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WO2025065106A1 - Procédés, milieux et suppléments pour expansion de cellules hématopoïétiques - Google Patents

Procédés, milieux et suppléments pour expansion de cellules hématopoïétiques Download PDF

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Publication number
WO2025065106A1
WO2025065106A1 PCT/CA2024/051295 CA2024051295W WO2025065106A1 WO 2025065106 A1 WO2025065106 A1 WO 2025065106A1 CA 2024051295 W CA2024051295 W CA 2024051295W WO 2025065106 A1 WO2025065106 A1 WO 2025065106A1
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hdaci
hspc
cells
cocktail
hspcs
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WO2025065106A9 (fr
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Javed Karim MANESIA
Albertus Wernerus Wognum
Marta Walasek
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Stemcell Technologies Canada Inc
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Stemcell Technologies Canada Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/065—Modulators of histone acetylation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • This disclosure relates to cell culture applications, and more specifically to cell culture applications using hematopoietic stem and progenitor cells (HSPCs), and still more specifically to cell culture applications related to expansion of CD34 + HSPCs and/or CD34 + malignant cells.
  • HSPCs hematopoietic stem and progenitor cells
  • HSCs Hematopoietic stem cells
  • HSPC hematopoietic stem and progenitor cells
  • One limitation of clinical use of HSCs and HSPCs has been the difficulty to obtain sufficient numbers of such cells.
  • HSCs and HSPCs are resistant to maintenance, propagation and expansion ex vivo.
  • Another challenge relates to the loss of HSC and HSPC multi-potency, which may arise when such cells are cultured ex vivo.
  • the present disclosure relates to the culture and/or expansion of target cells, which may be HSPCs, or CD34 + cells, and various subsets thereof, including primitive CD34 + subsets.
  • Target cells of this disclosure may be comprised in an initial population comprising the target cells and non-target cells, or as single cells.
  • the disclosed methods may be in vitro methods.
  • Methods of this disclosure may comprise contacting one or more of the target cells with a cocktail of a plurality of epigenetic modifiers in a culture medium, and culturing the target cells in the presence of the cocktail for a time sufficient to produce an expanded population of target cells, such as HSPCs or CD34 + cells.
  • the cocktail of a plurality of epigenetic modifiers may comprise one or more of, or two or more of, or three or more of, or each of a first inhibitor of a histone deacetylase (HDACi), a second HDACi, at least one inhibitor of a histone demethylase (HDMi), and at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • the first HDACi and the second HDACi are class I, II, or IV HDACi.
  • the first HDACi and the second HDACi are selected from: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin.
  • the at least one HDMi is a LSD1 or LSD2 inhibitor.
  • the at least one HMTi is a G9a/GLP HMTi.
  • Methods of this disclosure may comprise isolating or enriching the one or more HSPC from a sample before contacting the population with the cocktail.
  • a sample include whole blood, cord blood, peripheral blood, or bone marrow.
  • samples of this disclosure may be diseased or infected samples.
  • the target cells may comprise a primitive HSPC having a CD34 + CD45RA CD90 + phenotype, a CD34 + CD45RA CD90 + EPCR + phenotype, and/or a CD34 + CD45RA CD90 + EPCR + CD133 + CD49f + phenotype.
  • the expanded population of target cells e.g.
  • HSPCs, or CD34 + cells comprises more primitive cells having a CD34 + CD45RA CD90 + phenotype and/or a CD34 + CD45RA _ CD90 + EPCR + phenotype and/or a CD34 + CD45RA CD90 + EPCR + CD133 + CD49f + phenotype compared to when the one or more target cells are not cultured in the presence of the cocktail for the time sufficient to produce the expanded population of target cells.
  • the expanded population of target cells comprises more target cells compared to when the one or more target cells or the initial population of target cells are not cultured in the presence of one or more epigenetic modifiers (e.g. supplement or cocktail) for the time sufficient to produce the expanded population of target cells.
  • one or more epigenetic modifiers e.g. supplement or cocktail
  • Methods of this disclosure may further comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34 + cells) when the one or more target cells is contacted with and/or cultured in the presence of one or more epigenetic modifiers (of a cocktail or supplement) comprising each of the first and second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising only one, two or three of the foregoing.
