resuspended in 1 mL of fresh RB27.
  • DLL4hi cells were used, and for subsequent weeks, OP9 cells with a lower level of DLL4 were used (DLL41o).
  • One well was harvested weekly for flow cytometry analysis.
  • Initiation of culture was performed by adding 10 5 day 12 CD45 + CD34 + blood progenitor cells per well to produce a total volume of 1 mL of RB27 per well.
  • Media top up was performed in 3 - 4 days by adding another 1 mL of RB27.
  • an 80% media change was performed by aspirating and replacing 80% of the media from each well with fresh RB27.
  • One well was harvested weekly for flow cytometry analysis.
  • RM-RAGLGFP iPSCs were used to assess the B lineage differentiation potential of blood cell progenitors generated at differentiation day 12. Briefly, B cell differentiations using the non-adherent cell fraction from day 12 cultures were seeded onto a monolayer of MS 5 stromal cells in RB27 medium (as detailed above) supplemented with IL7 (1 ng/ml), SCF (5ng/ml), FGF2 (5 ng/ml), and IL3 (5 ng/ml), with medium changed every 3-4 days. Cultures were examined for the generation of B cell progenitors using flow cytometry analysis for expression of CD 19 in conjunction with RAG1 (GFP). Table 2.
  • a human arterial-haematopoietic culture (AHC) system representing a simple and efficient method to study and model human embryonic haematopoiesis/lymphopoiesis in vitro, is described. Unique to this system is the generation of a lawn of NOTCH-ligand expressing arterial endothelial cells effectively direct lymphoid commitment from emerging haematopoietic progenitors. This AHC system enabled the dissection of haematopoietic cell fate determination and the identification of optimal conditions for producing human lymphoid progenitors, providing new opportunities for experimental research and medical applications.
  • This disclosure provides the first time-series single-cell map of in vitro human haematopoiesis, representing a key reference for studying blood cell development.
  • the development of the highly reproducible AHC system disclosed herein has enabled the generation of a dataset that spans key stages of haematopoietic ontogeny in vitro, providing an important reference for the development of methods for blood cell production.
  • the PSC-derived haematopoietic differentiation platform disclosed herein generated a spectrum of blood cell types that have counterparts in the AGM and foetal liver, including those belonging to the erythroid, myeloid, and lymphoid lineages ( Figures 2 and 3).
  • RNAseq data sets that examine specific stages of haematopoietic differentiation have been described previously, the data of the present disclosure is the first to capture a substantial temporal window spanning ontogeny stages from endothelium to lymphoid commitment.
  • lymphoid commitment and cell-fate specification between the T and the NK-ILC lineages were explored.
  • the cultures generated by the methods of the present disclosure gave rise to a primitive lymphoid progenitor marked by the expression of the stem cell gene SPINK2 ( Figures 3C and 3D).
  • the de novo appearance of this progenitor is consistent with observations from animal studies that point to the possibility of a non-HSC derived lymphoid competent precursors that contribute to early lymphopoiesis and the formation of primary lymphoid organs during embryogenesis.
  • IL7R expression levels foreshadowed the differing potential of progenitors to form T cell lineages (IL7R high) or ILC lineages (IL7R low), and that levels of IL7R signalling could be manipulated to effect cell fate choices ( Figures 4A, 4B, and 4C).
  • IL7 is dispensable for early lymphoid commitment, including RAG1 activation and CD7 upregulation, this cytokine was indispensable for RAG1+ upregulation that further drives progenitors towards T cell development ( Figures 4C, 8C, and 8D).
  • results disclosed herein show that the IL7R signalling network is regulated in a dose-dependent manner, with minimal levels of IL7 maintaining a level of IL7R expression required for ongoing T cell differentiation.

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Abstract

The present application relates to methods for the generation of human pluripotent stem cell- derived cells involved in haematopoiesis and lymphopoiesis, such as arterial endothelial cells, innate lymphoid cells, natural killer (NK)-like cells, and T cells. The present application also relates to haemogenic arterial endothelial cells, innate lymphoid cells, natural killer (NK)-like cells, T cells and erythroid cells and myeloid cells, and progenitors of such cells, produced by such methods.

Description

METHODS AND COMPOSITIONS FOR IN VITRO HAEMATOPOIESIS AND LYMPHOPOIESIS
Field
[0001] The present application relates to methods for the generation of human pluripotent stem cell-derived cells involved in haematopoiesis and lymphopoiesis, such as arterial endothelial cells, innate lymphoid cells, natural killer (NK)-like cells, and T cells. The present application also relates to haemogenic arterial endothelial cells, innate lymphoid cells, natural killer (NK)- like cells, T cells and erythroid cells and myeloid cells, and progenitors of such cells, produced by such methods.
Background
[0002] The study of human haematopoietic development underpins understanding regarding congenital immune disorders and provides guidelines for the in vitro production of immune cells for applications in basic research and cell therapies. Studies in mice have contributed significantly to the understanding of embryonic haematopoiesis, including its location, its endothelial origin, and factors affecting cell fate determination of immune cell lineages. Nevertheless, there is increasing evidence showing divergent mechanisms in the regulation of haematopoiesis between humans and mice, suggesting caution needs to be exercised when extrapolating results between the two species. Therefore, a human system that recapitulates the key events of embryonic haematopoiesis will be an important tool for understanding haematopoiesis and immune development from a human perspective. Such a system would also facilitate de novo generation of immune cells with potential clinical applications.
[0003] Differentiation of human PSCs has enabled the generation of haematopoietic cells in vitro. However, PSC-derived haematopoietic cells often resemble those derived from extra- embryonic haematopoiesis in the yolk sac, a transient wave of haematopoiesis in early embryo which predominantly contributes to erythroid and myeloid lineages. By contrast, intra- embryonic definitive haematopoiesis, arising from the dorsal aorta within the Aorta-Gonad- Mesonephros (A GM) gives rise to multi-lineage development encompassing erythroid, myeloid, and lymphoid cells. Despite many previous attempts, differentiation of PSCs to an AGM-like haematopoiesis has been challenging, primarily because embryonic data corresponding to this stage has been limited. In this context, recent spatial and single-cell transcriptomic analysis of human embryos has been highly illuminating; this data indicates that AGM haematopoiesis is built upon haemogenic endothelial cells with a distinct arterial gene profile that provides a touchstone for the derivation of AGM-like haematopoiesis from PSCs in vitro.
[0004] Studying lymphoid commitment in vitro is an additional challenge, an event that is highly dependent on NOTCH ligands. In vitro lymphopoiesis, from either primary human haematopoietic progenitors or PSCs, requires exogenous supply of NOTCH ligands provided with immobilized recombinant proteins, or ectopic expression by mouse stromal cells, such as OP9 and MS5. Additionally, the relative opacity of many in vitro PSC differentiation platforms not only impacts the reproducibility of these methods, but also affects the exact type of cells that are generated. In turn, this lack of clarity also represents concerns for manufacturing lymphoid cells from PSCs for clinical applications, which may result in variable treatment outcomes. Current protocols can generate NK cells from PSCs which display effective cell killing activity and show promise for biomedical applications. In the case of NK cells, it is likely such cells arise from a population that resembles yolk-sac derived erythroid-myeloid progenitors.
However, relationship of these cells to established lymphoid developmental pathways remains unclear.
[0005] Accordingly, there is a need to provide PSC-based AGM-like haematopoiesis that recapitulates events associated with early human haematopoiesis and lymphopoiesis, enabling the efficient generation of NK cells that arise from a defined lymphoid developmental path.
Summary of Invention
[0006] The inventors have surprisingly identified novel culture methods that robustly generated NOTCH-ligand expressing haemogenic endothelial cells, whose gene profile resembles that of arterial haemogenic endothelial cells found the in AGM, and which permit sufficient priming of haematopoietic progenitors to the lymphoid lineage enabling the generation of PSC-derived innate lymphoid cells, natural killer (NK)-like cells, and T cells, erythroid cells and myeloid cells. In the development of the novel culture methods described herein, the inventors provide a simplified culture system that enables the generation of the aforementioned cell types without the provision of exogenous NOTCH ligands or co-culture with exogenous stromal cells.
[0007] According to a first aspect, the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway activator, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising at least one of SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells, and optionally cry opreserving the population of DLL4-expressing AECs.
[0008] According to a second aspect, the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor, a Wnt pathway activator, a BMP pathway inhibitor, FGF and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal- endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells, and optionally cryopreserving the population of DLL4-expressing AECs.
[0009] According to a third aspect, the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising A83-O1, CHIR99021, LDN-193189, FGF2, and which is free or essentially free of BMP4, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising A83-O1, VEGF, BMP4, FGF2, for a time sufficient for generating a population of CD13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, BMP4, FGF2, for a time sufficient for generating the population of CD34+ cells, and optionally cryopreserving the population of DLL4-expressing AECs.
[00010] According to a fourth aspect, the present invention provides method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of: 2a. generating a monolayer of DEE4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
2b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IE3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising at least one of Flt3E, VEGF, FGF and IE7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising at least one of Flt3L, VEGF, FGF, IE7 and IE 15 for a time sufficient for generating the mixed population of PSC- derived innate lymphoid cells (IFCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[00011] According to a fifth aspect, the present invention provides method for generating a mixed population of PSC-derived innate lymphoid cells (IFCs) and PSC-derived NK-like cells, comprising the sequential steps of:
2a. generating a monolayer of DEE4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
2b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3E, VEGF, FGF2, and IE7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells, 2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising Flt3L, VEGF, FGF2, IL7, and IL15 for a time sufficient for generating the mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[00012] According to a sixth aspect, the present invention provides a method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
3b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3 and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[00013] According to a seventh aspect, the present invention provides a method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3, 3b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[00014] According to an eighth aspect, the present invention provides a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
4b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[00015] According to a ninth aspect, the present invention provides a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3, 4b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[00016] According to a tenth aspect, the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
5al. generating a population of cells enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells; and
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IL-7 and at least one of Flt3L, VEGF, FGF and IL 15, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[00017] According to an eleventh aspect, the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
5a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
5b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
5c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating a cell suspension comprising a population of CD161+RAG1+ cells,
5d. separating the cell suspension from the monolayer and sorting the cell suspension for a cell population enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells;
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IL15, and optionally IL7, wherein the concentration of IL15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[00018] According to a twelfth aspect, the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
6a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
6b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
6c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating a cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
6d. separating the cell suspension from the monolayer, removing the sixth defined medium from the suspension, and adding a seventh defined medium comprising IL15, and optionally IL7, wherein the concentration of IL15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
[00019] According to a thirteenth aspect, the present application provides method for generating a cell population enriched in PSC-derived erythroid cells, wherein the PSC-derived erythroid cells are CD235a+CD14- cells, comprising the sequential steps of:
7a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
7b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF,
FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
7c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in an eighth defined medium comprising Erythropoietin (EPO), and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a+CD14- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
[00020] According to a fourteenth aspect, the present invention provides a method for generating a cell population enriched in PSC-derived myeloid cells, wherein the PSC-derived erythroid cells are CD235a-CD14+ cells, comprising the sequential steps of:
8a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
8b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF,
FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
8c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a ninth defined medium comprising one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), and IL34, and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a CD14+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
[00021] According to a fifteenth aspect, the present invention provides a population of DLL4- expressing arterial endothelial cells (AECs) obtained from the method of any one of the first to the third aspects, wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
[00022] According to a sixteenth aspect, the present invention provides a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells obtained from the method of any one of the fourth to the fifth aspects.
[00023] According to a seventeenth aspect, the present invention provides a cell population enriched in PSC-derived ILCs obtained from the method of any one of the sixth to the seventh aspects.
[00024] According to an eighteenth aspect, the present invention provides a cell population enriched in PSC-derived T cells obtained from the method of any one of the eighth to the ninth aspects.
[00025] According to a nineteenth aspect, the present invention provides a cell population enriched in PSC-derived NK-like cells obtained from the method of any one of the tenth to the twelfth aspects.
[00026] According to a twentieth aspect, the present invention provides a population of PSC- derived erythroid cells obtained from the method of the thirteenth aspect.
[00027] According to a twenty-first aspect, the present invention provides a population of PSC- derived myeloid cells obtained from the method of the fourteenth aspect.
[00028] According to a twenty-second aspect, the present invention provides a population of PSC-derived DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells. [00029] According to a twenty-third aspect, the present invention provides a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells.
[00030] According to a twenty-fourth aspect, the present invention provides a cell population enriched in PSC-derived ILCs.
[00031] According to a twenty-fifth aspect, the present invention provides a cell population enriched in PSC-derived T cells.
[00032] According to a twenty-sixth aspect, the present invention provides a cell population enriched in PSC-derived NK-like cells.
[00033] Numbered statements of the invention are as follows:
1. A method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway activator, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising at least one of SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells. 2. A method for generating a population of DEE4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor, a Wnt pathway activator, a BMP pathway inhibitor, FGF and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal- endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells.
3. A method for generating a population of DEE4-expressing arterial endothelial cells (AECs), wherein the DEE4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising A83-O1, CHIR99021, EDN-193189, FGF2, and which is free or essentially free of BMP4, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising A83-O1, VEGF, BMP4, FGF2, for a time sufficient for generating a population of CD13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, BMP4, FGF2, for a time sufficient for generating the population of CD34+ cells.
4. A method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of:
2a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
2b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising at least one of Flt3L, VEGF, FGF and IL7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising at least one of Flt3L, VEGF, FGF, IL7 and IL 15 for a time sufficient for generating the mixed population of PSC- derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
5. A method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of: 2a. generating a monolayer of DEE4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
2b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, IE3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3E, VEGF, FGF2, and IE7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising Flt3E, VEGF, FGF2, IE7, and IE15 for a time sufficient for generating the mixed population of PSC-derived innate lymphoid cells (IFCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
6. A method for generating a cell population enriched in PSC-derived innate lymphoid cells (IFCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
3b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IE3 and Flt3E, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IE7 and at least one of Flt3E, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mE, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
7. A method for generating a cell population enriched in PS C -derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
3b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
8. A method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
4b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
9. A method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
4b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3E, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3E, VEGF, FGF2, and IE7, wherein the concentration of IL7 is about 0.01 to about 1 ng/mE, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
10. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
5al. generating a population of cells enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells; and
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IE-7 and at least one of Flt3L, VEGF, FGF and IL 15, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
11. The method of statement 10, wherein the CD161+/RAG1+ cells are prepared according to the method of any one of statements 1 to 5.
12. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of: 5a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
5b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3E, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
5c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3E, VEGF, FGF2, and IE7, wherein the concentration of IL7 is about 1 to about 50 ng/mE, preferably about 20 ng/mL, for a time sufficient for generating a cell suspension comprising a population of CD161+RAG1+ cells,
5d. separating the cell suspension from the monolayer and sorting the cell suspension for a cell population enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells;
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IE15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IE 15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
13. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
6a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
6b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
6c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating a cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
6d. separating the cell suspension from the monolayer, removing the sixth defined medium from the suspension, and adding a seventh defined medium comprising IL15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IL15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
14. A method for generating a cell population enriched in PSC-derived erythroid cells, wherein the PSC-derived erythroid cells are CD235a+CD14- cells, comprising the sequential steps of:
7a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
7b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
7c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in an eighth defined medium comprising Erythropoietin (EPO), and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a+CD14- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
15. A method for generating a cell population enriched in PSC-derived myeloid cells, wherein the PSC-derived erythroid cells are CD235a-CD14+ cells, comprising the sequential steps of:
8a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3, 8b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
8c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a ninth defined medium comprising one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GMCSF), and IL34, and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a- CD14+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
16. The method of any one of statements 1 to 15, wherein the first defined medium further comprises Y-27263, preferably the concentration of Y-27263 is about 1 to 50 pM, preferably about 8 to 12 pM, preferably about 10 pM.
17. The method of any one of statements 1 to 16, wherein the method further comprising cryopreserving the population of DLL4-expressing arterial endothelial cells (AECs) following step Id.
18. The method of any one of statements 1 to 17, wherein the population of substantially undifferentiated pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).
19. The method of statement 18, wherein the iPSCs are selected from the group consisting of iPSC RM3.5 (male)77407 GFP, iPSC PB0-01 (male), iPSC PB0-04 (female), iPSC PB0-05 (female), iPSC PB0-06 (male), iPSC PB0-10 (male), and iPSC CRL2429 (ATCC).
20. The method of statement 18, wherein the ESCs are selected from the group consisting of ESC Hl (male), ESC H9 (female)77407 :GFP, ESC }^soxi7:tdTOMATO;Ruxic:GFP and ESC HES3(female)M7XL7;GF7>.
21. The method of any one of statements 1 to 20, wherein the concentration of Activin A in the first defined medium is about 10 to about 50 ng/mL, preferably about 30 ng/mL, the concentration of CHIR99021 in the first defined medium is about 1 to about 10 pM, preferably about 4 pM, the concentration of FGF2 in the first defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL, and the concentration of PIK90 in the first defined medium is about 10 to about 300 nM, preferably about 100 nM.
22. The method of any one of statements 1 to 21, wherein the concentration of A83-O1 in the second defined medium is about 0.1 to about 10 pM, preferably about IpM, the concentration of CHIR99021 in the second defined medium is about 1 to about 10 pM, preferably about 3 pM, the concentration of LDN-193189 in the second defined medium is about 50 to about 750 nM, preferably about 250 nM, and the concentration of FGF2 in the second defined medium is about
1 to about 100 ng/mL, preferably about 20 ng/mL.
23. The method of any one of statements 1 to 22, wherein the concentration of A83-O1 in the third defined medium is about 0.1 pM to about 10 pM, preferably about IpM, the concentration of VEGF in the third defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL, the concentration of BMP4 in the third defined medium is about 10 to about 100 ng/mL, preferably about 30 ng/mL, and the concentration of FGF2 in the third defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL.
24. The method of any one of statements 1 to 23, wherein the concentration of SCF in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL, the concentration of VEGF in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL, the concentration of BMP4 in the fourth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL, and the concentration of FGF2 in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL.
25. The method of any one of statements 1 to 24, wherein the time sufficient for generating the population of MIXL1+ cells at the end of step la is about 2 to about 72 hours, preferably about 24 hours.
26. The method of any one of statements 1 to 25, wherein the time sufficient for generating a population of CD13+ early mesoderm cells at the end of step lb is about 2 to about 72 hours, preferably about 24 hours.
27. The method of any one of statements 1 to 26, wherein the time sufficient for generating the population of CD13+ and KDR+ mesodermal-endothelial cells at the end of step 1c is about
2 to about 72 hours, preferably about 24 hours. 28. The method of any one of statements 1 to 27, wherein the time sufficient for generating the population of CD34+ cells at the end of step Id is about 24 to about 144 hours, preferably about 72 hours.
29. The method of any one of statements 1 to 28, wherein the population of MIXL1+ cells at the end of step la is at least 75% of total cells.
30. The method of any one of statements 1 to 29, wherein the population of CD 13+ early mesoderm cells at the end of step lb is at least 85% of total cells.
31. The method of any one of statements 1 to 30, wherein the population of CD 13+ and KDR+ mesodermal-endothelial cells at the end of step 1c is at least 20% of total cells.
32. The method of any one of statements 1 to 31, wherein the population of CD34+ cells at the end of step Id is at least 70% of total cells.
33. The method of any one of statements 1 to 32, wherein the population of CD34+ cells further co-expresses CXCR4 and/or CDH5 (VE-cadherin).
34. The method of statement 33, wherein the population of CD34+ cells further co-expressing CXCR4 is at least 70% of total cells.
35. The method of statement 33, wherein the population of CD34+ cells further co-expressing CDH5 (VE-cadherin) is at least 70% of total cells.
36. The method of any one of statements 4 to 35, wherein the concentration of SCF in the fifth defined medium is about 50 to about 500 ng/mL, preferably about 100 ng/mL, the concentration of VEGF in the fifth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL, the concentration of FGF2 in the fifth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL, the concentration of IL3 in the fifth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL, and the concentration of Flt3L in the fifth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL.
37. The method of any one of statements 4, 5, 13 and 16 to 36, wherein the concentration of Flt3L in the sixth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL, the concentration of VEGF in the sixth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL, the concentration of FGF2 in the sixth defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL, and the concentration of IL7 in the sixth defined medium is about 0.1 to about 10 ng/mL, preferably about 1 ng/mL.
38. The method of any one of statements 4, 5, 14 and 16 to 37, wherein the concentration of Flt3L in the seventh defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL, the concentration of VEGF in the seventh defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL, the concentration of FGF2in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL, the concentration of IL7 in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL, and the concentration of
IL 15 in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL.
39. The method of any one of statements 4 to 38, wherein the time sufficient for generating the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 2b, 3b, 4b, 5b, 6b, 7b or 8b is about 72 to about 288 hours, preferably about 144 hours.
40. The method of any one of statements 4, 5, 13 and 16 to 35, wherein the time sufficient for generating the population of CD34+CD7+ lymphoid haematopoietic progenitor cells at the end of step 2c or 6c is about 24 to about 144 hours, preferably about 72 hours.
41. The method of any one of statements 4, 5, and 16 to 35, wherein the time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells at the end of step 2d is about 48 to about 192 hours, preferably about 96 hours.
42. The method of any one of statements 4, 5, and 16 to 35, wherein the time sufficient for generating the mixed population of CD7+CD161+RAG1- cells and CD161+CD56+ cells at the end of step 2e is about 48 to about 192 hours, preferably about 96 hours.
43. The method of any one of statements 4 to 39, wherein the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 2b, 3b, 4b, 5b, 6b, 7b or 8b is at least 25% of total cells.
44. The method of any one of statements 4, 5, 13 and 16 to 35, wherein the population of CD34+CD7+ lymphoid haematopoietic progenitor cells at the end of step 2c or 6c is at least 40% of total cells.