  • target cells e.g. HSPCs, or CD34 + cells
  • epigenetic modifiers of a cocktail or supplement
  • Methods of this disclosure may further comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34 + cells) when the one or more target cells is contacted with and/or cultured in the presence of one or more epigenetic modifiers (of a cocktail or supplement) comprising a first HDACi, or a functional equivalent thereof, and two of the second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising two of the first and second HDACi, at least one HDMi, and at least one HMT, wherein the first HDACi is valproic acid.
  • target cells e.g. HSPCs, or CD34 + cells
  • Methods of this disclosure may further comprise yielding a more highly expanded population target cells (e.g. HSPCs, or CD34 + cells) when the one or more target cells is contacted with and cultured in the presence of one or more epigenetic modifiers (of a cocktail or supplement) comprising a first HDACi, or a functional equivalent thereof, and one of the second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising one of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMTi, wherein the first HDACi is valproic acid.
  • a more highly expanded population target cells e.g. HSPCs, or CD34 + cells
  • Methods of this disclosure may further comprise yielding a more highly expanded population of HSPCs with multi-lineage differentiation capacity when the one or more HSPC is contacted with and cultured in the presence of a cocktail comprising the first HDACi and the at least one HMTi, wherein the first HDACi is valproic acid.
  • the expanded population of target cells comprises between 2-fold to more than 100-fold more target cells compared to when the target cells are not cultured in the presence of the cocktail for the time sufficient to produce the expanded population of target cells.
  • the culture media may further comprise one or more of SCF, FLT- 3L, and/or TPO.
  • Different methods of this disclosure may comprise culturing or expanding a population of target cells (e.g. HSPC, or CD34 + cells) by contacting the target cells with a serum-free culture media comprising at least one epigenetic modifier and one or more of SCF, FLT-3L, and/or TPO, and culturing the target cells in the culture medium for a time sufficient to produce an expanded population of target cells.
  • target cells e.g. HSPC, or CD34 + cells
  • the expanded population may comprise target cells comprising primitive HSPCs or CD34 + cells having a CD34 + CD45RA CD90 + phenotype and/or a CD34 + CD45RA _ CD90 + EPCR + phenotype and/or a CD34 + CD45RA CD90 + EPCR + CD133 + CD49f phenotype.
  • the at least one epigenetic modifier is a histone deacetylase inhibitor (HDACi).
  • HDACi histone deacetylase inhibitor
  • the HDACi is valproic acid.
  • the modifiers may comprise one or more of a second histone deacetylase inhibitor (HDACi), at least one histone demethylase inhibitor (HDMi), and at least one histone methyltransferase inhibitor (HMTi).
  • HDACi include: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin.
  • TSA Trichostatin A
  • Entinostat Entinostat
  • Tacedinaline Tacedinaline
  • Panobinostat Panobinostat
  • LMK235 histone methyltransferase inhibitor
  • HMTi histone methyltransferase inhibitor
  • Exemplary HDACi include: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin.
  • HDMi include a LSD1 or LSD2 inhibitor.
  • HMTi
  • the cocktail of epigenetic modifiers comprises two or more of, three or more of, or all a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi.
  • any method of this disclosure may further comprise isolating an initial population of target cells (e.g. HSPC, or CD34 + cells) from a sample before contacting the population with the one or more epigenetic modifiers (or cocktail).
  • the sample is human cord blood, mobilized peripheral blood, or bone marrow.
  • the sample is a leukemia sample.
  • the initial population of CD34 + cells is isolated from CML sample or AML peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMCs).
  • PBMCs peripheral blood mononuclear cells
  • BMMCs bone marrow mononuclear cells
  • the expanded population comprises one or more of the following primitive subsets: CD34 + CD45RA CD90 + ; CD34 + CD45RA CD90 + EPCR + ; and/or CD34 + CD45RA CD90 + EPCR + CD133 + CD49f + .
  • the expanded population of target cells e.g. HSPC, or CD34 + cells
  • culturing the population of target cells is for between about 5 to 21 days.
  • Supplements for expanding target cells (e.g. HSPC, or CD34 + cells) in vitro.
  • Supplements may comprise a cocktail of a plurality of epigenetic modifiers comprising one or more of, two or more of, three or more of, or each of the following: a first inhibitor of a histone deacetylase (HDACi), a second HDACi, at least one inhibitor of a histone demethylase (HDMi), and at least one inhibitor of a histone methyltransferase (HMTi).
  • the cocktail may comprise a first HDACi and a second HDACi.
  • the first HDACi and the second HDACi are class I, II, or IV HDACi.