45. The method of any one of statements 4, 5, 16 to 35, wherein the population of CD34- CD7+ and CD7+RAG1+ lymphoid cells at the end of step 2d is at least 45% of total cells. 46. The method of any one of statements 4, 5, 16 to 35, wherein the population of PSC- derived innate lymphoid cells (ILCs) at the end of step 2e is at least 75% of total cells and/or the population of PSC-derived NK-like cells at the end of step 2e is at least 50% of total cells.
47. The method of any one of statements 4, 5 and 16 to 35, wherein PSC-derived innate lymphoid cells (ILCs) are CD161+CD7+ cells and/or CD161+RAG1- cells.
48. The method of statement 47, wherein the population of CD161+CD7+ cells is at least 70% of total cells.
49. The method of statement 47, wherein the population of CD161+RAG1- cells is at least 70% of total cells.
50. The method of any one of statements 4, 5, and 16 to 35, wherein PSC-derived NK-like cells are CD161+CD56+ cells.
51. The method of statement 50, wherein the population of CD161+CD56+ cells is at least 50% of total cells.
52. The method of any one of statements 6 to 13 and 16 to 35, wherein the concentration of Flt3L in the sixth defined medium is about 1 to about 100 ng/mL, preferably about 10 ng/mL, the concentration of VEGF in the sixth defined medium is about 5 to about 500 ng/mL, preferably about 50 ng/mL, and the concentration of FGF2 in the sixth defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL.
53. The method of any one of statements 6, 7, and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+RAG1- cells at the end of step 3c is about 7 to about 21 days, preferably about 11 days.
54. The method of any one of statements 6, 7, and 16 to 35, wherein the cell population enriched in CD161+RAG1- cells at the end of step 3c is at least 50% of total cells.
55. The method of any one of statements 8, 9, and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD4+CD8a+ cells at the end of step 4c is about 7 to about 21 days, preferably about 11 days.
56. The method of any one of statements 8, 9, and 16 to 35, wherein the cell population enriched in CD4+CD8a+ cells at the end of step 4c is at least 12% of total cells. 57. The method of any one of statements 10 to 33, wherein the concentration of Flt3L in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 10 ng/mL, the concentration of VEGF in the seventh defined medium is about 5 to about 500 ng/mL, preferably about 50 ng/mL, and the concentration of FGF2 in the seventh defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL.
58. The method of any one of statements 12, and 16 to 35, wherein the time sufficient for generating the cell suspension comprising the population of CD161+RAG1+ cells at the end of step 5c is about 3 to about 14 days, preferably about 7 days.
59. The method of any one of statements 12, and 16 to 35, wherein the sorting at step 5d is fluorescence-activated cell sorting (FACS).
60. The method of any one of statements 10, 11, 12 and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+CD56+ cells at the end of step 5e is about 48 to about 192 hours, preferably about 96 hours.
61. The method of any one of statements 10, 11, 12 and 16 to 35, wherein the cell population enriched in CD161+CD56+ cells at the end of step 5e is at least 70% of total cells.
62. The method of any one of statements 13 and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+CD56+ cells at the end of step 6d is about 48 to about 240 hours, preferably about 120 hours.
63. The method of any one of statements 13 to 33, wherein the cell population enriched in CD161+CD56+ cells at the end of step 6d is at least 80% of total cells.
64. A population of DLL4-expressing arterial endothelial cells (AECs) obtained from the method of any one of statements 1 to 3 and 16 to 35, wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
65. The population of DLL4-expressing AECs of statement 34, wherein the population of CD34+ cells co-expresses CXCR4.
66. The population of DLL4-expressing AECs of statement 64 or 65, wherein the population of CD34+ cells co-expresses CDH5 (VE-cadherin). 67. A mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK- like cells obtained from the method of any one of statements 4, 5, and 16 to 51.
68. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of statement 67, wherein the PSC-derived ILCs are CD161+CD7+ cells.
69. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of statement 67 or 66, wherein the PSC-derived ILCs are CD161+RAG1- cells.
70. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of any one of statements 67 to 69, wherein the NK-like cells are CD161+CD56+ cells.
71. A cell population enriched in PSC-derived ILCs obtained from the method of any one of statements 6, 7, 16 to 36, 39, 43, and 52 to 54.
72. The cell population enriched in PSC-derived ILCs of statement 71, wherein the PSC- derived ILCs are CD161+RAG1- cells.
73. A cell population enriched in PSC-derived T cells obtained from the method of any one of statements 8, 9, and 16 to 36, 39, 43, 52, 55, and 56.
74. The cell population enriched in PSC-derived T cells of statement 73, wherein the PSC- derived T cells are CD4+CD8a+ cells.
75. A cell population enriched in PSC-derived NK-like cells obtained from the method of any one of statements 10 tol3, 16 to 37, 39, 40, 43, 44, 52, and 57 to 63.
76. The cell population enriched in PSC-derived NK-like cells of statement 75, wherein the PSC-derived NK-like cells are CD161+CD56+ cells.
77. A population of CD34+CD43+ haematopoietic progenitor cells obtained from step 2b of any one of statements 4, 5, 16 to 36 and 39.
78. A population of CD34+CD7+ lymphoid haematopoietic progenitor cells obtained from step 2c of any one of statements 4, 5, 16 to 37, 39 and 40.
79. A population of CD34-CD7+ lymphoid cells obtained from step 2d of any one of statements 4, 5, 16 to 37, and 39 to 41. 80. A population of CD7+RAG1+ lymphoid cells obtained from step 2d of any one of statements 4, 5, 16 to 37, and 39 to 41.
81. A population of CD161+RAG1+ cells obtained from step 5c or 5d of any one of statements 12, 16 to 37, 39, 58 and 59.
82. An isolated population of PSC-derived DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
83. The population of DLL4-expressing AECs of statement 82, wherein the population of CD34+ cells co-expresses CXCR4.
84. The population of DLL4-expressing AECs of statement 82 or 83, wherein the population of CD34+ cells co-expresses CDH5 (VE-cadherin).
85. An isolated mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC- derived NK-like cells.
86. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of statement 85, wherein the PSC-derived ILCs are CD161+CD7+ cells.
87. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of statement 85 or 86, wherein the PSC-derived ILCs are CD161+RAG1- cells.
88. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of any one of statements 85 to 86, wherein the NK-like cells are CD161+CD56+ cells.
89. An isolated cell population enriched in PSC-derived ILCs.
90. The cell population enriched in PSC-derived ILCs of statement 89, wherein the PSC- derived ILCs are CD161+RAG1- cells.
91. An isolated cell population enriched in PSC-derived T cells.
92. The cell population enriched in PSC-derived T cells of statement 91, wherein the PSC- derived T cells are CD4+CD8a+ cells.
93. An isolated cell population enriched in PSC-derived NK-like cells. 94. The cell population enriched in PSC-derived NK-like cells of statement 93, wherein the PSC-derived NK-like cells are CD161+CD56+ cells.
95. A method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, consisting of the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway activator, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising at least one of SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells.
96. A method for generating a population of DEE4-expressing arterial endothelial cells (AECs), wherein the DEE4-expressing AECs are CD34+ cells, consisting of the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor, a Wnt pathway activator, a BMP pathway inhibitor, FGF and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal- endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells.
97. A method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, consisting of the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising A83-O1, CHIR99021, LDN-193189, FGF2, and which is free or essentially free of BMP4, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising A83-O1, VEGF, BMP4, FGF2, for a time sufficient for generating a population of CD13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, BMP4, FGF2, for a time sufficient for generating the population of CD34+ cells.
98. A method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, consisting of the sequential steps of: 2a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
2b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IE3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising at least one of Flt3E, VEGF, FGF and IE7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising at least one of Flt3L, VEGF, FGF, IE7 and IE 15 for a time sufficient for generating the mixed population of PSC- derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
99. A method for generating a mixed population of PSC-derived innate lymphoid cells (IFCs) and PSC-derived NK-like cells, consisting of the sequential steps of:
2a. generating a monolayer of DEE4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
2b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, IE3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3E, VEGF, FGF2, and IL7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells, 2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising Flt3L, VEGF, FGF2, IL7, and IL15 for a time sufficient for generating the mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
100. A method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, consisting of the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
3b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3 and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
101. A method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, consisting of the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3, 3b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
102. A method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, consisting of the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
4b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
103. A method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, consisting of the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
4b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells, 4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
104. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, consisting of the sequential steps of:
5al. generating a population of cells enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells; and
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IL-7 and at least one of Flt3L, VEGF, FGF and IL 15, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
105. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, consisting of the sequential steps of:
5a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
5b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
5c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating a cell suspension comprising a population of CD161+RAG1+ cells, 5d. separating the cell suspension from the monolayer and sorting the cell suspension for a cell population enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells;
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IL15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IL 15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
106. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, consisting of the sequential steps of:
6a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
6b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
6c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating a cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
6d. separating the cell suspension from the monolayer, removing the sixth defined medium from the suspension, and adding a seventh defined medium comprising IL15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IL15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting. 107. A method for generating a cell population enriched in PSC-derived erythroid cells, wherein the PSC-derived erythroid cells are CD235a+CD14- cells, consisting of the sequential steps of:
7a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
7b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
7c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in an eighth defined medium comprising Erythropoietin (EPO), and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a+CD14- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
108. A method for generating a cell population enriched in PSC-derived myeloid cells, wherein the PSC-derived erythroid cells are CD235a-CD14+ cells, consisting of the sequential steps of:
8a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of statements 1 to 3,
8b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
8c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a ninth defined medium comprising one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GMCSF), and IL34, and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a- CD14+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting. 109. A population of cells obtained from the method of any one of statements 95 to 108.
Brief Description of Drawings
[00034] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
[00035] Figures 1A to II illustrate differentiation of human pluripotent stem cells to DLL4+ AECs and lymphoid haematopoietic cells. Specifically, each figure provides the following:
[00036] Figure 1A is a schematic representation showing sequential stages of PSC differentiation towards DLL4+ AECs, haemogenic endothelium, haematopoietic progenitors, lymphoid commitment, and the generation of ILCs. The approximate corresponding days are indicated.
[00037] Figure IB is a bright field image of a monolayer of cells at differentiation day 6, comprising DLL4+ AECs. Scale bar, 100 [im.
[00038] Figure 1C is a set of flow cytometric analysis of day 6 cultures showing the coexpression of the AEC markers DLL4, CXCR4, CDH5 (VECAD) on CD34+ cells. Red dots indicate CD34+DLL4+ double positive populations in each plot. Frequency of cell fractions are indicated in the relevant quadrants.
[00039] Figure ID is a bar graph summarising the flow cytometry analysis of CD34 and DLL4 expression in day 6 samples derived from multiple independent differentiation experiments using 9 distinct parental PSC lines (and 2 additional subclones of RM3.5 iPSCs and H9 ESCs). Cell line details are provided in Methods section. The number of experiments for each line is shown. Data is shown in mean ± SD (n = 2 for PB004, PB006, PB0010 RM3.5RAG1 GFP (RAG1), RM3.5RAG1:GFP;GAPTRAP:tdTOMATO (tdT RAG1), H9; n=3 for PB005; n=4 for PB001, Hl, H9 RUNXlC:GFP;SOX17:mCHERRY(RX/SOX) HES3MIXI ('I P (MIXLI ))
[00040] Figure IE is a set of flow cytometry analysis tracing the progressive differentiation of haematopoietic populations, including CD43+CD34+ HSPCs at day 12, CD34+CD7+ lymphoid haematopoietic progenitor cells at day 15, CD34-CD7+ and CD1+RAG1+ lymphoid cells at day 19. Green dots indicate RAG1 :GFP+ cells. Frequencies of cell fractions are indicated. [00041] Figure IF is a set of bar graphs depicting quantification of /MG/:GFP+ cells showing the frequency of RAG1+ cells increases from day 15 to day 19. Statistics is calculated by a t-test; data is shown in mean +SEM; n=5 for RM3.5 day 15, n=5 for RM3.5 day 19, n=4 for H9 day 15, n=6 for H9 day 19.
[00042] Figure 1G is a set of bright field and fluorescence images showing the emergence and accumulation of RAG1:GFP cells at day 15 and day 19. Inset shows a higher magnification of the indicated rectangle. BF, Bright Field. Scale bar, 100 pm.
[00043] Figure 1H is a set of flow cytometric analysis of CD45+ cells at day 23 of differentiation showing the expression of lymphoid associated markers CD7, CD 161 and RAG1, and the NK cell marker CD56. Frequencies of cell fractions are indicated.
[00044] Figure II is a set of bar graphs depicting quantification of frequency of the CD7+CD161+ innate lymphoid cells and CD161+CD56+ NK-like cells in multiple independent experiments incorporating 3 distinct parental PSC lines.
[00045] Figures 2A to 2F illustrate that scRNA-seq reveals AEC and haematopoietic differentiation resembles human AGM and fetal liver haematopoiesis. Specifically, each figure provides the following:
[00046] Figure 2A is a graph of UMAP projection showing a comparison between cells isolated from the AGM and fetal liver tissues (left) and cells generated by PSC differentiation in vitro (right). Stromal cell, strom; Endothelial cell, Endo; haematopoietic stem/progenitor-like cell, HSPC; lymphoid progenitor expressing RAG genes, RAG+ Lymph; innate lymphoid cells, ILC; monocyte, mono; erythroid cells, Eryth; granulocyte, Granu; megakaryocyte, Mega; eosinophils, Eosino; epithelial cells, Epi.
[00047] Figure 2B is pair of dotplots showing differentially expressed genes of haemogenic and haematopoietic cells from PSC (right) and from fetal tissues (left).
[00048] Figure 2C is a set of UMAP projections showing the expression of AEC associated genes CD34, SOX17, CDH5, GJA4, CXCR4, and GJA5). and NOTCH ligand genes (DLL4, DLK1, JAG1, and JAG2) on PSC-derived cells in vitro. [00049] Figure 2D is a pie chart showing transcriptomics based ACTINN predication showing in vitro PSC-derived endothelial cells within the endothelial cell cluster projected to the arterial endothelial cells collected from the AGM of CS 14/15 embryos (AE-AGM CS 14/15).
[00050] Figure 2E is a set of UMAP projections showing the expression of key genes related to endothelial-to-haematopoietic transition and haemogenic endothelial cells in the fraction of day 12 and day 15 CD34+CDH5+RUNX1+ cells.
[00051] Figure 2F is set of UMAP projections (upper panel) and flow cytometric validation (lower panel) showing the persistent expression of DLL4 on CD34+ endothelial cells on day 12, day 15 and day 19.
[00052] Figures 3A to 3F illustrate lymphoid cell development in the PSC-derived arterial haematopoietic culture. Specifically, each figure provides the following:
[00053] Figure 3A is a UMAP projection showing haematopoietic cell types in the PSC-based arterial haematopoietic culture. Cells represent hematopoietic cell clusters in Figure 2A.
Haemogenic endothelial cells, HE; haematopoietic stem/progenitor-like cells, HSPC; common myeloid progenitor-like cells, CMP; erythroid progenitors, Eryth_Pro; myeloid progenitors, Mye_Pro; three lymphoid progenitor populations (Lymp_Prol, Lymph_Pro2, and Lymph_Pro3), T cell progenitor (T_Pro), T cell progenitor in cycling (T_Pro_cyc), innate lymphoid cells (ILC), and innate lymphoid cells in cycling (ILC_cyc). Colours indicate cell types.
[00054] Figure 3B is a set of UMAP projections showing the development of haematopoietic cell types in real-time (Day 6, 12, 15, 19 and 25) and in pseudo-time. Colours indicate cell types.
[00055] Figure 3C is a dot plot showing differentially expressed genes distinguish cell types generated in the PSC-based arterial haematopoietic culture.
[00056] Figure 3D is a set of violin plots showing the expression of a cohort of genes associate with T cell fate commitment.
[00057] Figure 3E is a UMAP projection showing three sub-clusters within the ILCs: NK-ILC1 like cells a (NK/ILCla), NK-ILC1 like cells b (NK/ILClb), and ILC2 like cells (ILC2-like). [00058] Figure 3F is a heatmap representation of the expression levels of selected ILC associated genes in cells assigned to clusters representing NK-ILC1 like cells a (NK/ILCla), NK-ILC1 like cells b (NK/ILClb), and ILC2 like cells (ILC2-like).
[00059] Figures 4A to 4F illustrate identification of IL7 as a determinant factor of fate choices between the T and the ILC lineages. Specifically, each figure provides the following:
[00060] Figure 4A is set of flow cytometry examination on expressions of CD7 and IL7R (top) with quantifications of frequencies (bottom) showing addition of IL7 increased the percentage of CD7+ cells but increased concentrations of IL7 downregulated the percentage of CD7+IL7R+ cells. Bar graph is shown in mean +SEM, statistics is calculated by a one-way ANOVA test, n= 5 independent of experiments.
[00061] Figure 4B is a set of flow cytometry examination on expressions of CD 161 and RAG1:GFP (top) with quantifications of frequencies (bottom) showing addition of IL7 increased the percentage of CD161-RAG1+ cells committed to the T lineage and CD161+RAG1- cells committed to the ILC lineage. Bar graph is shown in mean +SEM, statistics is calculated by a one-way ANOVA test, n= 5 independent of experiments.
[00062] Figure 4C is a set of flow cytometry examination of expression of CD4 and CD8a (top) with quantifications of frequencies (bottom) showing minimal addition of IL7 at 0.1 ng/ml is optimal for the generation of CD4+CD8a+ double positive T cell precursors. Bar graph showing different levels of RAGLGFP expression across the four populations marked CD4 and/or CD8a. Bar graph is shown in mean +SEM, statistics is calculated by a one-way ANOVA test, n= 5 independent of experiments.
[00063] Figure 4D is a set of fluorescent and bright field (BF) images showing the expression of RAGLGFP and cell growth of sorted GFP+ cells on the day of sorting and replating (day 0, sort) and after four days in culture (day 4) under the indicated conditions supplemented with IL7 (20ng/ml), IL15 (20ng/ml), or IL7 + IL15 (both 20ng/ml).
[00064] Figure 4E is a set of flow cytometry plots showing that IL15 (20 ng/ml) and IL7+IL15 (both 20ng/ml) support cell growth and the generation of CD161+CD56+ NK-like cells but IL7 (20ng/ml) does not.
[00065] Figure 4F is a bar graph showing quantification of output cell numbers per 5,000 input RAG1:GFP+ cells (as normalized to 1) showing IL7+IL15 robustly supported the generation of CD161+CD56+ NK-like cells from RAG1+ lymphoid progenitors. Bar graph is a representation of 2 independent experiments, data points represent technical replicates, data is shown in mean +SEM.
[00066] Figures 5A to 5F illustrate differentiation of PSCs to arterial endothelial cells. Specifically, each figure provides the following:
[00067] Figure 5A is a set of bright field images showing early days (day 1, 2 and 3) of PSC differentiation to arterial endothelial cells.
[00068] Figure 5B is a flow cytometry plot showing efficient generation of MIXL1 :GFP+ primitive streak cells on day 1 of PSC differentiation. Frequency of MIXL1+ cells is indicated.
[00069] Figure 5C is a set of flow cytometry plots tracking expressions of the mesodermal marker CD 13, and the endothelial cell marker KDR(VEGFR2). Differentiating cells are CD13+KDR- representing mesodermal progenitors, followed by upregulation of KDR on day 3.
[00070] Figure 5D is a set of flow cytometry plots showing reproducible generation of arterial endothelial cells from 10 PSC lines in one differentiation experiments.
[00071] Figure 5E is a bar graph showing quantification of the number of CD34+DLL4+ cells per input PSC on day 6 of differentiation. Data is shown with six technical replicates for four PSC lines of biologically independent backgrounds: PB005, iPSC; RM3.5, iPSC; H9, ESC; CRL-2429, iPSC.
[00072] Figure 5F is a set of flow cytometry analysis showing differentiation from cryopreserved PSC-derived arterial cell cultures at day 7 (24 hours after thaw), day 14 (CD45+CD34+, haematopoiesis), and day 20 (CD7+RAG1+, CD161+RAG1-, and CD161- RAG1+ lymphopoiesis). Frequencies of cells of each fraction are indicated.
[00073] Figures 6A to 6J illustrate scRNA-seq analysis and characterization of the PSC- derived arterial haematopoietic cultures. Specifically, each figure provides the following:
[00074] Figure 6A is a set of UMAP projections showing samples representing cells collected at different timepoints of PSC differentiation (PSC Day 6, 12, 15, 19 and 25), and samples representing cells collected from the AGM and foetal liver tissues. [00075] Figure 6B is a set of UMAP projections showing the expression cell type specific genes: stromal cell (COL3A F). arterial endothelial cell \ CD34 and GJA4); haematopoietic stem/progenitor-like cell (CD34. SPINK2), lymphoid haematopoietic cell (CD7), lymphoid progenitor expressing RAG genes (RAG I). innate lymphoid cell (KLRB1), monocyte (CSF/R). erythroid cell (HBA2). granulocyte (S100A9), megakaryocyte (PF4). eosinophils (CP A3).
[00076] Figure 6C is a set of immunofluorescence images for human DLK1 and CDH5 of arterial-haematopoietic culture on day 8. Red, DLK1. Green, CDH5; Blue, DAPI. Scale bar, 100 pm.
[00077] Figure 6D is a set of UMAP projections (upper panel) and flow cytometric validation (right panel) showing expression of Jagged 1 (JAG1) on CD34+ endothelial cells on day 6. CD34- stromal cells also expressed JAG1 with persistent expression to day 15 of PSC differentiation. Frequencies of cell fractions are indicated.
[00078] Figure 6E is a set of UMAP projections showing the expression of JAG1, CXCR4, and DLL4 in fetal tissues across developmental time, including AGM (week 4.5, 5, 5.5, and 6) and fetal liver (week 5.5, 6, 8, 11, 15).
[00079] Figure 6F is a bar graph showing quantification of JAG1+ cells in the arterial endothelial cell cluster of fetal tissues across development time.
[00080] Figure 6G is a CellChat analysis of NOTCH ligand-receptor pair commination between of PSC-derived cell types in the arterial haematopoietic cultures on day 12, 15 and 19: stromal cells, haematopoietic stem/progenitor-like cells (HSPC), endothelial cells, innate lymphoid cells (ILCs) and lymphoid progenitor expressing RAG genes.
[00081] Figure 6H is a set of flow cytometry analysis, bright field images, and cytospin analysis demonstrating erythroid and myeloid differentiation from day 12 AECs following addition of the indicated growth factors. For the flow cytometry plots, the percentage of cells in pertinent gates is indicated. Images are of single wells of a 96 well tray containing cells differentiated in the presence of indicated growth factors, noting the overt haemoglobinisation apparent in cell populations treated with Erythropoietin (EPO) for two weeks. Similarly, cytospin analysis shows cells with a distinctive macrophage morphology arising from cultures supplemented with M-CSF. [00082] Figure 61 is a set of flow cytometry analysis demonstrating T cell (TCR+CD3+) & B lymphoid (RAG1+ CD19+) differentiation from day 12 AECs following co-culture with stromal cells. For the flow cytometry plots, the percentage of cells in pertinent gates is indicated.
[00083] Figure 6J is a set of is a set of brightfield and immunofluorescence images showing the tube forming ability of differentiation day 6 RM-tTom endothelial cells following disaggregation and re-seeding on Matrigel. Scale bar, 200 pm.
[00084] Figures 7A to 7E illustrate scRNA-seq analysis of the lymphoid components in PSC- derived arterial haematopoietic cultures. Specifically, each figure provides the following:
[00085] Figure 7A is a set of UMAP projections showing samples representing haematopoietic cells collected at different timepoints of PSC differentiation (PSC Day 6, 12, 15, 19 and 25). This figure is corelated to Figure 3A. Colours indicate differentiation day.
[00086] Figure 7B is a set of UMAP projections showing rare cells expressing B cell development genes MME/CD10, CD19, MS4A1/CD20.
[00087] Figure 7C is a heatmap showing top 2000 differentially expressed genes across the four cell populations of haematopoietic stem/progenitor like cells (HSPC), three lymphoid progenitors (Lymph_Prol, Lymph_Pro2, and Lymph_Pro3).
[00088] Figure 7D is set of gene ontology analysis the cell type specific gene modules shown in the Figure 7C.
[00089] Figure 7E is a set of UMAP projections showing the expression of ILC genes and NK genes in the PSC-derived ILC sub-populations. This figure is related to Figure 3E.
[00090] Figures 8A to 8J illustrates IL7 concentrations determine cell fate choices between the T and the ILC lineages and RAG1 gene expression. Specifically, each figure provides the following:
[00091] Figure 8A is set of UMAP projections showing the expressions of IL7R, KLRB1 CD161), RAG1 and CD4 expression on PSC-derived haematopoietic cells.
[00092] Figure 8B is a UMAPC projection showing undetected IL7 mRNA in cells from the entire PSC-derived arterial haematopoietic culture. [00093] Figure 8C is set of flow cytometry plots (left) and quantification of the frequency RAG1:GFP+ cells (right) showing IL7 is required for the upregulation of RAG1. Data is shown in mean +SEM, statistics is calculated by a two-way ANOVA test, n=5 of independent experiments. p(RAGl-hi IL7 0.1 ) = 0.0321, p(RAGl-hi IL7 1 ) = 0.0107.
[00094] Figure 8D is a set of violin plots of scRNA-seq showing the expression of ILC-related genes (KLRB1, NKG7, NCAMI) expressed in /MG/-low cells, while /MG /-high cells show higher levels of expressions of T cell development related genes \ CD4. CD8A, CD8B, TCF7 and BCL11B} and antigen receptor related genes (RAG2 and JCHAIN). The mean of RAG1 average expression across all clusters was calculated by the AverageExpression function in R; cells that were above the mean average expression of 2.259 (4sf) were labelled as “7MG7-high” and those lower as “TMGJ-low”.
[00095] Figure 8E is a set of violin plots of scRNA-seq showing day 19 culture contains predominantly /?AG7-low cells and day 25 culture contains both RAG 1 -high and RAG 1 -low cells.
[00096] Figure 8F is a set of fluorescent and bright field (BF) images showing the expression of RAG1 :GFP and cell growth of sorted GFP+ cells on the day 1, 2 and 3 after replating and cultured under the indicated conditions supplemented with IL7 (20ng/ml), IL15 (20ng/ml), or IL7 + IL 15 (both 20ng/ml). This figure is related to Figure 4D.
[00097] Figure 8G is a set of flow cytometry analysis showing a flow cytometry characterization for CD56, CD7, CD 161 on isolated day 15 haematopoietic cells treated with IL7 and IL 15 (both 20 ng/ml) for 5 days (left image) and a quantification of the frequency of CD56+CD161+ cells from the different PSC lines (n=2 for PB005, n=2 for RM3.5, n=l for H9, n=5 for total; data is shown in mean ± SD) (right image).
[00098] Figure 8H is a set of flow cytometry analysis of CD45+ cells isolated from cultures at day 30 in which IL 15 had been added from day 15.
[00099] Figure 81 is graphical summary of data showing the viability of K562 target cells following 4 hours co-culture with cells derived from cultures subjected days IL15 treatment from day 15 or day 19 until at least differentiation day 28, as indicated. Data points represent technical replicates for each ratio of Effector:K562 shown. Two different cell lines were used in these experiments - derivatives RM3.2 RAG1:GFP (Motazedian et al., 2020) and = PB005 (Vlahos et al., 2019) expressing tandem tomato from the GAPDH locus (Kao et al., 2016).
Description of Embodiments
Definitions
[000100] Definitions of common terms in cellular and molecular biology, and biochemistry can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 9780911910421, 0911910425); Robert s. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 2008 (ISBN 3527305424, 9783527305421); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1- 56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; laneway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2016 (ISBN 9780815345510, 0815345518); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al , Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Laboratory Methods in Enzymology: RNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN: 9780124200371, 0124200370); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), Immunological Methods, Ivan Lefkovits, Benvenuto Pemis, (eds.) Elsevier Science, 2014 (ISBN: 9781483269993, 148326999X), the contents of which are all incorporated by reference herein in their entireties.
[000101] As used in this specification and the appended claims, terms in the singular and the singular forms "a," "an" and "the," for example, optionally include plural referents unless the content clearly dictates otherwise. For example, "a" cell includes one cell, one or more cells and a plurality of cells. [000102] The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
[000103] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.
[000104] The transitional phrase "consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
[000105] The transitional phrase "consisting essentially of" is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term "consisting essentially of" occupies a middle ground between "comprising" and "consisting of".
[000106] Where applicants have defined an invention or a portion thereof with an open-ended term such as "comprising", it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms "consisting essentially of' or "consisting of." In other words, with respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of’ or, alternatively, by “consisting essentially of’. [000107] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
[000108] The term “about” as used herein contemplates a range of values for a given number of ±25% the magnitude of that number. In other embodiments, the term “about” contemplates a range of values for a given number of ±30%, ±20%, ±15%, ±10%, or ±5% the magnitude of that number. For example, in one embodiment, “about 3 pM” indicates a value of 2.7 to 3.3 pM (i.e.
3 pM ±10%), and the like.
[000109] Similarly, while differentiation processes include ordered, sequential events, the timing of the events may be varied by at least 25%. For example, while a particular step may be disclosed in one embodiment as lasting one day, the event may last for more or less than one day. For example, “one day” may include a period of about 18 to about 30 hours. In other embodiments, periods of time may vary by ±20%, ±15%, ±10%, or ±5% of that period of time. Periods of time indicated that are multiple day periods may be multiples of “one day,” such as, for example, about two days may span a period of about 36 to about 60 hours, and the like. In another embodiment, time variation may be lessened, for example, where 1 day is 24±3 hours; 3 days is 72±3 hours; 4 days is 96±3 hours; 5 days is 120±3 hours; 6 days is 144±3 hours; 7 days is 168±3 hours; 11 days is 264±3. As used herein, about 3 days may be 2.5, 3 or 3.5 days, about
4 days may be 3.5, 4 or 4.5 days, about 5 days may be 4.5, 5 or 5.5 days, about 6 days may be
5.5, 6 or 6.5 days, about 7 days may be 6.5, 7 or 7.5 days, about 11 days may be 10, 10.5, 11,
11.5, or 12 days, about 21 days may be 20, 20.5, 21, 21.5, or 22 days.
[000110] Numeric ranges are inclusive of the numbers defining the range. It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
[000111] The headings provided herein are not intended to limit the disclosure. [000112] Throughout the specification, references to particular genes or proteins may be used interchangeably. A person skilled in the art will understand in the context whether the reference is intended to be a reference to the particular gene or the protein that is encoded by that gene.
[000113] The terms “pluripotent stem cell” and “PSC” refer to cells that display pluripotency. The terms “human pluripotent stem cell” and “hPSC” refer to cells derived, obtainable or originating from human tissue that display pluripotency. The hPSC may be a human embryonic stem cell or a human induced pluripotent stem cell.
[000114] Human pluripotent stem cells may be derived from inner cell mass or reprogrammed using Yamanaka factors from many fetal or adult somatic cell types. The generation of hPSCs may be possible using somatic cell nuclear transfer.
[000115] The terms “human embryonic stem cell”, “hES cell” and “hESC” refer to cells derived, obtainable or originating from human embryos or blastocysts, which are self-renewing and pluri- or toti-potent, having the ability to yield all of the cell types present in a mature animal. Human embryonic stem cells (hESCs) can be isolated, for example, from human blastocysts obtained from human preimplantation embryos, in vitro fertilized embryos, or onecell human embryos expanded to the blastocyst stage.
[000116] The terms “induced pluripotent stem cell” and “iPSC” and “hiPSC” (human iPSC) refer to cells derivable, obtainable or originating from adult somatic cells of any type reprogrammed to a pluripotent state through the expression of exogenous genes, such as transcription factors, including but not limited to a preferred combination of OCT4, SOX2, KLF4 and c-MYC. hiPSC show levels of pluripotency equivalent to hESC but can be derived from an individual for autologous therapy with or without concurrent gene correction prior to differentiation and cell delivery.
[000117] More generally, the method disclosed herein could be applied to any pluripotent stem cell derived from any individual or a hPSC subsequently modified to generate a mutant model using gene-editing or a mutant hPSC corrected using gene-editing. Gene-editing could be by way of CRISPR, TALEN or ZF nuclease technologies.
[000118] As used herein, the term “cell culture” refers to any in vitro culture of cells. The term “culturing” refers to the process of growing and/or maintaining and/or manipulating a cell Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos. As used herein, the terms “primary cell culture,” and “primary culture,” refer to cell cultures that have been directly obtained from cells in vivo, such as from a tissue specimen or biopsy from an animal or human. These cultures may be derived from adults as well as fetal tissue.
[000119] A "progenitor cell" is a cell which is capable of differentiating along one or a plurality of developmental pathways, with or without self-renewal. Typically, progenitor cells are unipotent or oligopotent and are capable of at least limited self- renewal.
[000120] The terms "differentiate", "differentiating" and "differentiated", relate to progression of a cell from an earlier or initial stage of a developmental pathway to a later or more mature stage of the developmental pathway. Thus, “undifferentiated”, in this context, relate to a cell from an earlier or initial stage of a developmental pathway or a cell that has not yet developed into a specialized cell type. It will be appreciated that in this context "differentiated' does not mean or imply that the cell is fully differentiated and has lost pluripotency or capacity to further progress along the developmental pathway or along other developmental pathways. Differentiation may be accompanied by cell division.
[000121] As will be well understood in the art, the stage or state of differentiation of a cell may be characterized by the expression and/or non-expression of one or more specific markers. In some embodiments, the expression of “signature” or “milestone” markers may be used in determining or defining the stage or state of differentiation instead of using the period of time defined in days and/or hours. In this context, by "markers" is meant nucleic acids or proteins that are encoded by the genome of a cell, cell population, lineage, compartment or subset, whose expression or pattern of expression changes throughout development. Nucleic acid marker expression may be detected or measured by any technique known in the art including nucleic acid sequence amplification (e.g. polymerase chain reaction) and nucleic acid hybridization (e.g. microarrays, Northern hybridization, in situ hybridization), although without limitation thereto. Protein marker expression may be detected or measured by any technique known in the art including flow cytometry, immunohistochemistry, immunoblotting, protein arrays, protein profiling (e.g. 2D gel electrophoresis), although without limitation thereto.
[000122] Such terms are commonplace and well-understood by the skilled person when characterizing cell phenotypes. By means of additional guidance, when a cell is said to be positive for or to express or comprise expression of a given marker, such as a given gene or gene product, a skilled person would conclude the presence or evidence of a distinct signal for the marker when carrying out a measurement capable of detecting or quantifying the marker in or on the cell. Suitably, the presence or evidence of the distinct signal for the marker would be concluded based on a comparison of the measurement result obtained for the cell to a result of the same measurement carried out for a negative control (for example, a cell known to not express the marker) and/or a positive control (for example, a cell known to express the marker). Where the measurement method allows for a quantitative assessment of the marker, a positive cell may generate a signal for the marker that is at least 1.5-fold higher than a signal generated for the marker by a reference cell (e.g. negative control cell) or than an average signal generated for the marker by a population of reference or negative control cells, e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher, at least 100-fold higher, or even higher. Further, a positive cell may generate a signal for the marker that is 3.0 or more standard deviations, e.g., 3.5 or more, 4.0 or more, 4.5 or more, or 5.0 or more standard deviations, higher than an average signal generated for the marker by a population of reference or negative control cells.
[000123] As used herein, the terms “culture medium,” “cell culture medium,” “defined medium,” “first defined medium,” “second defined medium,” “third defined medium,” “fourth defined medium,” “fifth defined medium”, “sixth defined medium”, and “seventh defined medium” refer to media that are suitable to support the growth of cells in vitro (i.e., cell cultures, cell lines, etc.). It is not intended that the term be limited to any particular culture medium. For example, it is intended that the definition encompass maintenance media as well as other media for the differentiation or specialization of cells. Indeed, it is intended that the term encompass any culture medium suitable for the growth of the cell cultures and cells of interest. In some embodiments, the cell culture medium used in various steps includes a basal medium which is supplemented. In some embodiments, the basal medium is an STAPEL Medium (Ng E.S., et al. Nat. Biotechnol. 2016;34: 1168-1179). (ODM). In one example, the STAPEL medium was prepared by mixing 0.5% OsrHSA, 0.5% BSA(bovostar), , 0.05% polyvinyl alcohol (Sigma- Aldrich), IxGlutaMAX, Ixascorbic acid-2-phosphate (Sigma- Aldrich), ITSE AF blood-free cell culture media supplement (50pgml-l; InVitria), linoleic and linolenic acid Soybean oil (125ngml-l; Sigma- Aldrich), synthetic cholesterol (4pgml-l; Sigma- Aldrich), and protein-free hybridoma mix II (5%) in IMDM/F12 media.
[000124] As used herein the term “enriched” is used to refer to a population of cells which contains a significant proportion of a specific subset or subtype of cell, wherein the set of cells may contain 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95% or 100% of the specific subset/subtype of cell. “Enriched”, as in an enriched population of cells, can be defined phenotypically based upon the increased number of a specific subset or subtype of cells having a particular marker, or combination of markers, or having one or more markers and lacking one or more other markers, in a fractionated, or expanded, set of cells as compared with the number of cells having the marker, combination of markers, or having one or more markers and lacking one or more other markers, in the unfractionated or unexpanded set of cells.
[000125] As used herein, “tissue” means an aggregate of cells. In some embodiments, the cells in the tissue are cohered or fused.
[000126] The terms “decrease”, “reduced”, “reduction”, “to a lesser extent,” or “inhibit” are all used herein to mean a decrease or lessening of a property, level, or other parameter, including by a statistically significant amount. In some embodiments, “reduced,” “reduction,” “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
[000127] The terms “increased”, “increase”, “increases”, or “enhance” or “activate” or “to a greater extent” are all used herein to generally mean an increase of a property, level, or other parameter, including by a statistically significant amount; for the avoidance of any doubt, the terms “increased”, “increase”, “to a greater extent,” “enhance" or “activate" can refer to an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold increase, at least about a 1000-fold increase or more as compared to a reference level.
[000128] As used herein, a “reference level” refers to the level of a marker or parameter in a normal, otherwise unaffected cell population or tissue (e.g., a cell, tissue, or biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, e.g., cell, tissue, or a biological sample obtained from a patient prior to being diagnosed with a disease, or a biological sample that has not been contacted with an agent or composition as disclosed herein). Alternatively, a reference level can also refer to the level of a given marker or parameter in a subject, organ, tissue, or cell, prior to administration of a treatment, e.g., with an agent or via administration of a composition.
[000129] As used herein, a “control” or an “appropriate control” refers to an untreated, otherwise identical cell, subject, organism, or population (e.g., a cell, tissue, or biological sample that was not contacted by an agent or composition described herein) relative to a cell, tissue, biological sample, or population contacted or treated with a given treatment. For example, an appropriate control can be a cell, tissue, organ or subject that has not been contacted with an agent or subjected to the same methods as described herein.
[000130] In one or more embodiments described herein, assessing the expression of various genes includes comparing the fold change. In one embodiment, the fold change is used to measure the change in the expression level of genes. In one embodiment the expression of a gene can be expressed as relative expression comparative to a housekeeping gene.
[000131] The term “agonist” or “activator” may be used interchangeably and as used herein means an activator, for example, of a pathway or signalling molecule. An agonist of a molecule can retain substantially the same, or a subset, of the biological activities of the molecule (e.g. FGF). For example, an FGF agonist or FGF activator means a molecule that selectively activates FGF signalling.
[000132] The term “inhibitor” as used herein means a selective inhibitor, for example of a pathway or signalling molecule. An inhibitor or antagonist of a molecule (e.g. BMP4 inhibitor) can inhibit one or more of the activities of the naturally occurring form of the molecule. For example, a BMP4 inhibitor is a molecule that selectively inhibits BMP signalling mediated by BMP4. [000133] The term “cell population” or “population of XXX cells” as used herein is meant as an in vitro or ex vivo collection of cells.
[000134] The term “sequential” as used herein means following in a logical order or sequence. Thus, the term “sequential steps” means that each step will follow one after the other. For example, step la is performed before step lb, step lb is performed before step 1c, and so on.
[000135] The term “monolayer” as used herein refers to a 2D adherent cell culture.
[000136] In some embodiments, the methods of the present invention comprise a TGF-beta pathway activator. In some embodiments, the TGF-beta pathway activator is selected from the group consisting of Activin A, TGF-betal, TGF-beta2, TGF-beta3, IDE1/2 (IDE1 (l-[2- [(2Carboxyphenyl)methylene]hydrazide]heptanoic acid), IDE2 (Heptanedioic acid-l-(249 cyclopentylidenehydrazide)), and Nodal. In a preferred embodiment the TGF-beta pathway activator is Activin A. In some embodiments, the concentration of Activin A in the medium used in the methods of the invention is about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29 ng/mL, about 30 ng/mL. about 31 ng/mL, about 32 ng/mL, about 33 ng/mL, about 34 ng/mL, about 35 ng/mL, about 36 ng/mL, about 37 ng/mL, about 38 ng/mL, about 39 ng/mL, about 40 ng/mL, about 41 ng/mL, about 42 ng/mL, about 43 ng/mL, about 44 ng/mL, about 45 ng/mL, about 46 ng/mL, about 47 ng/mL, about 48 ng/mL, about 49 ng/mL, about 50 ng/mL.
[000137] In some embodiments, the methods of the present invention comprise a WNT pathway activator or a WNT agonist. In some embodiments, the WNT pathway activator or WNT agonist is selected from the group consisting of CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)5-(5- methyl- 1 H-imidazol-2-yl)-2-pyrimidinyl] amino] ethyl] amino] -3 -pyridinecarbonitrile), Wnt 1 , Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a/b, Wnt-7b, Wnt48 8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-lOa, Wnt-lOb, Wnt-11, Wnt-16b, RSPO co-agonists, lithium chloride, TDZD8 (4-Benzyl-2-methyl-l, 2, 4-thiadiazolidine-3, 5-dione), BIO-Acetoxime ((2'Z,3'E)-6-Bromoindirubin-3 '-acetoxime), A1070722 (l-(7-Methoxyquinolin-4-yl)-3- [6(trifhioromethyl)pyridin-2-yl]urea), HLY78 (4-Ethyl-5,6-Dihydro-5-methyl- [l,3]dioxolo[4,5j]phenanthridine), CID 11210285 hydrochloride (2-Amino-4- (3,4(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride), WAY- 316606, (hetero)arylpyrimidines, IQ1, QS11, SB-216763, and DCA. In a preferred embodiment the Wnt pathway activator is CHIR99021. In one embodiment, the WNT agonist in the cell culture media is CHIR99021 ((CHIR) CAS 252917-06-9). In some embodiments, the concentration of CHIR99021 in the medium used in the methods of the invention is about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3 pM, about 3.1 pM, about 3.2 pM, about 3.3 pM, about 3.4 pM, about 3.5 pM, about 3.6 pM, about 3.7 pM, about 3.8 pM, about 3.9 pM, about 4 pM, about 4.1 pM, about 4.2 pM, about 4.3 pM, about 4.4 pM, about 4.5 pM, about 4.6 pM, about 4.7 pM, about 4.8 pM, about 4.9 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or about 10 pM.
[000138] In some embodiments, the methods of the present invention comprise FGF. In some embodiments, the FGF is selected from the group consisting of FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20 and FGF23. In a preferred embodiment the FGF is FGF2. In some embodiments, the concentration of FGF2, also known as basic fibroblast growth factor (bFGF), in the medium used in the methods of the invention is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29 ng/mL, about 30 ng/mL, about 35 ng/mL, about 41 ng/mL, about 42 ng/mL, about 43 ng/mL, about 44 ng/mL, about 45 ng/mL, about 46 ng/mL, about 47 ng/mL, about 48 ng/mL, about 49 ng/mL, about 50 ng/mL, about 51 ng/mL, about 52 ng/mL, about 53 ng/mL, about 54 ng/mL, about 55 ng/mL, about 56 ng/mL, about 57 ng/mL, about 58 ng/mL, about 59 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL, about 325 ng/mL, about 350 ng/mL, about 375 ng/mL, about 400 ng/mL about 425 ng/mL, about 450 ng/mL, about 475 ng/mL, or about 500 ng/mL.