  • Exemplary HDACi include: Valproic acid, Trichostatin A (TSA), Entinostat, Tacedinaline, Panobinostat, LMK235, and Romidepsin.
  • the at least one HDMi is a LSD1 or LSD2 inhibitor.
  • the at least one HMTi is a G9a/GLP HMTi.
  • media for culturing e.g. expanding a population of target cells (e.g. HSPCs, or CD34 + cells).
  • Media of this disclosure may comprise a basal medium and a supplement (comprising one or more epigenetic modifiers, as described herein).
  • Media of this disclosure may further comprise one or more of SCF, FLT- 3L, IL-3, IL-6, and/or TPO.
  • Figure 1 shows bar graphs quantifying expansion of human cord blood HSPC, and certain CD34 + subsets thereof (A-D) and colony forming potential (E-F) after 7-day culture in the presence of CD34 Supp (StemSpanTM CD34+ Expansion Supplement) + individual or various cocktails of epigenetic modifiers, as indicated.
  • Tested epigenetic modifiers included: a first HDACi (HDACi-1), a second HDACi (HDACi-2), a histone demethylase inhibitor (HDMi- 1), and a histone methyltransferase inhibitor (HMTi-1).
  • Ref a positive control comprising CD34 Supp + a published small molecule reference compound.
  • TNC total nucleated cells.
  • CFU-GEMM colony-forming unit, granulocyte, erythroid, macrophage, megakaryocyte;
  • CFU- G/M/GM colony-forming unit, granulocyte-macrophage;
  • BFU-E burst-forming unit, erythroid. Data shown are mean of two cord blood samples.
  • Figure 2 shows bar graphs quantifying expansion of human cord blood HSPC, and certain CD34 + subsets thereof (A-D) and colony forming potential (E-F) after 7-day culture essentially as performed in Figure 1 , except the 4i cocktail of epigenetic modifiers was assessed either in the presence (4i) or absence (4i-AO) of an anti-oxidizing agent.
  • TNC, Ref, CFU-GEMM, CFU-G/M/GM, and BFU-E are as defined in the above paragraph. Data shown are mean ⁇ SEM of 2 cord blood samples.
  • Figure 3 shows bar graphs quantifying expansion of human cord blood (CB), human mobilized peripheral blood (mPB), and human bone marrow (BM) HSPC and certain CD34 + subsets thereof after 7-day (A-D) and 14-day (E-H) culture, essentially as performed in Figure 1 .
  • Ref is as defined in the above paragraphs. Data shown are mean ⁇ SEM of cell samples from 2-8 separate donors.
  • FIG. 4 shows bar graphs quantifying expansion of CD34 + cells isolated from AML samples.
  • CD34 + AML cells were cultured for 7 days in a serum-free medium comprising CD34 Supp (as described in the above paragraphs) either alone or in combination with a reference compound (Ref, as described in the paragraphs above) or a 4i cocktail of epigenetic modifiers (as described in Figure 1).
  • Day 7 frequency (A) and cell numbers (B) of presumptive AML stem or progenitor cells are shown, including for the more primitive subsets: CD34 + CD45RA _ CD90 + and CD34 + CD45RA _ 90 + EPCR + .
  • Data shown are mean ⁇ SEM of PB samples from 5 AML patients.
  • Figure 5 shows bar graphs quantifying expansion of CD34 + cells isolated from CML samples.
  • CD34 + CML cells were cultured for 7 days in a serum-free medium comprising CD34 Supp (as described in the above paragraphs) either alone or in combination with a reference compound (Ref, as described in the paragraphs above) or a 4i cocktail of epigenetic modifiers (as described for Figure 1).
  • Day 7 frequency (A) and cell numbers (B) of presumptive CML stem or progenitor cells are shown, including for the more primitive subsets: CD34 + CD45RA _ CD90 + and CD34 + CD45RA _ 90 + EPCR + .
  • Data shown are mean ⁇ SEM of PB samples from 4 CML patients.
  • This disclosure relates to media compositions and/or supplements to be added into a medium, and to methods for culturing (e.g., expanding) hematopoietic stem and progenitor (HSPC) cells, specifically, CD34 + HSPCs, and/or CD34 + malignant cells.
  • HSPC hematopoietic stem and progenitor
  • hematopoietic stem and progenitor cell refers to a cell of the hematopoietic lineage that is capable of self-renewal and/or differentiating into a more specialized cell of the hematopoietic lineage including the myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and the lymphoid lineages (T cells, B cells, NK cells).
  • myeloid lineages myeloid lineages
  • monocytes and macrophages neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • T cells B cells, NK cells
  • HSPC may be obtained from bone marrow (BM), umbilical cord blood (CB), embryonic through to adult peripheral blood (PB), thymus, peripheral lymph nodes, gastrointestinal track, tonsils, gravid uterus, liver, spleen or any other tissue having localized populations of HSPC.
  • HSPC may also be differentiated from pluripotent stem cells such as induced pluripotent stem cells, embryonic stem cells, naive stem cells, extended stem cells, or the like.
  • pluripotent stem cells such as induced pluripotent stem cells, embryonic stem cells, naive stem cells, extended stem cells, or the like.
  • a hallmark of HSPC is the expression of the transmembrane phosphoglycoprotein CD34 on its surface, thus HSPC may be referred to as CD34 + cells, though cells of the non-hematopoietic lineage, or the like, may also express CD34.
  • a population of HSPCs may comprise a single population of CD34 + cells, that is, CD34 + cells of the same phenotype. Or, a population of HSPCs may comprise a plurality of CD34 + cells, that is, the cells may all express CD34 but variously other surface markers.
  • Human HSPC subsets may be further defined by expression of CD45, and may still further be defined by combinations of markers such as CD38, CD43, CD45RO, CD45RA, CD10, CD49f, CD59, CD90, CD109, CD117, CD133, CD166, HLA-DR, EPCR (CD201), and integrin-alpha3.
  • HSPCs may lack expression or have only low expression of markers such as Glycophorin A, CD3, CD4, CD8, CD14, CD15, CD19, CD20 and CD56; such markers may be characteristic of more mature blood cells.
  • markers such as Glycophorin A, CD3, CD4, CD8, CD14, CD15, CD19, CD20 and CD56; such markers may be characteristic of more mature blood cells.
  • markers such as Glycophorin A, CD3, CD4, CD8, CD14, CD15, CD19, CD20 and CD56; such markers may be characteristic of more mature blood cells.
  • markers of more common markers of HSPCs e.g. CD34 + HSPC
  • CD45RA which is absent or only weakly expressed on primitive cells
  • CD34, CD90 Thy-1
  • CD201 endothelial protein C receptor, EPCR
  • CD133 endothelial protein C receptor, and CD49f
  • epigenetic modifier or “epigenetic regulator”, used interchangeably herein, refer to an agent or a factor such as a protein, a small molecule, interfering RNA, messenger RNA, or other natural or synthetic compound (or catalytic domain thereof having enzymatic activity) that modify or cooperate with other factors to modify epigenetic marks of DNA and/or histones.
  • Epigenetic modifiers may be classified depending on their mechanism of action, such as DNA methyltransferases or inhibitors thereof, histone acetyltransferases or inhibitors thereof, histone methyltransferases or inhibitors thereof, histone deacetylases or inhibitors thereof, or histone demethylases or inhibitors thereof.
  • an expanded population refers to an output population of cells wherein a quantity of output cells (such as after a period of culture in the presence of one or more epigenetic modifiers) is greater than in a control population (such as one not cultured in the presence of the one or more epigenetic modifiers).
  • an expanded population of cells may specifically refer to a population of HSPC or CD34 + cells expanded in the presence of one or more epigenetic modifiers.
  • an expanded population of HSPC cells or CD34 + cells may more specifically refer to a progenitor or primitive population expanded in the presence of one or more epigenetic modifiers.
  • an expanded population may mean an increase of at least 10%, or 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 an input or control.
  • an expanded population may mean at least about a 2-fold, at least about a 3-fold, at least about a 5-fold, at least about a 10-fold increase, at least about a 15-fold increase, at least about a 20-fold, at least about a 25-fold increase, at least about a 30-fold increase, at least about a 35-fold increase, at least about a 40-fold increase, at least about a 45-fold increase, at least about a 50-fold increase, or any increase between 2-fold and 50- fold, or greater, as compared to an input or control.
  • the expanded population of the target cells e.g. HSPC, or CD34 + cells
  • the expanded population of the target cells e.g. HSPC, or CD34 + cells
  • whether gene edited or not may be used to engraft into a subject, such as a human subject or into an animal model such as a mouse.