[000139] In some embodiments, the methods of the present invention comprise a PI3K pathway inhibitor. In some embodiments, the PI3K pathway inhibitor is selected from the group consisting of AS 252424 (5-[[5-(4-Fluoro-2-hydroxyphenyl)-2-furanyl]methylene]-2,4- thiazolidinedione), AS 605240 (5-(6-Quinoxalinylmethylene)-2,4-thiazolidine-2, 4-dione), AZD 6482 ((-)-2-[[(lR)l-[7-Methyl-2-(4-morpholinyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-9- yl]ethyl]amino]benzoic acid), BAG 956 (a,a,-Dimethyl-4-[2-methyl-8-[2-(3-pyridinyl)ethynyl]- lH-imidazo[4,5c]quinolin-l-yl]-benzeneacetonitrile), CZC 24832 (5-(2-Amino-8- fluoro[l,2,4]triazolo[l,5a]pyridin-6-yl)-N-(l,l-dimethylethyl)-3-pyridinesulfonamide), GSK 1059615 (5-[[4-(4Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidenedione), KU 0060648 (4- Ethyl-N - [4- [2-(4morpholinyl)-4-oxo-4H- 1 -benzopyran- 8-yl] - 1 -dibenzothienyl] - 1 - piperazineacetamide), LY 294002 hydrochloride (2-(4-Morpholinyl)-8-phenyl-4H-l- benzopyran-4-one hydrochloride), 3 Methyladenine (3-Methyl-3H-purin-6-amine), PF 04691502 (2-Amino-8-[trans-4-(2hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl- pyrido[2,3-d]pyrimidin-7(8H) one), PF 05212384 (N-[4-[[4-(Dimethylamino)-l- piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4morpholinyl-l,3,5-triazin-2-yl)phenyl]urea), PI 103 hydrochloride (3-[4-(4Morpholinylpyrido[3l,2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride), PI 828 (2-(4Morpholinyl)-8-(4-aminophenyl)-4H-l-benzopyran-4-one), PP 121 (l-Cyclopentyl-3-(lHpyrrolo[2,3-b]pyridin-5-yl)-lH-pyrazolo[3,4-d]pyrimidin-4-amine), Quercetin, TG 100713 (3(2,4-Diamino-6-pteridinyl)-phenol), Wortmannin, PIK90, and GDC- 0941. In a preferred embodiment the PI3K pathway inhibitor is PIK90. In some embodiments, the concentration of PIK90, in the medium used in the methods of the invention is about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 82 nM, about 84 nM, about 86 nM, about 88 nM, about 90 nM, about 92 nM, about 94 nM, about 96 nM, about 98 nM, about 100 nM, 100 nM, about 102 nM, about 104 nM, about 106 nM, about 108 nM, about 110 nM, about 112 nM, about 114 nM, about 116 nM, about 118 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, or about 300 nM,.
[000140] In some embodiments, the methods of the present invention comprise a Rho kinase inhibitor (ROCKi). In a preferred embodiment the Rho kinase inhibitor is Thiazovivin or Y- 27263. In some embodiments, the concentration of Y-27263 in the medium used in the methods of the invention is about 1 pM, about 2 pM, about 5 pM, about 8 pM, about 8.2 pM, about 8.4 pM, about 8.6 pM, about 8.8 pM, about 9 pM, about 9.2 pM, about 9.4 pM, about 9.6 pM, about 9.8 pM, about 10 pM, about 10.2 pM, about 10.4 pM, about 10.6 pM, about 10.8 pM, about 11 pM, about 11.2 pM, about 11.4 pM, about 11.6 pM, about 11.8 pM, about 12 pM, about 15 pM, about 20 |iM, about 25 |iM or about 50 pM.
[000141] In some embodiments, the methods of the present invention comprise a TGF-beta pathway inhibitor. In some embodiments, the TGF-beta pathway inhibitor is selected from the group consisting of A-83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-lH-pyrazole- Icarbothioamide), D4476 (4-[4-(2,3-Dihydro-l,4-benzodioxin-6-yl)-5-(2-pyridinyl)- lHimidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]-2-pyridinyl]- N(tetrahydro-2H-pyran-4-yl)-benzamide), LY 364947 (4-[3-(2-Pyridinyl)-lH-pyrazol-4- yl]quinoline), RepSox (2-(3-(6-Methylpyridine-2-yl)-lH-pyrazol-4-yl)-l,5-naphthyridine), SB431542 (4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide), SB505124 (2-[4-(l,3-Benzodioxol-5-yl)-2-(l,l-dimethylethyl)-lH-imidazol-5-yl]-6- methylpyridine), SB 525334 (6-[2-(l,l-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-lH-imidazol- 4yl] quinoxaline), SD208 (2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine), ITD1 (4[l,l'-Biphenyl]-4-yl-l,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinecarboxylic acid ethyl ester), DAN/Fc, antibodies to TGF-beta and TGF-beta receptors, TGF-beta inhibitory nucleic acids. In a preferred embodiment the TGF-beta pathway inhibitor is A-83-01. In some embodiments, the concentration of A83-O1 in the medium used in the methods of the invention is about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.82 pM, about 0.84 pM, about 0.86 pM, about 0.88 pM, about 0.9 pM, about 0.92 pM, about 0.94 pM, about 0.96 pM, about 0.98 pM, about 1 pM, about 1.02 pM, about 1.04 pM, about 1.06 pM, about 1.08 pM, about 1.1 pM, about 1.12 pM, about 1.14 pM, about 1.16 pM, about 1.18 pM, about 1.2 pM, about 1.4 pM, about 1.6 pM, about 1.8 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or about 10 pM.
[000142] In some embodiments, the methods of the present invention comprise a BMP pathway inhibitor. In some embodiments, the BMP pathway inhibitor is selected from the group consisting of Chordin, soluble BMPRla, soluble BMPRlb, Noggin, LDN-193189, and Dorsomorphin. In a preferred embodiment the BMP pathway inhibitor is LDN-193189. In some embodiments, the concentration of LDN-193189 in the medium used in the methods of the invention is about 50 nM, about 80 nM, about 110 nM, about 140 nM, about 170 nM, about 200 nM, about 205 nM, about 210 nM, about 215 nM, about 220 nM, about 225 nM, about 230 nM, about 235 nM, about 240 nM, about 245 nM, about 250 nM, about 255 nM, about 260 nM, about 265 nM, about 270 nM, about 275 nM, about 280 nM, about 285 nM, about 290 nM, about 295 nM, about 300 nM, about 325 nM, about 350 nM, about 375 nM, about 400 nM, about 425 nM, about 450 nM, about 475 nM, about 500 nM, about 525 nM, about 550 nM, about 575 nM, about 600 nM, about 625 nM, about 650 nM, about 675 nM, about 700 nM, about 725 nM, or about 750 nM.
[000143] In some embodiments, the methods of the present invention comprise vascular endothelial growth factor (VEGF). In one embodiment the VEGF includes human VEGF family members such as VEGFA as well as no-human VEGF. In some embodiments, the concentration of VEGF in the medium used in the methods of the invention is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/mL, about 450 ng/mL, about 500 ng/mL.
[000144] In some embodiments, the methods of the present invention comprise a BMP pathway activator. In some embodiments, the BMP pathway activator is selected from the group consisting of BMP4, BMP2 and BMP7. In a preferred embodiment, the BMP pathway activator is BMP4. In some embodiments, the concentration of bone morphogenetic protein 4 (BMP4) in the medium used in the methods of the invention is about 5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL.
[000145] In some embodiments, the methods of the present invention comprise stem cell factor (SCF). The term “SCF” includes human SCF, non-human SCF and all naturally occurring variants thereof. In some embodiments, the concentration of SCF in the medium used in the methods of the invention is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 160 ng/mL, about 170 ng/mL, about 180 ng/mL, about 190 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL, about 325 ng/mL, about 350 ng/mL, about 375 ng/mL, about 400 ng/mL about 425 ng/mL, about 450 ng/mL, about 475 ng/mL, or about 500 ng/mL.
[000146] In some embodiments, the concentration of interleukin 3 (IL3) in the medium used in the methods of the invention is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 8.2 ng/mL, about 8.4 ng/mL, about 8.6 ng/mL, about 8.8 ng/mL, about 9 ng/mL, about 9.2 ng/mL, about 9.4 ng/mL, about 9.6 ng/mL, about 9.8 ng/mL, about 10 ng/mL, about 10.2 ng/mL, about 10.4 ng/mL, about
10.6 ng/mL, about 10.8 ng/mL, about 11 ng/mL, about 11.2 ng/mL, about 11.4 ng/mL, about
11.6 ng/mL, about 11.8 ng/mL, about 12 ng/mL, about 14 ng/mL, about 16 ng/mL, about 18 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, or about 50 ng/mL.
[000147] In some embodiments, the concentration of fms-related tyrosine kinase 3 ligand (FLT3-L) in the medium used in the methods of the invention is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 8.2 ng/mL, about 8.4 ng/mL, about 8.6 ng/mL, about 8.8 ng/mL, about 9 ng/mL, about 9.2 ng/mL, about 9.4 ng/mL, about 9.6 ng/mL, about 9.8 ng/mL, about 10 ng/mL, about 10.2 ng/mL, about 10.4 ng/mL, about 10.6 ng/mL, about 10.8 ng/mL, about 11 ng/mL, about 11.2 ng/mL, about 11.4 ng/mL, about 11.6 ng/mL, about 11.8 ng/mL, about 12 ng/mL, about 14 ng/mL, about 16 ng/mL, about 18 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, or about 50 ng/mL.
[000148] In some embodiments, the concentration of Interleukin- 15 (IL15) in the medium used in the methods of the invention is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, or about 90 ng/mL, about 95 ng/mL, or about 100 ng/mL.
[000149] In some embodiments, the concentration of Interleukin-7 (IL7) in the medium used in the methods of the invention is about 0.1 ng/mL, about 0.2 ng/mL, about 0.3 ng/mL, about 0.4 ng/mL, about 0.5 ng/mL, about 0.6 ng/mL, about 0.7 ng/mL, about 0.8 ng/mL, about 0.9 ng/mL, 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, or about 90 ng/mL, about 95 ng/mL, or about 100 ng/mL.
[000150] In one embodiment, the concentration of Macrophage Colony-Stimulating Factor (MCSF) is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, or about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL, about 325 ng/mL, about 350 ng/mL, about 375 ng/mL, about 400 ng/mL, about 425 ng/mL, about 450 ng/mL, about 475 ng/mL, or about 500 ng/mL. In a preferred embodiment, the concentration of MCSF is about 50ng /mL.
[000151] In one embodiment, the concentration of Granulocyte Macrophage Colony- Stimulating Factor (GM-CSF) is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, or about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL, about 325 ng/mL, about 350 ng/mL, about 375 ng/mL, about 400 ng/mL, about 425 ng/mL, about 450 ng/mL, about 475 ng/mL, or about 500 ng/mL. In a preferred embodiment, the concentration of Granulocyte Macrophage Colony- Stimulating Factor (GM-CSF) is about 50ng /mL.
[000152] In one embodiment, the concentration of interleukin- 34 (IL34) is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, or about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL, about 325 ng/mL, about 350 ng/mL, about 375 ng/mL, about 400 ng/mL, about 425 ng/mL, about 450 ng/mL, about 475 ng/mL, or about 500 ng/mL. In a preferred embodiment, the concentration of IL34 is about lOOng /mL.
[000153] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may refer to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination. Any example or embodiment herein shall be taken to apply mutatis mutandis to any other example or embodiment unless specifically stated otherwise.
[000154] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent methods and systems are clearly within the scope of the disclosure, as described herein.
[000155] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
[000156] The disclosure is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying drawings. Although the examples herein concern humans and the language is primarily directed to human concerns, the concepts described herein are applicable to other animals. These and other aspects and features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the disclosure as set forth hereinafter.
[000157] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.
Methods for generation of DLL4-expressing arterial endothelial cells (AECs)
[000158] The inventors of the present application have identified a need to develop a new monolayer differentiation method that robustly generated NOTCH-ligand expressing haemogenic endothelial cells, whose gene profile resembles that of arterial haemogenic endothelial cells found the in AGM. The inventors surprisingly found that patterning differentiating PSCs resulted in the rapid transit of cells through stages indicative of MIXL1+ primitive streak and CD13+ early mesoderm. Subsequently, treatment of said mesoderm population with BMP4 signalling antagonist whilst simultaneously inhibiting Activin signalling resulted in the generation of cells that can further differentiate in the presence of BMP4 to yield a cell population highly enriched for expression of the mesodermal-endothelial markers CD 13 and KDR. Subsequent culturing of said cells generates CD34+ cells population that uniformly co-expresses the AEC markers VE-cadherin, CXCR4 and DLL4. Without wishing to be bound by theory, it is believed that the inhibition of the bone morphogenetic (BMP) pathway (i.e. inhibiting ALK1, ALK2, ALK3, and ALK6) early on followed by inhibition of activin receptorlike kinase (ALK) and addition of BMP4 allows for the generation of the population of DLL4- expressing arterial endothelial cells (AECs). This approach is in contrast with methods known in the art which typically require early activation of BMP4.
[000159] Thus, the present invention provides a method for generating a population of DLL4- expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD13+ early mesoderm cells, lc. incubating the population of CD 13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway activator, for a time sufficient for generating a population of CD13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD 13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising at least one of SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells, and optionally cryopreserving the population of DLL4-expressing AECs.
[000160] In another embodiment, the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor, a Wnt pathway activator, a BMP pathway inhibitor, FGF and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD 13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating a population of CD13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD 13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells, and optionally cry opreserving the population of DLL4-expressing AECs.
[000161] In another embodiment, the present invention provides a method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising A83- 01, CHIR99021, LDN-193189, FGF2, and which is free or essentially free of BMP4, for a time sufficient for generating a population of CD13+ early mesoderm cells, lc. incubating the population of CD 13+ early mesoderm cells in a third defined medium comprising A83-O1, VEGF, BMP4, FGF2, for a time sufficient for generating a population of CD13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD 13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, BMP4, FGF2, for a time sufficient for generating the population of CD34+ cells, and optionally cry opreserving the population of DLL4-expressing AECs.
[000162] In one embodiment, the population of substantially undifferentiated pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In a preferred embodiment, the iPSCs are selected from the group consisting of iPSC RM3.5 (ma e)RAGI:GFP, iPSC PB0-01 (male), iPSC PB0-04 (female), iPSC PB0-05 (female), iPSC PB0-06 (male), iPSC PB0-10 (male), iPSC CRL2429 (ATCC), and the like. In another preferred embodiment, the ESCs are selected from the group consisting of ESC Hl (male), ESC H9 (female)FAG/;GF/>, ESC ^soxn.-tdTOMATo.-Ruxi^GFP^ ESC HES3(female)Mm7;GFF, and the like.
[000163] In one embodiment, the culturing in step la is performed in a first defined medium capable of and for a time sufficient for generation of a population of MIXL1+ cells. In a preferred embodiment, the first defined medium comprises Activin A, CHIR99021 , FGF2, and PIK90. In another preferred embodiment, the first defined medium is free of or is essentially free of or does not comprise BMP4. In a more preferred embodiment, the concentration of Activin A in the first defined medium is about 30 ng/mL (preferably, from 10 to 50 ng/mL), the concentration of CHIR99021 in the first defined medium is about 4 pM (preferably, from 1 to 10 pM), the concentration of FGF2 in the first defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL), and the concentration of PIK90 in the first defined medium is about 100 nM (preferably, from 10 to 300 nM). In another preferred embodiment, the time sufficient for generation of a population of MIXL1+ cells is about 24 hours (preferably, from 2 to 72 hours). In yet another preferred embodiment, the population of MIXL1+ cells at the end of step la is at least 75% of total cells.
[000164] In one embodiment, the culturing in step la is optionally performed in a first defined medium further comprising Y-27263. In a preferred embodiment, the concentration of Y-27263 is about 1 to 50 pM, preferably about 8 to 12 pM, more preferably about 10 pM
[000165] In one embodiment, the culturing in step lb is performed in a second defined medium capable of and for a time sufficient for generation of a population of CD 13+ early mesoderm cells. In a preferred embodiment, the second defined medium comprises A83-O1, CHIR99021, LDN-193189, and FGF2. In another preferred embodiment, the second defined medium is free of or is essentially free of or does not comprise BMP4. In a more preferred embodiment, the concentration of A83-01 in the second defined medium is about 1 pM (preferably, from 0.1 to 10 pM), the concentration of CHIR99021 in the second defined medium is about 3 pM (preferably, from 1 to 10 pM), the concentration of LDN-193189 in the second defined medium is about 250 nM (preferably, from 50 to 750 nM), and the concentration of FGF2 in the second defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL). In another preferred embodiment, the time sufficient for generation of a population of CD 13+ early mesoderm cells is about 24 hours (preferably, from 2 to 72 hours). In yet another preferred embodiment, the population of CD13+ early mesoderm cells at the end of step lb is at least 85% of total cells.
[000166] In one embodiment, the culturing in step 1c is performed in a third defined medium capable of and for a time sufficient for generation of a population of CD 13+ and KDR+ mesodermal-endothelial cells. In a preferred embodiment, the third defined medium comprises A83-01, VEGF, BMP4, and FGF2. In a more preferred embodiment, the concentration of A83- 01 in the third defined medium is about 1 pM (preferably, from 0.1 to 10 pM), the concentration of VEGF in the third defined medium is about 50 ng/mL (preferably, from 10 to 100 ng/mL), the concentration of BMP4 in the third defined medium is about 30 ng/mL (preferably, from 10 to 100 ng/mL), and the concentration of FGF2 in the third defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL). In another preferred embodiment, the time sufficient for generation of a population of CD13+ and KDR+ mesodermal-endothelial cells is about 24 hours (preferably, from 2 to 72 hours). In yet another preferred embodiment, the population of CD13+ and KDR+ mesodermal-endothelial cells at the end of step lc is at least 20% of total cells.
[000167] In one embodiment, the culturing in step Id is performed in a fourth defined medium capable of and for a time sufficient for generation of CD34+ cells. In another embodiment, the CD34+ cells are DLL4-expressing arterial endothelial cells (AECs). In yet another embodiment, the CD34+ cells uniformly co-express the AEC markers VE-cadherin, CXCR4, and DLL4. In a preferred embodiment, the fourth defined medium comprises SCF, VEGF, BMP4, and FGF2. In a more preferred embodiment, the concentration of SCF in the fourth defined medium is about 50 ng/mL (preferably, from 10 to 100 ng/mL), the concentration of VEGF in the fourth defined medium is about 50 ng/mL (preferably, from 10 to 100 ng/mL), the concentration of BMP4 in the fourth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of FGF2 in the fourth defined medium is about 50 ng/mL (preferably, from 10 to 100 ng/mL). In another preferred embodiment, the time sufficient for generation of a population of CD34+ cells is about 72 hours (preferably, from 24 to 144 hours). In yet another preferred embodiment, the population of CD34+ cells at the end of step Id is at least 70% of total cells. In yet a further preferred embodiment, one input pluripotent stem cell gives rise to approximately 6 DLL4+ CD34+ AECs at the and of step Id.
[000168] In one embodiment, there is provided a population of DLL4-expressing arterial endothelial cells (AECs) obtained from the method disclosed herein, wherein the DLL4- expressing arterial endothelial cells (AECs) are CD34+ cells. In a preferred embodiment, the population of CD34+ cells co-expresses CXCR4. In another preferred embodiment, the population of CD34+ cells co-expresses CDH5 (VE-cadherin).
[000169] The present invention provides a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells. In a preferred embodiment, the population of CD34+ cells co-expresses CXCR4. In another preferred embodiment, the population of CD34+ cells co-expresses CDH5 (VE-cadherin).
[000170] The population of DLL4-expressing AECs can be optionally cryopreserved and subsequently thawed and further cultured. Thus, in a preferred embodiment, the method further comprises cryopreserving the population of DLL4-expressing AECs following step Id. In yet a further embodiment there is provided a cryopreserved population of DLL4-expressing AECs obtained according to the methods described herein. That is, in one embodiment, there is provided a cryopreserved population of DLL4-expressing AECs produced according to steps la to Id as described above.
Methods for generation of a mixed population of PSC-derived innate lymphoid cells ) and
Figure imgf000066_0001
PSC-derived NK-like cells
[000171] The inventors surprisingly found that the DLL4-expressing arterial endothelial cells (AECs) as described above can efficiently generate a mixture of lymphoid cells with characteristics of the ILC lineage and natural killer-like (NK-like) cells. Unlike in previously described lymphoid differentiation methods, the method developed by the inventors surprisingly does not require addition of an agent which activates NOTCH signalling such as exogeneous NOTCH ligands. Again, without wishing to be bound by theory, the inventors consider that due to the frequency and arrangement of NOTCH ligands arising from the monolayer culture of PSC-derived haemogenic arterial endothelial cells, NOTCH signalling is increased in emerging haematopoietic progenitor cells thereby obviating a requirement for addition of exogenous NOTCH ligands, and leading to lymphoid commitment by the haematopoietic progenitors and to enhanced generation of ILCs.
[000172] Thus, the present invention provides a method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of:
2a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
2b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising at least one of Flt3L, VEGF, FGF and IL7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells, 2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising at least one of Flt3L, VEGF, FGF, IL7 and IL 15 for a time sufficient for generating the mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[000173] In another embodiment the present invention provides a method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of:
2a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
2b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising Flt3L, VEGF, FGF2, IL7, and IL15 for a time sufficient for generating the mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells,
[000174] wherein the method does not comprise addition of an agent which activates NOTCH signalling. In one embodiment, step 2a is carried out for a period of time and under conditions suitable for generation of a monolayer of the DLL4-expressing arterial endothelial cells (AECs) produced according to the methods described herein. In another embodiment, the population of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id and utilised in step 2a were cryopreserved and thawed prior to the commencement of step 2a.
[000175] In one embodiment, the incubating in step 2b is performed in a fifth defined medium capable of and for a time sufficient for generation of CD34+CD43+ haematopoietic progenitor cells. In a preferred embodiment, the fifth defined medium comprises SCF, VEGF, FGF2, IL3, and Flt3L. In a more preferred embodiment, the concentration of SCF in the fifth defined medium is about 100 ng/mL (preferably, from 50 to 500 ng/mL), the concentration of VEGF in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of IL3 in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of Flt3L in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL). In another preferred embodiment, the time sufficient for generation of a CD34+CD43+ haematopoietic progenitor is about 144 hours (preferably, from 72 to 288 hours). In yet another preferred embodiment, the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 2b is at least 25% of total cells.
[000176] In one embodiment, the incubating in step 2c is performed in a sixth defined medium capable of and for a time sufficient for generation of CD34+CD7+ lymphoid haematopoietic progenitor cells. In a preferred embodiment, the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7. In a more preferred embodiment, the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL), and the concentration of IL7 in the sixth defined medium is about 1 ng/mL (preferably, from 0.1 to 10 ng/mL). In another preferred embodiment, the time sufficient for generation of CD34+CD7+ lymphoid haematopoietic progenitor cells is about 72 hours (preferably, from 24 to 144 hours). In yet another preferred embodiment, the population of CD34+CD7+ lymphoid haematopoietic progenitor cells at the end of step 2c is at least 40% of total cells.
[000177] In one embodiment, the incubating in step 2d is performed in a sixth defined medium capable of and for a time sufficient for generation of CD34-CD7+ and CD7+RAG1+ lymphoid cells. In a preferred embodiment, the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7. In a more preferred embodiment, the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL), and the concentration of IL7 in the sixth defined medium is about 1 ng/mL (preferably, from 0.1 to 10 ng/mL). In another preferred embodiment, the time sufficient for generation of CD34-CD7+ and CD7+RAG1+ lymphoid cells is about 96 hours (preferably, from 48 to 192 hours). In yet another preferred embodiment, the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells at the end of step 2d is at least 45% of total cells.
[000178] In one embodiment, the incubating in step 2e is performed in a seventh defined medium capable of and for a time sufficient for generation of mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells. In a preferred embodiment, the seventh defined medium comprises Flt3L, VEGF, FGF2, IL7, and IL15. In a more preferred embodiment, the concentration of Flt3L in the seventh defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), the concentration of VEGF in the seventh defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the seventh defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of IL7 in the seventh defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL), and the concentration of IL 15 in the seventh defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL). In another preferred embodiment, the time sufficient for generation of mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells is about 96 hours (preferably, from 48 to 192 hours). In yet another preferred embodiment, the population of PSC-derived innate lymphoid cells (ILCs) at the end of step 2e is at least 75% of total cells and the population of PSC-derived NK-like cells at the end of step 2e is at least 50% of total cells. In yet another preferred embodiment, the population of PSC-derived innate lymphoid cells (ILCs) at the end of step 2e is at least 75% of total cells or the population of PSC-derived NK-like cells at the end of step 2e is at least 50% of total cells. In yet another preferred embodiment, the PSC-derived innate lymphoid cells (ILCs) are CD161+CD7+ cells and CD161+RAG1- cells. In yet another preferred embodiment, the PSC-derived innate lymphoid cells (ILCs) are CD161+CD7+ cells or CD161+RAG1- cells. In yet another preferred embodiment, the population of CD161+CD7+ cells is at least 70% of total cells. In yet another preferred embodiment, the population of CD161+RAG1- cells is at least 70% of total cells. [000179] In one embodiment, there is provided a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells obtained from the method disclosed herein. In a preferred embodiment, the PSC-derived ILCs are CD161+CD7+ cells. In another preferred embodiment, the PSC-derived ILCs are CD161+RAG1- cells. In another preferred embodiment, the NK-like cells are CD161+CD56+ cells.
[000180] The present invention provides a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells. In a preferred embodiment, the PSC-derived ILCs are CD161+CD7+ cells. In another preferred embodiment, the PSC-derived ILCs are CD161+RAG1- cells. In another preferred embodiment, the NK-like cells are CD161+CD56+ cells.
Methods for generation of a cell population enriched either in PSC-derived innate lymphoid cells (ILCs) or PSC-derived T cells
[000181] The inventors surprisingly found that controlling the IL7 concentrations in the sixth defined medium used in the method of the present invention can regulate T-cell versus NK-cell lineage commitment. Surprisingly, the inventors found that high concentration of IL7 favours the development of innate lymphoid lineages. On the other hand, low concentration of IL7 generates a cell profile that more closely resembles early T cell differentiation in the human thymus.
[000182] Thus, the present invention provides a method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
3b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3 and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[000183] In another embodiment, the present invention provides a method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
3b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[000184] Thus, the present invention provides a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
4b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling. [000185] In another embodiment, the present invention provides a method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
4b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[000186] Steps 3a and 4a are identical. Thus, in one embodiment, step 3a or step 4a is carried out for a period of time and under conditions suitable for generation of a monolayer of the DLL4-expressing arterial endothelial cells (AECs) produced according to the methods described herein. In another embodiment, the population of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id and utilised in step 3 a or 4a were cryopreserved and thawed prior to the commencement of step 3 a or 4a.
[000187] Steps 3b and 4b are identical. Thus, in one embodiment, the incubating in step 3b or 4b is performed in a fifth defined medium capable of and for a time sufficient for generation of CD34+CD43+ haematopoietic progenitor cells. In a preferred embodiment, the fifth defined medium comprises SCF, VEGF, FGF2, IL3, and Flt3L. In a more preferred embodiment, the concentration of SCF in the fifth defined medium is about 100 ng/mL (preferably, from 50 to 500 ng/mL), the concentration of VEGF in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of IL3 in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of Flt3L in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL). In another preferred embodiment, the time sufficient for generation of a CD34+CD43+ haematopoietic progenitor is about 144 hours (preferably, from 72 to 288 hours). In yet another preferred embodiment, the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 3b or 4b is at least 25% of total cells.
[000188] Steps 3c and 4c are identical except for the concentration of IL7 and for the types of cells generated. Thus, in one embodiment, the incubating in step 3c is performed in a sixth defined medium capable of and for a time sufficient for generation of cell population enriched in CD161+RAG1- cells. In another embodiment, the PSC-derived innate lymphoid cells (ILCs) are CD161+RAG1- cells. In a preferred embodiment, the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 20 ng/mL (preferably, from 10 to 50 ng/mL). In a more preferred embodiment, the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), and the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 10 to 20 ng/mL). In another preferred embodiment, the time sufficient for generation of cell population enriched in CD161+RAG1- cells is about 11 days (preferably, from 7 to 21 days). In yet another preferred embodiment, the cell population enriched in CD161+RAG1- cells at the end of step 3c is at least 50% of total cells.
[000189] In another embodiment the sixth defined medium employed in step 3c is supplemented with IL15 starting from about 72h to about 168h following commencement of step 3c until completion of step 3c. In another embodiment, the concentration of IL 15 is about 20 ng/mL (preferably, from 1 to 100 ng/mL). In yet another embodiment, following supplementation of the sixth defined medium with IL 15 as described above, one input pluripotent stem cell gives rise to from 5 to 76 CD161+ CD7+ cells at the and of step 3c. In one embodiment, the incubating in step 4c is performed in a sixth defined medium capable of and for a time sufficient for generation of cell population enriched in CD4+CD8a+ cells. In another embodiment, the PSC- derived T cells are CD4+CD8a+ cells. In a preferred embodiment, the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 0.1 ng/mL (preferably, from 0.05 to 1 ng/mL). In a more preferred embodiment, the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), and the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL). In another preferred embodiment, the time sufficient for generation of cell population enriched in CD4+CD8a+ cells is about 11 days (preferably, from 7 to 21 days). In yet another preferred embodiment, the cell population enriched in CD4+CD8a+ cells at the end of step 4c is at least 12% of total cells.
[000190] In one embodiment, there is provided a cell population enriched in PSC-derived ILCs obtained from the method disclosed herein. In a preferred embodiment, the PSC-derived ILCs are CD161+RAG1- cells. In another embodiment, there is provided a cell population enriched in PSC-derived T cells obtained from the method disclosed herein. In another preferred embodiment, the PSC-derived T cells are CD4+CD8a+ cells.
[000191] The present invention provides a cell population enriched in PSC-derived ILCs. In a preferred embodiment, the PSC-derived ILCs are CD161+RAG1- cells. In another embodiment, there is provided a cell population enriched in PSC-derived T cells obtained from the method disclosed herein. In another preferred embodiment, the PSC-derived T cells are CD4+CD8a+ cells.
Methods for generation of a cell population enriched in PSC-derived NK-like cells
[000192] The inventors surprisingly found that the method disclosed herein can be used to efficiently generate NK-like cells from human RAG1+ lymphoid progenitors. When NK-like cells are generated using such method, there is a need to obtain a cell population enriched in CD161+RAG1+ cells. The inventors also surprisingly found an alternative method for efficiently generating NK-like cells from CD34+CD7+ lymphoid haematopoietic progenitor cells. When said alternative method is used, a cell sorting step is not required.
[000193] Without wishing to be bound by theory, the inventors have found that RAG1 expression levels presaged gene expression profiles indicative of further commitment to the adaptive lymphoid lineage (RAGl-high) or the innate lymphoid lineage (RAG 1 -low). Thus, the inventors surprisingly found that RAG1 expression may be indicative of fate decisions between different branches of the ILC lineages and that PSC-derived NK-like cells can be generated from a RAG1+ intermediate.
[000194] Thus, the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising:
5al. generating a population of cells enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells; and 5e. incubating the cell population enriched in CD161+RAG1+ cells in a defined medium comprising IL7 and at least one of Flt3L, VEGF, FGF and IL15, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[000195] In some embodiments, the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
5a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
5b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF2, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
5c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF2, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating a cell suspension comprising a population of CD161+RAG1+ cells,
5d. separating the cell suspension from the monolayer and sorting the cell suspension for a cell population enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells;
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IL15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IL 15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when present the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL and, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
[000196] Thus, the present invention provides a method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of: 6a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
6b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF2, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
6c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF2, for a time sufficient for generating a cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
6d. separating the cell suspension from the monolayer, removing the sixth defined medium from the suspension, and adding a seventh defined medium comprising IL15 and at least one of Flt3L, VEGF, FGF2 and IL7, wherein the concentration of IL15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when present the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
[000197] Steps 5a and 6a are identical. Thus, in one embodiment, step 5a or step 6a is carried out for a period of time and under conditions suitable for generation of a monolayer of the DLL4-expressing arterial endothelial cells (AECs) produced according to the method described herein. In another embodiment, the population of DLL4-expressing AECs produced according to the method as previously described in steps la to Id and utilised in step 5a or 6a were cryopreserved and thawed prior to the commencement of step 5a or 6a.
[000198] In one embodiment, step 5a 1 is carried out for a period of time and under conditions suitable for generation of a population of cells enriched in CD161+RAG1+ cells. In one embodiment the population of cells enriched in CD161+RAG1+ cells is prepared according to the methods described herein.
[000199] Steps 5b and 6b are identical. Thus, in one embodiment, the incubating in step 5b or 6b is performed in a fifth defined medium capable of and for a time sufficient for generation of CD34+CD43+ haematopoietic progenitor cells. In a preferred embodiment, the fifth defined medium comprises SCF, VEGF, FGF2, IL3, and Flt3L. In a more preferred embodiment, the concentration of SCF in the fifth defined medium is about 100 ng/mL (preferably, from 50 to 500 ng/mL), the concentration of VEGF in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of IL3 in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of Flt3L in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL). In another preferred embodiment, the time sufficient for generation of a CD34+CD43+ haematopoietic progenitor is about 144 hours (preferably, from 72 to 288 hours). In yet another preferred embodiment, the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 5b or 6b is at least 25% of total cells.
[000200] The two methods for generation of a cell population enriched in PSC-derived NK-like cells diverges from step c, onwards. That is, steps 5c to 5e are different from steps 6c to 6d.
[000201] Thus, in one embodiment, the incubating in step 5c is performed in a sixth defined medium capable of and for a time sufficient for generation of a cell suspension comprising a population of CD161+RAG1+ cells. In a preferred embodiment, the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 20 ng/mL (preferably, from 10 to 50 ng/mL). In a more preferred embodiment, the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), and the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL). In another preferred embodiment, the time sufficient for generation of the cell suspension comprising the population of CD161+RAG1+ cells at the end of step 5c is about 7 days (preferably, from 3 to 14 days).
[000202] A person skilled in the art is aware of the typical or standard procedures used for separating a cell suspension from a cell monolayer. A person skilled in the art is also aware of the typical or standard procedures used for sorting a cell suspension for or to obtain a cell population enriched in a particular cell type (such as through the expression of one or more genes or proteins, typically one or more markers expressed on the cell surface). In some embodiments, antibodies or similar agents specific for a given marker, or set of markers, can be used to separate and isolate the desired cells using fluorescent activated cell sorting (FACS), panning methods, magnetic particle selection, particle sorter selection and other methods known to persons skilled in the art. In one embodiment, the sorting at step 5d is fluorescence-activated cell sorting (FACS).
[000203] In one embodiment, the incubating in step 5e is performed in a seventh defined medium capable of and for a time sufficient for generation of cell population enriched in CD161+CD56+ cells. In another embodiment, the PSC-derived NK-like cells are CD161+CD56+ cells. In a preferred embodiment, the seventh defined medium comprises Flt3L, VEGF, FGF2, and IL15, wherein the concentration of IL15 is about 20 ng/mL (preferably, from 1 to 100 ng/mL). In another preferred embodiment, the seventh defined medium comprises Flt3L, VEGF, FGF2, IL7, and IL15, wherein the concentration of IL7 is about 20 ng/mL (preferably, from 10 to 50 ng/mL) and the concentration of IL15 is about 20 ng/mL (preferably, from 1 to 100 ng/mL). In a more preferred embodiment, the concentration of Flt3L in the seventh defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the seventh defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), and the concentration of FGF2 in the seventh defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL). In another preferred embodiment, the time sufficient for generation of cell population enriched in CD161+CD56+ cells is about 96 hours (preferably, from 48 to 192 hours). In yet another preferred embodiment, the cell population enriched in CD161+CD56+ cells at the end of step 5e is at least 70% of total cells. In yet another preferred embodiment, one input pluripotent stem cell gives rise to approximately 1 to 2.5 CD161+ CD56+ cells at the and of step 5e.
[000204] In one embodiment, the incubating in step 6c is performed in a sixth defined medium capable of and for a time sufficient for generation of a cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells. In a preferred embodiment, the sixth defined medium comprises Flt3L, VEGF, FGF2, and IL7. In a more preferred embodiment, the concentration of Flt3L in the sixth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the sixth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), the concentration of FGF2 in the sixth defined medium is about 20 ng/mL (preferably, from 10 to 50 ng/mL), and the concentration of IL7 in the sixth defined medium is about 1 ng/mL (preferably, from 0.1 to 10 ng/mL). In another preferred embodiment, the time sufficient for generation of cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells is about 72 hours (preferably, from 24 to 144 hours). In yet another preferred embodiment, the population of CD34+CD7+ lymphoid haematopoietic progenitor cells at the end of step 6c is at least 40% of total cells. [000205] A person skilled in the art is aware that step 6d comprises three separate sub-steps namely the sub-step of separating a cell suspension from a monolayer, the sub-step of removing the sixth defined medium from the suspension thereby obtaining cells from the suspension that is free from or essentially free from the sixth defined medium, and the sub-step of adding a seventh defined medium to the cells obtained from the previous sub-step. A person skilled in the art is aware of the typical or standard procedures used for performing each of the three separate sub-steps. In one embodiment, the final sub-step of step 6d is performed in a seventh defined medium capable of and for a time sufficient for generation of cell population enriched in CD161+CD56+ cells. In another embodiment, the PSC-derived NK-like cells are CD161+CD56+ cells. In a preferred embodiment, the seventh defined medium comprises Flt3L, VEGF, FGF2, IL7, and IL15, wherein the concentration of IL7 is about 20 ng/mL (preferably, from 1 to 100 ng/mL) and the concentration of IL 15 is about 20 ng/mL (preferably, from 1 to 100 ng/mL). In a more preferred embodiment, the concentration of Flt3L in the seventh defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), the concentration of VEGF in the seventh defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), and the concentration of FGF2 in the seventh defined medium is about 20 ng/mL (preferably, from 1 to 100 ng/mL). In another preferred embodiment, the time sufficient for generation of cell population enriched in CD161+CD56+ cells is about 120 hours (preferably, from 48 to 240 hours). In yet another preferred embodiment, the cell population enriched in CD161+CD56+ cells at the end of step 6d is at least 80% of total cells.
[000206] In another embodiment, the cells in step 6d are maintained in said seventh defined medium for a time sufficient for generation of cell population enriched in CD161+CD16+ cells, preferably wherein the time is from 10 to 17 days, even more preferably 15 days. After being maintained in said seventh defined medium the cells obtained after 15 days display an enhanced cytotoxic function compared to cells obtained maintained in said medium for 5 days.
[000207] In one embodiment, there is provided a cell population enriched in PSC-derived NK- like cells obtained from the method disclosed herein. In a preferred embodiment, the PSC- derived NK-like cells are CD161+CD56+ cells.
[000208] The present invention provides a cell population enriched in PSC-derived NK-like cells. In a preferred embodiment, the PSC-derived NK-like cells are CD161+CD56+ cells.
Methods for generation of a cell population enriched in PSC-derived erythroid and myeloid cells [000209] The inventors surprisingly found that the method disclosed herein can be used to efficiently generate cells representing a broad spectrum of linages include erythroid and myeloid lineages.
[000210] Thus, in another embodiment, there is provided a method for generating a cell population enriched in PSC-derived erythroid cells, wherein the PSC-derived erythroid cells are CD235a+CD14- cells, comprising the sequential steps of:
7a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
7b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3E, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
7c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in an eighth defined medium comprising Erythropoitein (EPO), optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a+CD14- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
[000211] Thus, in another embodiment, there is provided a method for generating a cell population enriched in PSC-derived myeloid cells, wherein the PSC-derived erythroid cells are CD235a-CD14+ cells, comprising the sequential steps of:
8a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method as previously described in steps la to Id,
8b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
8c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a ninth defined medium comprising one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), and IL34, and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a-CD14+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
[000212] Steps 7a and 8a are identical. Thus, in one embodiment, step 7a or step 8a is carried out for a period of time and under conditions suitable for generation of a monolayer of the DLL4-expressing arterial endothelial cells (AECs) produced according to the method described herein. In another embodiment, the population of DLL4-expressing AECs produced according to the method as previously described in steps la to Id and utilised in step 7a or 8a were cryopreserved and thawed prior to the commencement of step 7a or 8a.
[000213] Steps 7b and 8b are identical. Thus, in one embodiment, the incubating in step 7b or 8b is performed in a fifth defined medium capable of and for a time sufficient for generation of CD34+CD43+ haematopoietic progenitor cells. In a preferred embodiment, the fifth defined medium comprises SCF, VEGF, FGF2, IL3, and Flt3L. In a more preferred embodiment, the concentration of SCF in the fifth defined medium is about 100 ng/mL (preferably, from 50 to 500 ng/mL), the concentration of VEGF in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of FGF2 in the fifth defined medium is about 50 ng/mL (preferably, from 10 to 500 ng/mL), the concentration of IL3 in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL), and the concentration of Flt3L in the fifth defined medium is about 10 ng/mL (preferably, from 1 to 50 ng/mL). In another preferred embodiment, the time sufficient for generation of a CD34+CD43+ haematopoietic progenitor is about 144 hours (preferably, from 72 to 288 hours). In yet another preferred embodiment, the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 7b or 8b is at least 25% of total cells.
[000214] In one embodiment, the incubating in step 7c is performed in an eighth defined medium capable of and for a time sufficient for generation of cell population enriched in CD235a+ erythroid cells. In another embodiment, the PSC-derived CD235a+ erythroid cells are CD235a+CD14- cells. In a preferred embodiment, the eighth defined medium comprises EPO, optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, wherein the concentration of EPO is about 2 units/mL (preferably, from 1 to 5 units/mL). In one embodiment, when included, the concentration of Flt3L in the eighth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the eighth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), and the concentration of FGF2 in the eighth defined medium is about 20 ng/mL (preferably, from 10 to 20 ng/mL). In another preferred embodiment, the time sufficient for generation of cell population enriched in CD235a+ cells is about 14 days (preferably, from 7 to 21 days). In yet another preferred embodiment, the cell population enriched in CD235a+ cells at the end of step 7c is at least 80% of total cells.
[000215] In one embodiment, there is provided a cell population enriched in PSC-derived erythroid cells obtained from the method disclosed herein. In a preferred embodiment, the PSC- derived erythroid cells are CD235a+CD14- cells.