  • supplements which may be added to culture media for culturing or generating or expanding a population of target cells, such as HSPC (e.g. CD34 + cells) and/or progenitors or primitive subsets thereof.
  • HSPC e.g. CD34 + cells
  • a composition/formulation of a supplement of this disclosure is not limited in any particular way, provided that it is suitable for contacting one or more HSPC, or a population thereof, and facilitating their culture.
  • Supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules. More specifically, supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that modulate epigenetic marks (e.g. epigenetic modifiers). While supplements of this disclosure may be entirely small molecule-, compound-, and/or macromolecule-based, they may comprise or be combined with one or more macromolecules (e.g. lipids, proteins, carbohydrates) that does not have a primary function as an epigenetic modifier.
  • macromolecules e.g. lipids, proteins, carbohydrates
  • supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that are epigenetic modifiers, and one or more macromolecules that does not have a primary function as an epigenetic modifier, or supplements of this disclosure may comprise one or more small molecules, compounds, or macromolecules that are epigenetic modifiers and be combined with one or more macromolecules that does not have a primary function as an epigenetic modifier (as may be comprised in a separate supplement or culture medium).
  • supplements of this disclosure may support the expansion of target cells, such as HSPC cells (e.g., CD34 + HSPCs and/or CD34 + malignant cells).
  • supplements of this disclosure may comprise one or more epigenetic modifiers.
  • supplements of this disclosure comprise a plurality of epigenetic modifiers (e.g. a cocktail of epigenetic modifiers).
  • supplements of this disclosure may comprise either purely small molecule epigenetic modifiers, purely non-small molecule epigenetic modifiers (e.g. compounds), purely macromolecule modifiers (e.g. fatty acids), or any combination of the foregoing.
  • Supplements (and as applicable a cocktail) of this disclosure may comprise one or more of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) of this disclosure may comprise two or more of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) of this disclosure may comprise each of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) of this disclosure may comprise one or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) of this disclosure may comprise two or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) of this disclosure may comprise three or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) of this disclosure may comprise each of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • a supplement (and as applicable a cocktail) comprises at least a first HDACi (e.g. valproic acid). In some embodiments, a supplement (and as applicable a cocktail) comprises at least a first HDACi (e.g. valproic acid) and/or at least one HMTi.
  • HDAC inhibitors are known and widely available, and are thus not particularly limited in supplements of this disclosure.
  • one HDACi is comprised in a supplement of this disclosure.
  • two HDACi are comprised in a supplement of this disclosure.
  • more than two HDACi are comprised in a supplement of this disclosure.
  • the one or more HDACi comprised in a supplement may be class I, II, or IV HDACi. If only one HDACi is comprised in a supplement, then such HDACi may belong to a single class of HDACi or to multiple HDACi classes. If more than one (e.g. two or more) HDACi are comprised in a supplement, then each HDACi may belong to the same or different class(es). In one embodiment, a supplement of this disclosure comprises at least two class I HDACi.
  • HDACi examples include Valproic acid (HDAC class I & II inhibitor), Trichostatin A (TSA) (HDAC class I, II & IV inhibitors), Romidepsin (HDAC class I inhibitor), Entinostat (HDAC class I inhibitor), Tacedinaline (HDAC class I inhibitor), Panobinostat (HDAC class I, II & IV inhibitors), and LMK235 (HDAC class II inhibitors).
  • the supplement comprises Valproic acid.
  • the supplement comprises Romidepsin.
  • the supplement comprises one or both of Valproic acid and Romidepsin.
  • a concentration of the one or more HDACi in the supplement is not particularly limited, except that the concentration should not be lethal or toxic to cells. If the HDACi is a small molecule, then its concentration may be in the nanomolar to micromolar range. For example, the concentration may range between about 0.1 nM to 100 uM, or between about 0.5 nM to 50 uM, or between about 1 nM to 10 uM, or between about 5 nM to 1 uM, or between about 10 nM to 0.5 uM, or between about 25 nM and 250 uM. If the HDACi is not a small molecule, then its concentration may be in the micromolar to millimolar range.
  • the concentration may range between about 1 uM to 1000 mM, or between about 10 uM to 500 mM, or between about 100 uM to 250 mM, or between about 250 uM to 200 mM, or between about 500 uM to 150 mM, or between about 1 mM and 100 mM.
  • HDM inhibitors are known and widely available, and are thus not particularly limited in supplements of this disclosure.
  • one HDMi is comprised in a supplement of this disclosure.