[000216] In another embodiment, the incubating in step 8c is performed in a ninth defined medium capable of and for a time sufficient for generation of cell population enriched in CD 14+ myeloid cells. In another embodiment, the PSC-derived CD 14+ erythroid cells are CD235a- CD14+ cells. In a preferred embodiment, the ninth defined medium comprises one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), and IL34, and optionally further comprises one or more of Flt3L, VEGF, FGF2, and IL7. In a preferred embodiment, the concentration of MCSF is about 50ng /mL (preferably, from 10 to 500 ng/mL). In a preferred embodiment, the concentration of GM-CSF is about 50ng /mL (preferably, from 10 to 500 ng/mL). In a preferred embodiment, the concentration of IL-34 is about lOOng /mL (preferably, from 10 to 500 ng/mL). In another embodiment, when included, the concentration of Flt3L in the ninth defined medium is about 10 ng/mL (preferably, from 1 to 100 ng/mL), the concentration of VEGF in the ninth defined medium is about 50 ng/mL (preferably, from 5 to 500 ng/mL), and the concentration of FGF2 in the ninth defined medium is about 20 ng/mL (preferably, from 10 to 20 ng/mL). In another preferred embodiment, the time sufficient for generation of cell population enriched in CD 14+ cells is about 42 days (preferably, from 35 to 49 days). In yet another preferred embodiment, the cell population enriched in CD235a+ cells at the end of step 7c is at least 50% of total cells.
[000217] In another embodiment, there is provided a cell population enriched in PSC-derived myeloid cells obtained from the method disclosed herein. In a preferred embodiment, the PSC- derived myeloid cells are CD14+CD235a- cells. Other Cell Populations
[000218] In employing the methods described herein, a skilled person will understand that a cell population may be obtained following any of the individual steps recited in any of the methods described herein. That is, by pausing or arresting the method, or obtaining a sample of cells after a first or any subsequent step of one of the methods described herein a population of cells may be obtained. Accordingly in one embodiment, the cell population obtained may be AECs obtained from step Id, or mesodermal-endothelial cells obtained from step 1c, or early mesoderm cells obtained from step lb, or MIXL1+ cells obtained from step la . Alternatively, in another embodiment, the cell population obtained may be population of CD34+CD43+ haematopoietic progenitor cells obtained from step 2b, a population of CD34+CD7+ lymphoid haematopoietic progenitor cells obtained from step 2c, a population of CD34-CD7+ lymphoid cells obtained from step 2d, a population of CD7+RAG1+ lymphoid cells obtained from step 2d, or a population of CD161+RAG1+ cells obtained from step 5c or 5d. Accordingly, in another one embodiment, there is provided a cell population obtained from any of steps la, lb, 1c, Id, 2a, 2b, 2c, 2d, 2e, 3a, 3b, 3c, 4a, 4b, 4c, 5a, 5b, 5c, 5d, 5e, 6a, 6b, 6c, 6d, 7a, 7b, 7c, 8a, 8b, or 8c.
Examples
[000219] Example 1 - Materials and Methods
[000220] 1.1 Human pluripotent stem cell lines and culture
[000221] Pluripotent stem cells (PSCs) used in study is all human origin. Work related to human pluripotent stem cell lines was conducted in accordance with RCH Human Research Ethics Committee approval 33OO1A. Human PSC lines, including ESCs and iPSCs, used in this study are summarized as the follows: ESC Hl (male) (see, Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-7 (1998).), iPSC RM3.5 (male)ffAG2;GFP, ESC H9 (female)R4G2;GF (see, Motazedian, A. et al. Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell- derived haematopoietic organoids. Nat Cell Biol 22, (2020).), ^^OX17MTOMAT°;RVX1C:GFP (see, Ng, E. S. et al. Differentiation of human embryonic stem cells to HOXA + hemogenic vasculature that resembles the aorta-gonad-mesonephros. Nat Biotechnol 34, 1168-1179 (2016).), ESC HES3(female)M2X£2 GF (see, Davis, R. P. et al. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood 111, 1876-1884 (2008).), iPSC PB0-01 (male)/ -04 (female)/ -05 (female)/ -06 (male)/ -10 (male) (see, Vlahos, K. et al. Generation of iPSC lines from peripheral blood mononuclear cells from 5 healthy adults. Stem Cell Res 34, 101380 (2019).), iPSC CRL2429 (ATCC). For all differentiation experiments, the inventors used PSCs that were less than 50 passages since their last karyotype analysis. Karyotyping was performed using the standard Infinium CoreExome-24 SNP array. Reagents for PSC culture and differentiation medium were purchased from ThermoFisher unless otherwise specified. PSC were grown in E8 medium as previously described (see, Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8, 424- 429 (2011).). Once cultures reached a confluency of approximately 80-90%, PSCs were passaged by detaching cells using dissociation buffer comprising phosphate buffer saline without calcium and magnesium (PBS-/ ) supplemented with 100 mM NaCl and 0.5 mM EDTA. The resultant small clumps of cells were transferred to new flasks that had been previously coated with Geltrex™cell culture plate coating matrix (ThermoFisher). Geltrex™cell was dissolved in cold PBS-/- at a dilution rate of 1:100. Cells were routinely passaged at a ratio of 1:3 to 1:5 relative to the starting flask surface area. Medium was refreshed daily. As an alternative, hESCs and hiPSCs were sometimes grown in the presence of inactivated mouse embryonic fibroblasts in PSC media consisting of DMEM-F12, 20% knock-out serum replacement, Ixnon-essential amino acids, IxGlutaMAX, 0.11 mM -mercaptoethanol and FGF2 (10 ng/ml) as previously described (see, Costa, M., Sourris, K., Hatzistavrou, T., Elefanty, A. G. & Stanley, E. G. Expansion of human embryonic stem cells in vitro. Curr Protoc Stem Cell Biol Chapter 1, Unit 1C.1.1-1C.1.7 (2008).).
[000222] 1.2 Monolayer differentiation of arterial endothelial cells and lymphoid progenitor cells from PSCs
[000223] Formulation of the basal medium is as previously described (see, Motazedian, A. et al. Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids. Nat Cell Biol 22, (2020).), and supplement factors on the indicated days are listed below. On the day of differentiation, referred to as day 0, PSCs were dissociated using EDTA dissociation buffer and resuspended in day 0 medium (basal cell differentiation medium containing day 0 supplements). The cells were then plated onto Geltrex-coated 6-well plates at a low density at about 5% of confluency, approximately 10000 cells per cm2. After 26 hours, cell culture was refreshed by day 1 medium, which was followed by additional medium change on day 2 (after 24 hours) using day 2 medium. During day 3-6, cells were maintained in the day 3-6 medium that was replenished on day 5.
[000224] On day 6 when the culture reached confluency (over 95% coverage of the surface), the culture was dissociated using TrypLE (3 minutes at 37°C) and the cells re-plated at the ratio of 1:3 onto Geltrex-coated 6-well plates as described above. From day 6-12, cells were cultured in the day 6 medium, and the medium was completely changed every 2-3 days, 2 ml/well. From day 12, the cells were cultured in day 12 medium and the medium was changed every 2-3 days. From day 19, the cells were culture in day 19 medium and the medium was changed every 2-3 days. Flow cytometry characterization for CD161 and CD56 was performed at day 23-25.
[000225] Day 0: Activin A, 30ng/ml; CHIR99021, 4pM; fibroblast growth factor (FGF) 2, 20ng/ml; PIK90, lOOnM; Y-27263 10 pM (optional for cell lines with poor viability after dissociation). Day 1: A83-01, 1 pM; CHIR99021, 3pM; LDN-193189, 250 nM; FGF2, 20ng/ml. Day 2: A83-01, 1 pM; vascular endothelium growth factor (VEGF), 50ng/ml; bone morphogenic protein (BMP4), 30ng/ml; FGF2, 20ng/ml. Day 3: stem cell factor (SCF), 50ng/ml; VEGF, 50ng/ml; BMP4, lOng/ml; FGF2, 50ng/ml. Day 6: SCF, lOOng/ml; VEGF, 50ng/ml; FGF2, 50ng/ml; interleukin 3 (IL3), lOng/ml; FLT3 ligand (FLT3-L), 10 ng/ml. Day 12: FLT3-L, 10 ng/ml; VEGF, 50ng/ml; FGF2, 20ng/ml; IL7 1 ng/ml; Day 19: FLT3-L, 10 ng/ml; VEGF, 50ng/ml; FGF2, 20ng/ml; IL7 20 ng/ml; IL15 20ng/ml.
[000226] 1.3 Cytokines and small molecules
[000227] Activin A (338-AC, R&D Systems), BMP4 (314-BP, R&D Systems ), FGF2 (100- 18B, Peprotech), FLT3-L (300-19, Peprotech), IL3 (200-03, Peprotech), IL7 (200-07, Peprotech), IL15 (200-15, Peprotech), VEGF (100-20, Peprotech), SCF (synthesized by CSIRO), Y-27632 (72304, Stem Cell Technologies), CHIR99021 (4423, Tocris), PIK-90 (SI 187, Selleckchem), A83-01 (2939, Tocris), LDN-193189 (TB6053, Tocris).
[000228] 1.4 IL7 titration experiment
[000229] On day 6 of differentiation, the endothelial cell culture derived from the RM3.5/'>'G/'G/ /J iPSC line was dissociated (as above) and replated onto 12-well Geltrex™ plates at the ratio of 1:3. Media for day 6-12 was as the above. From day 12, IL7 concentration was titrated based on the Day 12 and Day 19 (as above) factors without IL15: FLT3-L, 10 ng/ml; VEGF, 50ng/ml; FGF2, 20ng/ml; IL7, 0 /0.1 /I /20 ng/ml. Media was refreshed every two days from day 12-23, and flow cytometry analysis for NK and T cell associated markers was performed at day 23.
[000230] 7.5 Generation ofCD56+ cells from RAG1 + cells
[000231] 7 G7. GFP+ hematopoietic cells were sorted using an Influx FACS sorter (BD) on day 19 of PSC differentiation. 5000 sorted RAG1 + cells were plated per well of 96-well round bottom plates, which enabled cells to be positioned in the centre of wells to visualise RAG! :GFP fluorescence. Cells were cultured with IL7 20ng/ml, IL15 20ng/ml, or IL7 and IL15 both 20ng/ml. Medium was carefully changed 2 days after replating (as day 2) and flow cytometry characterization was performed on day 4 after replating. Fluorescence images were taken using a Zeiss Observer Z1 fluorescent microscope and processed using Fiji for Mac OS X.
[000232] 1.6 Flow cytometry and cell sorting
[000233] Anti-human conjugated bodies used for flow cytometry are as the follows. CD4-PE (BioLegend, 300508, RPA-T4, 1:30), CD7-APC (BD Pharmingen, 561604, MT-701, 1:50), CD8a-PE-Cy7 (BioLegend, 344712, SKI), CD13-PE-Cy7 (BioLegend, 301712, WM15, 1:100), CD34-BV421 (BioLegend, 343610, 581, 1:50), CD34-PE-Cy7 (BioLegend, 343516, 581, 1:100), CD43-PE (BioLegend, 343204, 10G7, 1:50), CD45-BV421 (BioLegend, 304032, HI30; 1:30), CD56-PE (BD Pharmingen, 555516, B 159, 1:50), CD127/IL7R-PE (BioLegend, 351304, A019D5, 1:50), CD161-PE-Viol770 (MACS, 130-113-594, 191B8, 1:50), CDH5/V-Cadherin- FFTC (BD Pharmingen, 560411, 55-7H1, 1:20), CXCR4-PE (BioLegend, 306506, 12G5, 1:50), DLL4-APC (BioLegend, 346508, MHD4-46, 1:40), JAG1-APC (BioLegend, 399105, W16199B, 1:30), KDR-AF647 (BioLegend, 359910, 7D4-6, 1:50).
[000234] Conjugated antibodies were diluted in FACS wash buffer (PBS supplemented with 5% fetal bovine serum) and incubated with cells for 20 minutes on ice. The cell suspension was washed twice with FACS wash solution to remove unbound antibodies and resuspended in FACS wash solution containing 1 pg/ml propidium iodide. Cell surface staining was examined by Becton Dickenson (BD) LSRFortessa Cell Analyzer. Flow cytometry data was analyzed using the FlowLogic program (7.2.1, DataNova). Alternatively, cell purification was performed using a BD FACSaria FUSION or Infux cell sorter based on cell surface staining or the expression of a fluorescent reporter. Cells were collected using a 5ml FACS tube containing 0.5ml cold fetal calf serum. [000235] 1.7 Immunofluorescence and antibodies
[000236] Differentiated cells were fixed with 4% paraformaldehyde solution for 5 minutes at room temperature. Cells were blocked in blocking buffer (PBS + 10% FCS + 0.1% Triton-X) for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. Cells were washed three times with PBS, then stained with secondary antibodies diluted in blocking buffer for 1 hour at room temperature. Stained cells were imaged using a Zeiss confocal LSM 780 inverted microscope. Image analysis was performed using ImageJ software.
Table 1.
Figure imgf000087_0001
[000237] 1.8 51 Chromium release assay
[000238] K562 target cells were labelled with 100 pCi Chromium-51 (51Cr, PerkinElmer) for one hour at 37°C and subsequently co-cultured with PSC-derived NK cells or NK cells freshly isolated from healthy donors’ peripheral blood mononuclear cells (PBMC) by NK Cell Isolation Kit (Miltenyi Biotec). NK cells were added in triplicate wells at effector: target ratios from 4:1 to 1:1. Wells with target cells alone (spontaneous release) and target cells with 10% Triton X 100 (maximum release) were included as controls. After 4-hour or 16-hour co-culture, cells were spun down and supernatants were collected. The amount of 5 ICr released in the supernatants was detected using a gamma counter (Wallac Wizard 1470). The %specific lysis was calculated by [(experimental release - spontaneous release)/(maximum release - spontaneous release)] * 100. [000239] 1.8.1 K562 killing assay using flow cytometry.
[000240] K562 cells were maintained in RPMI +10% FCS +1% Pen Strep (medium changed weekly). For killing experiments, cells were passaged the day before and then given fresh medium on the morning of the experiment. Approximately 1 million K562 target cells suspended in 1 ml of phosphate buffered saline (PBS) were labelled with 1 pM carboxyfluorescein succinimidyl ester (CFSE) for 10 minutes at 37°, in the dark. Cells were pelleted and resuspended in 1 ml of PBS + 10% Fetal calf serum - and incubated for a further 30 mins at 37°. Cells were then concentrated by centrifugation, washed twice with PBS 4°C degrees and then resuspended in day 19 medium (including growth factors) at 1 - 3 million cells per ml. Flow cytometry K562 killing assays were performed essentially .Labelled K562 cells distributed into each well of a round bottomed 96 well tray at between 25,000 - 150,000 cells per well - with each well in any given experiment receiving the same number of K562 cells. Non-adherent (NK-effector) cells from day 28-32 cultures that had been grown in medium supplemented with IL 15 from day 15 were added to each well to give effector: target (K562) cell ratios as indicated. Following 4 hours incubation at 37°C, counting beads were added to each well in medium containing Ipg/ml 4',6-diamidino-2-phenylindole (DAPI). Flow cytometry was used to assess the number of viable (DAPI negative, CSFE+ cells). The flow cytometer was programmed to acquire a set volume uniformly for each separate experimental set up. Counting beads were used as an independent measure of consistency. The number of viable K562 cells per given volume / bead count was used calculate the overall K562 viability for each sample. Percentage viability was calculated as the fraction of viable K562 cells in the test well relative to the number of viable K562 cells present in the corresponding control wells (or the average of control wells) X 100. Each assay for a given effector / target ratio was performed at least in triplicate.
[000241 ] 1.9 Single Cell RNA sequencing and Bioinformatics
[000242] Single cell suspension samples were prepared at 1 ,000,000 cells/ml with at least 90% cell viability. The Victorian Clinical Genetics Service performed the library preparation and sequencing following the lOx Genomics Cell Preparation Guide ( ww, 1 Oxgenomics .com) . Sequencing was performed with the Illumina Novaseq-6000 system for a target of 300 million reads per sample with 6000 cells and 50,000 read depth. [000243] Fastq files generated from the Illumina sequencing were mapped against the human reference genome GRCh38- 1.2.0 with CellRanger software using the count function [2], An additional CellRanger aggregation with the aggr function was applied to group all samples into one output ‘filtered_gene-bc_matrices’ folder. The R platform along with its suite of single cell bioinformatic packages with R version 4.2.1 was used to for single cell data analysis (www.R- project.org). Seurat (v4.1.1) was used for single cell data pre-processing and subsequent downstream analysis and visualizations. Cells that were not within the quality control boundaries (see in GitHub) were excluded. The standard Seurat pipeline including log normalization at a scale factor of 10000, scale data to centre gene expression values and principal component analysis (PCA) to reduce dimensions. The FindCluster function was used to identify clusters within each sample.
[000244] PSC derived cells were integrated with fetal human embryonic AGM and fetal liver data at developmental stages week 4.5 to 15 (see, Calvanese, V. et al. Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature 604, 534-540 (2022).). The integration was performed with Seurat’s FindlntegrationAnchors and IntegrateData function based on a list of genes identified by the SelectlntegrationFeatures function. The human embryonic data, as a reference along with canonical markers was used for the identification of cell identities of each cluster. The FindAllMarkers function was used to produce a list of genes that were specific to each cluster which were then used in visualization plots such as heatmaps, violin plots, dot plots and feature plots. Differential gene per cluster was performed with the FindMarkers function.
[000245] Downstream analysis including machine learning-based analysis and neural networks to predict PSC cluster identities was completed with ACTINN version 2 (see, Ma, F. & Pellegrini, M. ACTINN: Automated identification of cell types in single cell RNA sequencing. Bioinformatics 36, 533-538 (2020).) (see GitHub to access the test and reference datasets used). Gene ontology was completed with Metascape (see, Zhou, Y. et al. Metascape provides a biologist-oriented resource for the analysis of systems -level datasets. Nat Commun 10, 1523 (2019).) and inputting the gene list identified by the FindAllMarkers function. CellChat version 1.4.0 (see, Jin, S. et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun 12, 1088 (2021).) was used for the ligand and receptor pair analysis following the standard CellChat vignette. Pseudotime analysis to track the hematopoiesis and lymphopoiesis within the cultures utilized the SeuratWrappers package version 0.3.0 as well as following the Monocle3 vignette (Cao, J. et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 566, 496-502 (2019).) to select the root starting cells as the “HE (hemogenic endothelial cell)” cluster.
[000246] Analysis of endothelial to hematopoietic transition in Figure 2E utilize PSC derived cells from day 12 and 15 based on expression of CD31 or CD34 and RUNX1 or CDH5. The analysis of blood cells from HSPC to lymphoid cells utilize a subset of PSC derived cells based on the “HSPC”, “ILC” and “RAG+Lymph” clusters identified in Figure 2A. To investigate the heterogeneity and different ILC clusters in Figure 3E, cells from “ILC” and “ILC_cyc” clusters were pooled from the Figure 3A. Additionally cell cycling genes were regressed out to avoid influence by ILC cells’ cycling state. Genes displayed on Figure 8 heatmap ware the specific genes of each cluster based on reclustered and pooled cells identified as the “HSPC”, Lymph_prol, pro2 and pro3 clusters in Figure 3A. Cells in Figure 8D were based on pooling of RAG1+ cells from day 19 and 25 samples. The AverageExpression function was used to calculate the mean of RAG1 average expression across all clusters. Cells that were above the mean average expression of 2.259 (4sf) were labelled as “RAG-high” and those lower “RAG- low”. All subsets described above were re-clustered with the standard Seurat pipeline.
[000247] 1.10 Data Availability
[000248] RNA sequencing raw data is available in the public GEO data repository under the Geo accession number: GSE217705.
[000249] 1.11 Statistics
[000250] Statistical analysis was performed in Prism (Graphpad, Version 8.0.2) by t-test, oneway ANOVA or two-way ANOVA tests as indicated in figure legends.
[000251] Example 2 - Efficient generation of arterial endothelial cells and haematopoiesis in vitro
[000252] Through detailed studies the inventors have developed a novel method that robustly generated DLL4-expressing arterial endothelial cells (AECs) (Figure 1A). Patterning differentiating PSCs over the first two days resulted in the rapid transit of cells through stages indicative of MIXL1+ primitive streak (dl) and CD13+ early mesoderm (d2). Treatment of this mesoderm population with BMP4 whilst simultaneously inhibiting Activin signalling (A83O1) resulted in the generation of cells highly enriched for expression of the mesodermal-endothelial markers CD13 and KDR(d3) (Figures 5A to 5C). On day 6, the CD34+ population (80 ± 2 %, mean ± SEM, n=31 experiments) in the differentiation cultures uniformly co-expressed the AEC markers VE-cadherin, CXCR4 and DLL4 (Figures IB to ID). Importantly, this method for generating arterial endothelial cells was reproducible across 11 different human iPSC lines or ESC lines (Figures ID and 5D), with one PSC giving rise to approximately 6 CD34+DLL4+ cells (Figure 5E). On day 6, the confluent endothelial cell monolayer was passaged to enable further expansion or in preparation for cryopreservation, the latter option providing a significant logistical advantage of this method (Figure 5F). Between day 6 and day 12, cells proliferated further to re-establish a confluent monolayer and began to produce haematopoietic cells.
[000253] By day 12, CD34+CD43+ haematopoietic progenitor cells represented a substantial fraction of the culture, as assessed by flow cytometry (Figure IE). Over the ensuing 3 days, CD45+ blood cells begin to upregulate expression of the lymphoid lineage marker CD7, in response to the introduction of IL7 into the culture medium (Figure IE). On day 15, using RAG1 :GFP reporter lines, a small fraction of RAGI+ cells were detected within the CD34+CD7+ haematopoietic cell population (Figures IE and IF). The frequency this RAG1+ population increased over the next four days, an increase that was accompanied by downregulation of CD34 (Figures IE and IF). Fluorescence images also showed an accumulation of RAG1 :GFP+ cells from day 15 to day 19, suggestive of ongoing lymphoid differentiation (Figure 1G). At day 23, the majority of CD7+ cells were negative for RAG1 but positive for CD 161 (KLRB1), a cell surface marker frequently associated with innate lymphoid cells (Figures 1H and II). Indeed, approximately half of the CD161+ cells also expressed the natural killer cell marker CD56. Additional experiments with two independent iPSC lines, PB0- 011 and PB0-05, showed each input iPSC yielded 35 +1-3.1, and 59 +/- 17 CD161+CD7+ cells for cultures in which IL15 was added at day 15, and 7.3 +/- 2.3 and 12 +/-1.8 CD161+CD7+ cells for cultures when IL15 was added at day 19 (average +/- S.E.M for 3 technical replicates). Collectively, these results show that the DLL4-epxressing arterial-haematopoietic culture, designated AHC, efficiently generates lymphoid cells with characteristics of the ILC lineage. This process occurs without the requirement for exogenously added NOTCH ligands, which is a mandatory characteristic of previously described lymphoid differentiation methods.
[000254] Example 3 - PSC-derived arterial haematopoietic culture (AHC) models human embryonic haematopoiesis.
[000255] To explore the differentiation trajectories of cells subjected to this protocol, the inventors performed time-series single cell RNA-sequencing (scRNA-seq) analysis of AHCs at day 6, 12, 15, 19, and 25 (haematopoietic cells only on day 25), encompassing the period from the generation of haemogenic endothelium to lymphoid commitment (Figure 6A). The results from this experiment were cross-mapped to a recently reported dataset derived from early human embryonic haematopoietic tissues, including the AGM and the fetal liver (Figures 2A and 6A).
[000256] This comparative analysis indicated that the in vitro PSC differentiation method developed herein generated a diverse spectrum of cell lineages that mirror those arising during early human intraembryonic haematopoiesis in vivo (Figures 2A and 2B). This spectrum includes fibroblast-like stromal cells (COL3A1, COL1 Al), AECs (CDH5, and PECAM1), haematopoietic stem/progenitor cell-like cells/HSPCs (CD34. SPINK2 and MLLT3), erythroid cells (HBZ, HBA1, and HBA2), a variety of myeloid haematopoietic lineages, and lymphoid lineages marked by RAG genes or ILC-associated genes (Figures 2A, 2B, and 6B).
[000257] To examine tube forming ability of cells generated during differentiation, cryopreserved differentiation day 6 RM-tTom endothelial cells were thawed into T cell medium (supplemented with day 6 growth factors, as described in 1.2 above) and seeded onto adherent tissue culture plates to enable recovery. The following day, cells were detached from the plates using TrypLE™ Select (ThermoFisher), counted, and then 150,000 cells were resuspended in T cell media containing VEGF 50ng/ml, EGF lOng/ml, FGF2 lOng/ml, hydrocortisone at 10 ng/ml. Resuspended cells were then gently layered over a film of Matrigel (Merk) (300 pl/each well of a 24 well plate) and incubated for 24 hours. Cultures were imaged using a LSM900 confocal microscope and analysed using ImageJ.
[000258] Disaggregation of day 6 cultures and re-seeding of cells in Matrigel revealed the capacity of cells to form tubes, suggestive of a bone fide endothelial identity (Figure 6J).
[000259] On day 25, cultures contained predominantly lymphoid cells (Figure 6A), an outcome most likely due to the provision of IL7 and IL 15 as the key haematopoietic cytokines over the prior 6 days. Importantly, the analysis showed that PSC-derived endothelial cells expressed a cohort of AEC associated genes, including SOX17, GJA4, CXCR4 and GJA5 (Figure 6C). Based on transcriptomic features, ACTINN, an artificial intelligence-based computation program, predicated that these endothelial cells resemble the AECs found in the AGM of Carnegie stage (CS) 14/15 human embryos (Figure 2D). Consistently, the inventors detected activation of specific arterial-haemogenic genes, such as MECOM (EVI1 ), KCNK17, and SPINK2 (Figure 2E). Additionally, these AECs expressed a group of NOTCH-ligand genes, including DLL4, DLK1, JAG1 and JAG2 (Figure 2C). Flow cytometry and immunofluorescence validated protein expression of these genes (Figures 2F, 6C, and 6D). The inventors found CD34+ cells persistently expressed DLL4 from day 6 to day 19, creating conditions favourable for lymphopoiesis (Figure 2F). Interestingly, although JAG1 was expressed by both endothelial cells and stromal cells, its endothelial expression gradually diminished from day 6 to day 19 (Figure 6D). In the human embryo dataset, JAG1+ cells were more prevalent in the AGM-AEC population of early embryos, while fewer JAG1+ AECs were found in the fetal liver (Figures 6E and 6F), suggesting that JAG1 expression, instead of DLL4, may be an important indicator of arterial specification in the AGM. Ligand-receptor pair analysis, using CellChat, suggested endothelial cells within these cultures may represent a major source of NOTCH ligands, signalling to NOTCH 1+ haematopoietic progenitors and lymphoid cells (Figure 6G). Collectively, these results suggest the PSC-derived endothelial cells disclosed herein acquire a cellular identity that closely resembles arterial endothelial cells present at a stage of human embryogenesis during which intra embryonic haemopoiesis is initiated.
[000260] Example 4 - Lymphoid commitment and lineage specification
[000261] The endogenous expression of NOTCH-ligands by the AHC disclosed herein provides an environment conducive for lymphoid commitment. As such, the analysis was focused on the emergence of lymphoid cell populations within the haematopoietic cell pool (Figures 3A and 7A). UMAP clustering of scRNA-seq data showed that although this pool included a small fraction of haematopoietic progenitor cells and myeloid cells, the vast majority of cells belonged to the lymphoid lineages (Figures 3A, 3B and 3C). Time series analysis showed that myeloid compartments emerged on day 12, earlier than the wave of lymphoid commitment on day 15 (Figure 3B). This analysis also captured three transient lymphoid progenitor populations; lymph_prol on day 15, and lymph_pro2 and lymph_pro3 on day 19 (Figure 3B). By day 25, ILC-like cells KLRB1, NKG7 and GNLY) and T cell progenitors (RAG/, CD5 and TCF7) were the predominant cell types; both populations included a sub-faction enriched with cell-cycle related genes (CDK! . MKI67, and CENPF) (Figures 3B and 3C). However, cells representing the B cell lineage were not detected (Figure 7B), reminiscent of observations of human foetal liver haematopoiesis, where the NK/ILC and T cell lineages emerge together as a separate branch of lymphopoiesis from that giving rise to B cells. Pseudo-time analysis using Monocle is consistent with the real-time analysis disclosed herein (Figure 3B), suggesting that the dataset disclosed herein can be used to address other questions relating the dynamics and kinetics of the lymphoid differentiation process. [000262] Cluster-specific gene expression distinguished the three lymphoid progenitor populations (Figure 3C, 7C, and 7D). Lymph_prol upregulated NOTCH 1 and CD7 but downregulated CD34, indicative of lymphoid commitment. Retained expression of the stem cell gene, SPINK2, on lymph_prol also distinguished this population from lymph_pro2 and lymph_pro3. Lymph_pro2 showed a high-level expression of IL7 receptor gene (IL7R) and an upregulation of the NOTCH-pathway genes, NOTCH1 and HES4. However, lympho_pro3 showed a downregulation of IL7R but increased expression of KLRB1, which may suggest a cell fate potentially directed towards an ILC-like phenotype. Furthermore, analysis of a cohort of T cell differentiation genes indicated the lymph_pro2 and lympho_pro3 clusters showed characteristics consistent with differentiation trajectories towards T cell lineage and the ILC lineage, respectively (Figure 3D). The transcription factors BCL11B and TCF7, key drivers of T cell commitment, were enriched in lymph_pro2 and T cell progenitors, consistent with the expression patterns of RAG1, RAG2, and PTCRA. Additionally, the elevated expression of IL7R in lymph_pro2 and T progenitor clusters, but not in lymph_pro3 and ILC populations, suggests IL7 signalling might be important for ILC versus T cell lineage specification.
[000263] To further characterise the ILC-like populations, cell-cycle related genes were regressed and clustered for cell type annotation (Figure 3E). This analysis showed the ILC-like cells could be subdivided into three clusters: NK/ILCla, NK/ILClb, ILC2-like cells. All ILC- like cells expressed NKG7 and KLRB1, while the NK/ILC1 groups expressed typical genes encoding cytotoxic proteins, including perforin PKEI). granzyme B (GZMB). granlysin (GNLY), as well as the NK cell surface marker gene NCAM1/CD56 (Figure 7E). However, the ILC2-like cells expressed PTGDR2 and 1L7R, suggesting that the IL7 signalling might be also important for further sub-lineage specification within ILC compartment.
[000264] Collectively, these results suggest that the AHC system expressing intrinsic NOTCH- ligands disclosed herein efficiently patterned lymphoid lineages, in which 1L7R was shown to be a differential marker for different lymphoid progenitors and their committed progeny.
[000265] Example 5 - IL7 regulates T versus NK cell lineage commitment
[000266] Consistent with results summarised in the Dot and Violin plots in Figures 3C and 3D, UMAP projections showed that the pattern of IL7R expression contrasted with that of KLRB1 (CD161), and that clusters enriched with RAG1 and CD4 also expressed a higher level of IL7R expression (Figure 8A). Analysis of the entire AHC did not detect IL7 transcripts (Figure 6B), facilitating dissection of the consequences of IL7 supplementation on differentiation outcomes. Therefore, the inventors titrated the concentration of IL7 and examined how this influenced the appearance of different lymphoid populations that expressed the IL7 receptor. For these experiments, IL15 was also removed from the AHC because of its defined function in supporting human NK cell differentiation.
[000267] Using the RAG1 :GFP PSC reporter line, four concentrations of IL7 (0, 0.1, 1, and 20 ng/ml) were examined from day 12 to day 23, spanning a period between the emergence of haematopoietic progenitors and lymphoid lineage commitment. Flow cytometry analysis showed that IL7 was not necessary for the generation of CD7+ cells or RAG1 :GFP+ cells, suggesting this cytokine is dispensable for haematopoietic progenitors to commit to the lymphoid lineage (Figure 4C). Nevertheless, addition of even low concentrations of IL7 (0.1 ng/ml) significantly increased the frequency of CD7+ cells. Conversely, increased IL7 concentrations reduced the frequency of IL7R+ cells; the highest IL7 concentration (IL7-hi, 20 ng/ml) completed blocked the generation of CD7+ lymphoid cells with surface expression of IL7R whilst the lowest IL7 concentration (IL7-lo, 0.1 ng/ml) produced cultures with the highest frequency of IL7R+ lymphoid cells. These latter results are consistent with previous findings showing IL7 downregulates the surface expression of its receptor.
[000268] Addition of IL7 increased the frequency of RAG1+CD161- cells and the RAG1- CD161+ population, representing the T and the innate lymphoid lineages, respectively (Figure 4B). Importantly, IL7 was required to upregulate /?.4G/:GFP expression, which reflects RAG1 mRNA levels, confirming IL7 as an important driver lymphoid differentiation (Figures 4B and 8C). CD4 and CD8a expression as markers of further commitment to the T cell lineage was also examined. The inventors found the IL7-lo conditions promoted the generation of CD4+CD8a+ double positive cells that emerged from the immature CD4+ single positive population (Figure 4C). Moreover, these conditions did not generate an atypical immature CD8a+ single positive population which is often observed in in vitro T cell differentiation systems, but rarely seen in thymopoiesis in vivo. In this regard, IL7-lo conditions generate a cell profile that more closely resembles early T cell differentiation in the human thymus. Indeed, like in vivo thymopoiesis, CD4+CD8a+ cells were found to show the highest level of RAG1 expression. Of the remaining fractions, CD4+CD8a- cells showed the next highest level of RAG1 expression, while the CD4- CD8a+ and CD4-CD8a- populations showed minimal levels of RAG1. Interestingly, analysis of the scRNA-seq data disclosed herein suggested that RAGl- i cells were enriched with T cell associated genes, while /MG /-Io cells showed expression of NK cell related genes (Figure 8D). Collectively, these results support the conclusion that lymphoid differentiation within the AHC system disclosed herein is highly sensitive to IL7 concentrations and reaffirms that high levels of IL7 favours the development of innate lymphoid lineages. Importantly, the highly defined nature the AHC system provides myriad opportunities to dissect key events in lymphoid lineage commitment and differentiation.
[000269] In the above experiments, CDI 61+/ G/+ cells in the IL7-hi condition were observed, prompting the inventors to investigate whether human RAG1+ cells could give rise to cells of the NK ILC lineage. On day 19 when AHCs were enriched with /MG/ -low cells (Figure 8E), haematopoietic cells were separated from the AHC and purified GFP+ cells using FACS. The sorted GFP+ cells were cultured in medium supplemented with IL7, IL15, or IL7+IL15, at 20ng/ml. Examining these cultures after four days revealed that IL7 alone could not support continued cell growth, while IL 15 and IL7+IL15 promoted an increase in cell numbers (Figures 4D and 8F). Flow cytometry analysis showed that these latter two conditions yielded a high frequency of CD161+CD56+RAG1- NK-like cells, with each GFP+ input cell giving rise to 1.2 ± 0.2 (IL15) and 2.4 ± 0.2 (IL7+IL15) CD161+CD56+ NK-like cells after 4 days (Figure 4E). These results indicate that human RAG1+ lymphoid progenitors can efficiently generate NK-like cells.
[000270] Given the expansion of lymphoid derived NK cells in response to IL7 and IL 15, the inventors next examined if these same conditions were able generate NK cells from haematopoietic progenitors that arise at earlier stages of the AHC system disclosed herein, without a requirement for prior enrichment of progenitors using FACS. Capitalising on the high frequency of CD7+CD34+ lymphoid progenitors at day 15, suspension cells were separated from the AHC on this day and these cells were cultured in media supplemented with only IL7 and IL15. On day 20, flow cytometry analysis showed the efficient generation of CD161+CD56+ cells (Figure 8G). This was accompanied by a reduced number of RAG1+ cells.
[000271] Notably, after only 5 days of IL15 treatment these cells possessed a rudimentary cytotoxic function (Figure 8H) that was comparable to cells isolated from peripheral blood (PBMCs). Extending the time in which cells were exposed to IL15 for a further 15-17 days (from day 15) enabled further maturation of this population, evidenced by the up-regulation of CD16 on CD161+ cells, a phenotype conventionally associated with enhanced cytotoxity (Figure 81). Consistent with this, cells from cultures subjected to 15 or more days of IL 15 treatment exhibited an ability to kill K562 erythroleukemia cells (-50% after 4 hours at an Effector: Target ratio of 8: 1 or -40% for ratios of 4: 1) that was comparable with that reported for both donor-derived or iPSC derived NK populations reported in the literature (Figure 8 J) . Taken together, these data strongly support the proposition that CD161+CD56+ cells generated using the AHC culture system described herein represent early cells of the NK lineage.
[000272] Example 6 - Erythroid, Myeloid, T and B lymphoid lineage specification
[000273] In this example, PSCs were cultured according to the methods described in Examples 1 and 2 so as to give rise to a population of CD34+CD45+ haematopoietic progenitor cells by day 12 of culturing. The generation of a population of cells enriched in erythroid, myeloid, T and B lymphoid lineage cells, was undertaken by supplementing day 12 cultures with growth factors capable of erythroid or myeloid linage, or by transferring day 12 or day 15 cells into conditions enabling T and B lymphoid development.
[000274] For the generation of erythroid lineage cells, cultures were grown in a medium comprising EPO (2 units/ml). For the generation of myeloid lineage cells, cultures were grown in a medium comprising MCSF (50 ng/ml). For the generation of T lineage cells, day 12 haematopoietic cells were transferred to monolayers of MS 5 cells expressing high levels of human DLL4 (MS5-hDLL4) or OP9-DLL4 cells; or day 15 CD34+CD45+ haematopoietic progenitor cells were co-cultured with MS5-hDLL4 cells using an air-liquid interface culture to generate artificial thymic organoids.
[000275] For OP9 DLL4 Monolayer differentiations, one day prior to initiation of T-cell cultures, a tissue-culture treated 12-well plate was plated with 3 - 5 x 104 OP9 cells expressing high levels of human DLL4 (henceforth DLL4hi) per well in a-MEM with 10% FCS, lx GlutaMAX and lx Penicillin/Streptomycin. Half an hour prior to the start of culture, wells were washed with PBS and media changed to RPMI 1640 with 4% B27, 30 pM L-ascorbic acid 2- phosphate sesquimagnesium salt hydrate, lx GlutaMAX and lx Penicillin/Streptomycin with added cytokines (henceforth RB27) as detailed in Table 2 below. Initiation of culture was performed by adding 105 day 12 CD45+CD34+ blood progenitor cells per well to give a total volume of 1 mL of RB27 per well. Media top up was performed after 3 - 4 days by adding another 1 mL of RB27. After one week, cells were passaged onto a fresh layer of OP9 cells as described before. Cell passage was performed by harvesting and mechanically-dissociating the whole cells within each well and passing the cell mixture through a 40 pm membrane to exclude cell clumps. The flowthrough fraction was collected by centrifugation and resuspended in 1 mL of fresh RB27. On weeks 1 and 2, DLL4hi cells were used, and for subsequent weeks, OP9 cells with a lower level of DLL4 were used (DLL41o). One well was harvested weekly for flow cytometry analysis.
[000276] For MS5 DLL4 Monolayer differentiations, one day prior to initiation of T-cell culture, a tissue-culture treated 12-well plate was seeded with 5 x 104 MS5-hDLL4 cells per well in a-MEM with 10% FCS, lx GlutaMAX and lx Penicillin/Streptomycin. Half an hour prior to the start of culture, wells were gently washed with PBS and media changed to RPMI 1640 with 4% B27, 30 pM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, lx GlutaMAX and lx Penicillin/Streptomycin with added cytokines (henceforth RB27). Initiation of culture was performed by adding 105 day 12 CD45+ CD34+ blood progenitor cells per well to produce a total volume of 1 mL of RB27 per well. Media top up was performed in 3 - 4 days by adding another 1 mL of RB27. After a week, an 80% media change was performed by aspirating and replacing 80% of the media from each well with fresh RB27. One well was harvested weekly for flow cytometry analysis.
[000277] The same MS5 cells expressing DLL4 described above were used to generate artificial thymic organoids. Half an hour prior to the start of the experiment, a 40 pm Millicell 6-well transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) was placed on top of 1 mL of T-cell media with cytokines to create an air-liquid interface (cytokines summarised in Table 2 below). Each ATO was formed by mixing 1.5 x 105 MS5-hDLL4 cells and 104 day 15 CD45+ CD34+ blood progenitor cells. The cell mixture was concentrated by centrifugation and the cells resuspended in 5 pl medium to create a slurry that was then plated at the air-liquid interface. Each transwell insert was plated with a maximum of 5 ATOs. Media change was performed every two days with fresh T-cell media for the first two weeks, followed by RB27 for the remainder of the differentiation. ATOs were harvested every week for FACS analysis.
[000278] RM-RAGLGFP iPSCs were used to assess the B lineage differentiation potential of blood cell progenitors generated at differentiation day 12. Briefly, B cell differentiations using the non-adherent cell fraction from day 12 cultures were seeded onto a monolayer of MS 5 stromal cells in RB27 medium (as detailed above) supplemented with IL7 (1 ng/ml), SCF (5ng/ml), FGF2 (5 ng/ml), and IL3 (5 ng/ml), with medium changed every 3-4 days. Cultures were examined for the generation of B cell progenitors using flow cytometry analysis for expression of CD 19 in conjunction with RAG1 (GFP). Table 2.
Figure imgf000099_0001
[000279] Results
[000280] These experiments showed that supplementing cultures with EPO or MCSF led to the generation of CD235a+ erythroid and CD 14+ myeloid cells respectively (Figure 6H).
[000281] Assessment of cells directed to T and B cell differentiation showed development of CD3+TCR+ T cells (TCR+, 33+/-9.8%, 4 experiments), and RAG1+ CD19+ CD10+ B lymphoid lineage cells (Figure 61). [000282] Collectively, these experiments indicate the haematopoietic progenitors generated using the methods described herein can generate a broad spectrum of lineages, including some of those identified by RNAseq analysis.
[000283] Discussion
[000284] A human arterial-haematopoietic culture (AHC) system, representing a simple and efficient method to study and model human embryonic haematopoiesis/lymphopoiesis in vitro, is described. Unique to this system is the generation of a lawn of NOTCH-ligand expressing arterial endothelial cells effectively direct lymphoid commitment from emerging haematopoietic progenitors. This AHC system enabled the dissection of haematopoietic cell fate determination and the identification of optimal conditions for producing human lymphoid progenitors, providing new opportunities for experimental research and medical applications.
[000285] This disclosure provides the first time-series single-cell map of in vitro human haematopoiesis, representing a key reference for studying blood cell development. There are existing scRNA-seq datasets describing in vitro PSC-based embryonic models for the development of many organ systems, including the heart, pancreases, and brain. The development of the highly reproducible AHC system disclosed herein (Figures 1C and ID) has enabled the generation of a dataset that spans key stages of haematopoietic ontogeny in vitro, providing an important reference for the development of methods for blood cell production.
[000286] Detailed comparison with human embryonic and foetal tissues indicated that the endothelial cells generated according to the methods disclosed herein acquired an identity that has similarities to haemogenic arterial endothelial cells found in the AGM region (Figures 2A and 2B). In addition to classical AEC markers, previous studies show that using exogenous NOTCH ligands could specify definitive hemogenic endothelial cells during early PSC differentiation in vitro. As such, the expression of different NOTCH ligands on the endothelial cells generated according to the methods disclosed herein is also indicative of definitive hemogenic endothelial cells (Figures 2C, 2F, 6C, and 2D). Thus, the PSC-derived haematopoietic differentiation platform disclosed herein generated a spectrum of blood cell types that have counterparts in the AGM and foetal liver, including those belonging to the erythroid, myeloid, and lymphoid lineages (Figures 2 and 3). Although RNAseq data sets that examine specific stages of haematopoietic differentiation have been described previously, the data of the present disclosure is the first to capture a substantial temporal window spanning ontogeny stages from endothelium to lymphoid commitment. Interestingly, a rare population of cells showing the progressive expression of genes that span stages corresponding to the endothelial-to-haematopoietic transition and the formation of pre-haematopoietic stem cells was observed, including SPINK2, RUNX1, MLLT3, HLF and MECOM(EVH) (Figure 2E). These characteristics indicate that the AHC system disclosed herein is a tractable and relevant method for studying human embryonic haematopoiesis in vitro and for helping define conditions that might give rise to haematopoietic stem cells, a holy grail in the hematology and regenerative medicine fields.