  • two HDMi are comprised in a supplement of this disclosure.
  • more than two HDMi are comprised in a supplement of this disclosure.
  • the one or more HDMi comprised in a supplement may be an inhibitor of lysine specific demethylases, such as LSD1 and LSD2 inhibitors.
  • the HDMi is a LSD1 inhibitor.
  • LSD1 inhibitors that may be comprised in a supplement are 2-PCPA (tranylcypromine), RN-1 (LSD1 inhibitor IV), SP2509, ORY-100, and GSK-LSD1.
  • the LSD1 inhibitor is 2-PCPA (tranylcypromine).
  • the LSD1 inhibitor is tranylcypromine hydrochloride or a functional derivative thereof.
  • a concentration of the one or more HDMi in the supplement is not particularly limited, except that the concentration should not be lethal or toxic to cells. If the HDMi is a small molecule, then its concentration may be in the nanomolar to micromolar range. For example, the concentration may range between about 1 nM to 1 mM, or between about 25 nM to 750 uM, or between about 100 nM to 500 uM, or between about 250 nM to 400 uM, or between about 500 nM to 300 uM, or between about 1 uM and 250 uM. If the HDMi is not a small molecule, then its concentration may be in the micromolar to millimolar range.
  • the concentration may range between about 1 uM to 1000 mM, or between about 10 uM to 500 mM, or between about 100 uM to 250 mM, or between about 250 uM to 200 mM, or between about 500 uM to 150 mM, or between about 1 mM and 100 mM.
  • HMT inhibitors are known and widely available, and are thus not particularly limited in supplements of this disclosure.
  • one HMTi is comprised in a supplement of this disclosure.
  • two HMTi are comprised in a supplement of this disclosure.
  • more than two HMTi are comprised in a supplement of this disclosure.
  • the one or more HMTi comprised in a supplement may be histone methyltransferase G9a (also known as EHMT2 or KMTIC) and/or G9a-like protein (GLP, also known as EHMT1).
  • G9a histone methyltransferase G9a
  • GLP G9a-like protein
  • the at least one HMTi comprised in a supplement is a G9a/GLP HMTi.
  • the G9a/GLP HMTi is UNC0638.
  • a concentration of the one or more HMTi in the supplement is not particularly limited, except that the concentration should not be lethal or toxic to cells. If the HMTi is a small molecule, then its concentration may be in the nanomolar to micromolar range. For example, the concentration may range between about 1 nM to 1 mM, or between about 25 nM to 750 uM, or between about 100 nM to 500 uM, or between about 250 nM to 400 uM, or between about 500 nM to 300 uM, or between about 1 uM and 250 uM. If the HMTi is not a small molecule, then its concentration may be in the micromolar to millimolar range.
  • the concentration may range between about 1 uM to 1000 mM, or between about 10 uM to 500 mM, or between about 100 uM to 250 mM, or between about 250 uM to 200 mM, or between about 500 uM to 150 mM, or between about 1 mM and 100 mM.
  • a HSPC (e.g. CD34 + cell) expansion supplement of this disclosure comprises at least one HDACi, and at least one of an HDMi and HMTi.
  • a HSPC (e.g. CD34 + cell) expansion supplement of this disclosure comprises at least one HDACi, at least one HDMi, and at least one HMTi.
  • a HSPC (e.g. CD34 + cell) expansion supplement of this disclosure comprises at least two HDACi, and at least one of an HDMi and HMTi.
  • a HSPC (e.g. CD34 + cell) expansion supplement of this disclosure comprises at least two HDACi, at least one HDMi, and at least one HMTi.
  • a supplement (and/or media to which the supplement is added) further comprises an antioxidant, such as ascorbic acid, retinoic acid, glutathione, or any derivative, analog, or functional equivalent thereof.
  • an antioxidant such as ascorbic acid, retinoic acid, glutathione, or any derivative, analog, or functional equivalent thereof.
  • Supplements of this disclosure may be concentrated.
  • the supplement is at about a 2X concentration, about a 5X concentration, about a 10X concentration, about a 15X concentration, about a 20X concentration, about a 25X concentration, about a 50X concentration, about a 100X concentration, about a 200X concentration, or greater.
  • Supplements of this disclosure may be added to cell culture media.
  • a supplement comprising one or more epigenetic modifier may be further combined with one or more cytokines or growth factors.