[000287] Using the AHC system, lymphoid commitment and cell-fate specification between the T and the NK-ILC lineages were explored. Without using exogenous NOTCH ligands or animal cells, mandatory components for previous lymphoid differentiation systems, the cultures generated by the methods of the present disclosure gave rise to a primitive lymphoid progenitor marked by the expression of the stem cell gene SPINK2 (Figures 3C and 3D). The de novo appearance of this progenitor is consistent with observations from animal studies that point to the possibility of a non-HSC derived lymphoid competent precursors that contribute to early lymphopoiesis and the formation of primary lymphoid organs during embryogenesis. Furthermore, the inventors found that IL7R expression levels foreshadowed the differing potential of progenitors to form T cell lineages (IL7R high) or ILC lineages (IL7R low), and that levels of IL7R signalling could be manipulated to effect cell fate choices (Figures 4A, 4B, and 4C). Although IL7 is dispensable for early lymphoid commitment, including RAG1 activation and CD7 upregulation, this cytokine was indispensable for RAG1+ upregulation that further drives progenitors towards T cell development (Figures 4C, 8C, and 8D). The results disclosed herein show that the IL7R signalling network is regulated in a dose-dependent manner, with minimal levels of IL7 maintaining a level of IL7R expression required for ongoing T cell differentiation. These results provide an example of how this AHC system can be used to dissect early lymphopoietic events and to identify optimal conditions for the efficient generation of innate and adaptive lymphoid immune cells.
[000288] An unconventional developmental pathway in which RAG1+ lymphoid cells give rise to NK cells was also documented. The RAG genes are responsible for gene rearrangement of antigen- specific receptors during T and B cell differentiation but are not required for the genesis of ILC lineages. However, experiments in mice identified an NK cell population that transiently expressed Ragl during development. Moreover, mice lacking RAG recombinase generate lymphoid NK cells with compromised fitness, alluding to a yet undiscovered role for RAG1 in the genesis of the innate immune cell repertoire. Importantly, clinical reports indicate that patients with severe combined immunodeficiency caused by RAG1 deficiency show an abnormal distribution of NK cells. Thus, despite caveats attached to making comparisons between the development of mouse and human immune system, these observations, coupled with the findings disclosed herein suggest grounds for further examination of the role of RAG1 in NK cell development. In the present work, experiments using a RAG1 :GFP PSC reporter line confirmed that, under defined conditions, RAG1+ lymphoid progenitors effectively gave rise to CD161+CD56+ cells representing the NK cell lineage (Figures 4D and 4E). Furthermore, it was found that RAG1 expression levels presaged gene expression profiles indicative of further commitment to the adaptive lymphoid lineage (RA G1 -high) or the innate lymphoid lineage (TMGT-low) (Figure 8D). The disclosures herein suggest that RAG1 expression is indicative of fate decisions between different branches of the ILC lineages. Overall, the methodology disclosed herein provides an insight into a key juncture in the genesis of innate and adaptive immunity.

Claims

1. A method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising at least one of a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor and a BMP pathway activator, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising at least one of SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells.
2. A method for generating a population of DEE4-expressing arterial endothelial cells (AECs), wherein the DEE4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising a TGF-beta pathway activator, a WNT pathway activator, FGF and a PI3 kinase inhibitor, and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising a TGF-beta pathway inhibitor, a Wnt pathway activator, a BMP pathway inhibitor, FGF and which is free or essentially free of BMP pathway activators, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising a TGF-beta pathway inhibitor, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating a population of CD 13+ and KDR+ mesodermal- endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, a BMP pathway activator and FGF, for a time sufficient for generating the population of CD34+ cells.
3. A method for generating a population of DLL4-expressing arterial endothelial cells (AECs), wherein the DLL4-expressing AECs are CD34+ cells, comprising the sequential steps of: la. culturing or maintaining a population of substantially undifferentiated pluripotent stem cells in a first defined medium comprising Activin A, CHIR99021, FGF2, and PIK90, and which is free or essentially free of BMP4, for a time sufficient for generating a population of MIXL1+ cells, lb. incubating the population of MIXL1+ cells in a second defined medium comprising A83-O1, CHIR99021, LDN-193189, FGF2, and which is free or essentially free of BMP4, for a time sufficient for generating a population of CD 13+ early mesoderm cells, lc. incubating the population of CD13+ early mesoderm cells in a third defined medium comprising A83-O1, VEGF, BMP4, FGF2, for a time sufficient for generating a population of CD13+ and KDR+ mesodermal-endothelial cells, ld. incubating the population of CD13+ and KDR+ mesodermal-endothelial cells in a fourth defined medium comprising SCF, VEGF, BMP4, FGF2, for a time sufficient for generating the population of CD34+ cells.
4. A method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of:
2a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
2b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising at least one of Flt3L, VEGF, FGF and IL7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising at least one of Flt3L, VEGF, FGF, IL7 and IL 15 for a time sufficient for generating the mixed population of PSC- derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
5. A method for generating a mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, comprising the sequential steps of:
2a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
2b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells, 2c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating a population of CD34+CD7+ lymphoid haematopoietic progenitor cells,
2d. incubating the monolayer and the population of CD34+CD7+ lymphoid haematopoietic progenitor cells in the sixth defined medium, for a time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells, and
2e. incubating the monolayer and the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells in a seventh defined medium comprising Flt3L, VEGF, FGF2, IL7, and IL15 for a time sufficient for generating the mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK-like cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
6. A method for generating a cell population enriched in PSC-derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
3b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3 and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
7. A method for generating a cell population enriched in PS C -derived innate lymphoid cells (ILCs), wherein the PSC-derived ILCs are CD161+RAG1- cells, comprising the sequential steps of:
3a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
3b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
3c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+RAG1- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
8. A method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
4b. incubating the monolayer in a fifth defined medium comprising at least one of SCF, VEGF, FGF, IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising IL7 and at least one of Flt3L, VEGF and FGF, wherein the concentration of IL7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mL, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
9. A method for generating a cell population enriched in PSC-derived T cells, wherein the PSC-derived T cells are CD4+CD8a+ cells, comprising the sequential steps of:
4a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
4b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3E, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
4c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3E, VEGF, FGF2, and IE7, wherein the concentration of IE7 is about 0.01 to about 1 ng/mL, preferably about 0.1 ng/mE, for a time sufficient for generating the cell population enriched in CD4+CD8a+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
10. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
5al. generating a population of cells enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells; and
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IE7 and at least one of Flt3E, VEGF, FGF and IE15, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
11. The method of claim 10, wherein the CD161+/RAG1+ cells are prepared according to the method of any one of claims 1 to 5.
12. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
5a. generating a monolayer of DLE4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3, 5b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
5c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, wherein the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating a cell suspension comprising a population of CD161+RAG1+ cells,
5d. separating the cell suspension from the monolayer and sorting the cell suspension for a cell population enriched in CD161+RAG1+ cells, optionally wherein the CD161+RAG1+ cells are CD161+RAGl-low cells;
5e. incubating the cell population enriched in CD161+RAG1+ cells in a seventh defined medium comprising IL15, and optionally IL7, wherein the concentration of IL15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling.
13. A method for generating a cell population enriched in PSC-derived NK-like cells, wherein the PSC-derived NK-like cells are CD161+CD56+ cells, comprising the sequential steps of:
6a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
6b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
6c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a sixth defined medium comprising Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating a cell suspension comprising a population of CD34+CD7+ lymphoid haematopoietic progenitor cells, 6d. separating the cell suspension from the monolayer, removing the sixth defined medium from the suspension, and adding a seventh defined medium comprising IL15, and optionally IL7, wherein the concentration of IL15 is about 1 to about 100 ng/mL, preferably about 20ng/mL and when IL7 is present the concentration of IL7 is about 1 to about 50 ng/mL, preferably about 20 ng/mL, for a time sufficient for generating the cell population enriched in CD161+CD56+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
14. A method for generating a cell population enriched in PSC-derived erythroid cells, wherein the PSC-derived erythroid cells are CD235a+CD14- cells, comprising the sequential steps of:
7a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3,
7b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
7c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in an eighth defined medium comprising Erythropoietin (EPO), optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a+CD14- cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
15. A method for generating a cell population enriched in PSC-derived myeloid cells, wherein the PSC-derived erythroid cells are CD235a-CD14+ cells, comprising the sequential steps of:
8a. generating a monolayer of DLL4-expressing arterial endothelial cells (AECs) produced according to the method of any one of claims 1 to 3, 8b. incubating the monolayer in a fifth defined medium comprising SCF, VEGF, FGF2, and IL3, and Flt3L, for a time sufficient for generating a population of CD34+CD43+ haematopoietic progenitor cells,
8c. incubating the monolayer and the population of CD34+CD43+ haematopoietic progenitor cells in a ninth defined medium comprising one or more of human Macrophage Colony-Stimulating Factor (MCSF), human Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), and IL34, and optionally further comprising one or more of Flt3L, VEGF, FGF2, and IL7, for a time sufficient for generating the cell population enriched in CD235a- CD14+ cells, wherein the method does not comprise addition of an agent which activates NOTCH signalling and does not comprise cell sorting.
16. The method of any one of claims 1 to 15, wherein the first defined medium further comprises Y-27263, preferably the concentration of Y-27263 is about 1 to 50 pM, preferably about 8 to 12 pM, preferably about 10 pM.
17. The method of any one of claims 1 to 16, wherein the method further comprising cryopreserving the population of DLL4-expressing arterial endothelial cells (AECs) following step Id.
18. The method of any one of claims 1 to 17, wherein the population of substantially undifferentiated pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).
19. The method of claim 18, wherein the iPSCs are selected from the group consisting of iPSC RM3.5 (male)77467 GFP, iPSC PB0-01 (male), iPSC PB0-04 (female), iPSC PB0-05 (female), iPSC PB0-06 (male), iPSC PB0-10 (male), and iPSC CRL2429 (ATCC).
20. The method of claim 18, wherein the ESCs are selected from the group consisting of ESC Hl (male), ESC H9 (female)RAG7 GF7>, ESC W)Soxi7:tdTOMATO;Ruxic:GFP and ESC
HES3(female)M7XL7;GF7>.
21. The method of any one of claims 1 to 20, wherein the concentration of Activin A in the first defined medium is about 10 to about 50 ng/mL, preferably about 30 ng/mL, the Ill concentration of CHIR99021 in the first defined medium is about 1 to about 10 |1M, preferably about 4 pM, the concentration of FGF2 in the first defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL, and the concentration of PIK90 in the first defined medium is about 10 to about 300 nM, preferably about 100 nM.
22. The method of any one of claims 1 to 21, wherein the concentration of A83-01 in the second defined medium is about 0.1 to about 10 pM, preferably about IpM, the concentration of CHIR99021 in the second defined medium is about 1 to about 10 pM, preferably about 3 pM, the concentration of LDN-193189 in the second defined medium is about 50 to about 750 nM, preferably about 250 nM, and the concentration of FGF2 in the second defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL.
23. The method of any one of claims 1 to 22, wherein the concentration of A83-O1 in the third defined medium is about 0.1 pM to about 10 pM, preferably about IpM, the concentration of VEGF in the third defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL, the concentration of BMP4 in the third defined medium is about 10 to about 100 ng/mL, preferably about 30 ng/mL, and the concentration of FGF2 in the third defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL.
24. The method of any one of claims 1 to 23, wherein the concentration of SCF in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL, the concentration of VEGF in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL, the concentration of BMP4 in the fourth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL, and the concentration of FGF2 in the fourth defined medium is about 10 to about 100 ng/mL, preferably about 50 ng/mL.
25. The method of any one of claims 1 to 24, wherein the time sufficient for generating the population of MIXL1+ cells at the end of step la is about 2 to about 72 hours, preferably about 24 hours.
26. The method of any one of claims 1 to 25, wherein the time sufficient for generating a population of CD13+ early mesoderm cells at the end of step lb is about 2 to about 72 hours, preferably about 24 hours.
27. The method of any one of claims 1 to 26, wherein the time sufficient for generating the population of CD13+ and KDR+ mesodermal-endothelial cells at the end of step 1c is about 2 to about 72 hours, preferably about 24 hours.
28. The method of any one of claims 1 to 27, wherein the time sufficient for generating the population of CD34+ cells at the end of step Id is about 24 to about 144 hours, preferably about 72 hours.
29. The method of any one of claims 1 to 28, wherein the population of MIXL1+ cells at the end of step la is at least 75% of total cells.
30. The method of any one of claims 1 to 29, wherein the population of CD 13+ early mesoderm cells at the end of step lb is at least 85% of total cells.
31. The method of any one of claims 1 to 30, wherein the population of CD 13+ and KDR+ mesodermal-endothelial cells at the end of step 1c is at least 20% of total cells.
32. The method of any one of claims 1 to 31, wherein the population of CD34+ cells at the end of step Id is at least 70% of total cells.
33. The method of any one of claims 1 to 32, wherein the population of CD34+ cells further co-expresses CXCR4 and/or CDH5 (VE-cadherin).
34. The method of claim 33, wherein the population of CD34+ cells further co-expressing CXCR4 is at least 70% of total cells.
35. The method of claim 33, wherein the population of CD34+ cells further co-expressing CDH5 (VE-cadherin) is at least 70% of total cells.
36. The method of any one of claims 4 to 35, wherein the concentration of SCF in the fifth defined medium is about 50 to about 500 ng/mL, preferably about 100 ng/mL, the concentration of VEGF in the fifth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL, the concentration of FGF2 in the fifth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL, the concentration of IL3 in the fifth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL, and the concentration of Flt3L in the fifth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL.
37. The method of any one of claims 4, 5, 13 and 16 to 36, wherein the concentration of Flt3L in the sixth defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL, the concentration of VEGF in the sixth defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL, the concentration of FGF2 in the sixth defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL, and the concentration of IL7 in the sixth defined medium is about 0.1 to about 10 ng/mL, preferably about 1 ng/mL.
38. The method of any one of claims 4, 5, 14 and 16 to 37, wherein the concentration of Flt3L in the seventh defined medium is about 1 to about 50 ng/mL, preferably about 10 ng/mL, the concentration of VEGF in the seventh defined medium is about 10 to about 500 ng/mL, preferably about 50 ng/mL, the concentration of FGF2 in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL, the concentration of IL7 in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL, and the concentration of
IL 15 in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 20 ng/mL.
39. The method of any one of claims 4 to 38, wherein the time sufficient for generating the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 2b, 3b, 4b, 5b, 6b, 7b or 8b is about 72 to about 288 hours, preferably about 144 hours.
40. The method of any one of claims 4, 5, 13 and 16 to 35, wherein the time sufficient for generating the population of CD34+CD7+ lymphoid haematopoietic progenitor cells at the end of step 2c or 6c is about 24 to about 144 hours, preferably about 72 hours.
41. The method of any one of claims 4, 5, and 16 to 35, wherein the time sufficient for generating a population of CD34-CD7+ and CD7+RAG1+ lymphoid cells at the end of step 2d is about 48 to about 192 hours, preferably about 96 hours.
42. The method of any one of claims 4, 5, and 16 to 35, wherein the time sufficient for generating the mixed population of CD7+CD161+RAG1- cells and CD161+CD56+ cells at the end of step 2e is about 48 to about 192 hours, preferably about 96 hours.
43. The method of any one of claims 4 to 39, wherein the population of CD34+CD43+ haematopoietic progenitor cells at the end of step 2b, 3b, 4b, 5b, 6b, 7b or 8b is at least 25% of total cells.
44. The method of any one of claims 4, 5, 13 and 16 to 35, wherein the population of CD34+CD7+ lymphoid haematopoietic progenitor cells at the end of step 2c or 6c is at least 40% of total cells.
45. The method of any one of claims 4, 5, 16 to 35, wherein the population of CD34-CD7+ and CD7+RAG1+ lymphoid cells at the end of step 2d is at least 45% of total cells.
46. The method of any one of claims 4, 5, 16 to 35, wherein the population of PSC-derived innate lymphoid cells (ILCs) at the end of step 2e is at least 75% of total cells and/or the population of PSC-derived NK-like cells at the end of step 2e is at least 50% of total cells.
47. The method of any one of claims 4, 5 and 16 to 35, wherein PSC-derived innate lymphoid cells (ILCs) are CD161+CD7+ cells and/or CD161+RAG1- cells.
48. The method of claim 47, wherein the population of CD161+CD7+ cells is at least 70% of total cells.
49. The method of claim 47, wherein the population of CD161+RAG1- cells is at least 70% of total cells.
50. The method of any one of claims 4, 5, and 16 to 35, wherein PSC-derived NK-like cells are CD161+CD56+ cells.
51. The method of claim 50, wherein the population of CD161+CD56+ cells is at least 50% of total cells.
52. The method of any one of claims 6 to 13 and 16 to 35, wherein the concentration of Flt3L in the sixth defined medium is about 1 to about 100 ng/mL, preferably about 10 ng/mL, the concentration of VEGF in the sixth defined medium is about 5 to about 500 ng/mL, preferably about 50 ng/mL, and the concentration of FGF2 in the sixth defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL.
53. The method of any one of claims 6, 7, and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+RAG1- cells at the end of step 3c is about 7 to about 21 days, preferably about 11 days.
54. The method of any one of claims 6, 7, and 16 to 35, wherein the cell population enriched in CD161+RAG1- cells at the end of step 3c is at least 50% of total cells.
55. The method of any one of claims 8, 9, and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD4+CD8a+ cells at the end of step 4c is about 7 to about 21 days, preferably about 11 days.
56. The method of any one of claims 8, 9, and 16 to 35, wherein the cell population enriched in CD4+CD8a+ cells at the end of step 4c is at least 12% of total cells.
57. The method of any one of claims 10, 11 and 16 to 35, wherein the concentration of Flt3L in the seventh defined medium is about 1 to about 100 ng/mL, preferably about 10 ng/mL, the concentration of VEGF in the seventh defined medium is about 5 to about 500 ng/mL, preferably about 50 ng/mL, and the concentration of FGF2 in the seventh defined medium is about 10 to about 50 ng/mL, preferably about 20 ng/mL.
58. The method of any one of claims 12, and 16 to 35, wherein the time sufficient for generating the cell suspension comprising the population of CD161+RAG1+ cells at the end of step 5c is about 3 to about 14 days, preferably about 7 days.
59. The method of any one of claims 12, and 16 to 35, wherein the sorting at step 5d is fluorescence-activated cell sorting (FACS).
60. The method of any one of claims 10, 11, 12 and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+CD56+ cells at the end of step 5e is about 48 to about 192 hours, preferably about 96 hours.
61. The method of any one of claims 10, 11, 12 and 16 to 35, wherein the cell population enriched in CD161+CD56+ cells at the end of step 5e is at least 70% of total cells.
62. The method of any one of claims 13 and 16 to 35, wherein the time sufficient for generating the cell population enriched in CD161+CD56+ cells at the end of step 6d is about 48 to about 240 hours, preferably about 120 hours.
63. The method of any one of claims 13 to 33, wherein the cell population enriched in CD161+CD56+ cells at the end of step 6d is at least 80% of total cells.
64. A population of DLL4-expressing arterial endothelial cells (AECs) obtained from the method of any one of claims 1 to 3 and 16 to 35, wherein the DLL4-expressing arterial endothelial cells (AECs) are CD34+ cells.
65. The population of DLL4-expressing AECs of claim 34, wherein the population of CD34+ cells co-expresses CXCR4.
66. The population of DLL4-expressing AECs of claim 64 or 65, wherein the population of CD34+ cells co-expresses CDH5 (VE-cadherin).
67. A mixed population of PSC-derived innate lymphoid cells (ILCs) and PSC-derived NK- like cells obtained from the method of any one of claims 4, 5, and 16 to 51.
68. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of claim 67, wherein the PSC-derived ILCs are CD161+CD7+ cells.
69. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of claim 67 or 66, wherein the PSC-derived ILCs are CD161+RAG1- cells.
70. The mixed population of PSC-derived ILCs and PSC-derived NK-like cells of any one of claims 67 to 69, wherein the NK-like cells are CD161+CD56+ cells.
71. A cell population enriched in PSC-derived ILCs obtained from the method of any one of claims 6, 7, 16 to 36, 39, 43, and 52 to 54.
72. The cell population enriched in PSC-derived ILCs of claim 71, wherein the PSC-derived ILCs are CD161+RAG1- cells.
73. A cell population enriched in PSC-derived T cells obtained from the method of any one of claims 8, 9, and 16 to 36, 39, 43, 52, 55, and 56.
74. The cell population enriched in PSC-derived T cells of claim 73, wherein the PSC-derived T cells are CD4+CD8a+ cells.
75. A cell population enriched in PSC-derived NK-like cells obtained from the method of any one of claims 10 to 13, 16 to 37, 39, 40, 43, 44, 52, and 57 to 63.
76. The cell population enriched in PSC-derived NK-like cells of claim 75, wherein the PSC- derived NK-like cells are CD161+CD56+ cells.
77. A population of CD34+CD43+ haematopoietic progenitor cells obtained from step 2b of any one of claims 4, 5, 16 to 36 and 39.
78. A population of CD34+CD7+ lymphoid haematopoietic progenitor cells obtained from step 2c of any one of claims 4, 5, 16 to 37, 39 and 40.
79. A population of CD34-CD7+ lymphoid cells obtained from step 2d of any one of claims 4, 5, 16 to 37, and 39 to 41.
80. A population of CD7+RAG1+ lymphoid cells obtained from step 2d of any one of claims 4, 5, 16 to 37, and 39 to 41.
81. A population of CD161+RAG1+ cells obtained from step 5c or 5d of any one of claims 12, 16 to 37, 39, 58 and 59.
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