  • HSPCs e.g., CD34 + cells
  • media for culturing/expanding target cells in vitro such as HSPCs (e.g., CD34 + cells).
  • HSPCs expanded in media of this disclosure may retain the ability to differentiate into a multitude of downstream cell types in the hematopoietic lineage.
  • Media of this disclosure may also be used to expand CD34 + malignant cells isolated from leukemias (e.g. AML and/or CML PBMCs) or other myelodysplastic syndromes.
  • Media of this disclosure may contain serum or may be serum-free.
  • media of this disclosure are serum-free. If serum-free, it may be necessary to include in such media a serum replacement supplement, such as BIT 9500 Serum Substitute (STEMCELL Technologies), or other commercially available serum replacement solutions. Alternatively, components ordinarily present in serum that are needed for culturing or differentiating any cells of this disclosure may be individually added at an acceptable concentration to the media.
  • a serum replacement supplement such as BIT 9500 Serum Substitute (STEMCELL Technologies)
  • components ordinarily present in serum that are needed for culturing or differentiating any cells of this disclosure may be individually added at an acceptable concentration to the media.
  • Culture media of this disclosure will comprise a basal medium.
  • the basal medium may be any medium that, when appropriately supplemented, supports the expansion of target cells, such as mammalian HSPC (e.g. CD34 + cells, or subsets thereof).
  • the basal medium may be any medium capable of supporting the expansion of mammalian CD34 + malignant cells.
  • the basal medium may be any basal medium which is supportive of culturing cells of the hematopoietic lineage.
  • the basal medium may be StemSpanTM SFEM (STEMCELL Technologies), StemSpanTM SFEM II (STEMCELL Technologies), StemSpanTM-XF (STEMCELL Technologies), StemSpanTM AOF (STEMCELL Technologies), or any other commercially available basal medium fit for the purpose.
  • Media of this disclosure may comprise one or more epigenetic modifiers, or more than one epigenetic modifier.
  • epigenetic modifiers or more than one epigenetic modifier.
  • Media of this disclosure may further comprise one or more additional cytokines or growth factors, or small molecules, or other proteins that provide critical signals for HSPC expansion.
  • critical signals required for HSPC expansion may include one or more of: interleukin(s) (e.g. IL-1 , IL-2, IL-3, IL-6), VEGF, G-CSF, GM-CSF, SCF, FLT3-L, TPO, and EPO.
  • Supplements and media of this disclosure may support fold expansion of target cells (e.g. HSPCs) on the order of at least 10%, or 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 control condition (lacking the supplement of one or more epigenetic modifier, as described above and fully incorporated herein by reference).
  • Supplements and media of this disclosure may support fold expansion of target cells (e.g.
  • HSPC on the order of at least about a 2-fold, or at least about a 3-fold, or at least about a 5- fold, or at least about a 10-fold increase, or at least about a 15-fold increase, or at least about a 20-fold, or at least about a 25-fold increase, or at least about a 30-fold increase, or at least about a 35-fold increase, or at least about a 40-fold increase, or at least about a 45-fold increase, or at least about a 50-fold increase or any increase between 2-fold and 50-fold, or greater, as compared to a control condition (lacking the supplement of one or more epigenetic modifier, as described above and fully incorporated herein by reference).
  • Enhanced expansion of HSPCs in the presence of supplements of one or more epigenetic modifier, and media of this disclosure comprising such supplements and one or more cytokines/growth factors beneficial to HSPC expansion may more specifically relate to expansion of more primitive HSPC subsets (e.g. CD34 + CD45RACD90 + , CD34 + CD45RA _ CD90 + EPCR + and CD34 + CD45RACD90 + EPCR + CD133 + CD49f + ), relative to a commercially available cytokine supplement alone (e.g. StemSpanTM CD34 Expansion Supplement, STEMCELL Technologies) or in combination with a reference published small molecule (e.g. a pyrimidoindole compound).
  • a commercially available cytokine supplement alone e.g. StemSpanTM CD34 Expansion Supplement, STEMCELL Technologies
  • a reference published small molecule e.g. a pyrimidoindole compound
  • higher expansion of primitive HSPC subsets are supported by (i) a supplement comprising each of the first and the second HDACi, the at least one HDMi, and the at least one HMTi, as compared to a cocktail comprising only one, two or three of the foregoing, (ii) a supplement comprising the first HDACi, or a functional equivalent thereof, and two of the second HDACi, the at least one HDMi, and the at least one HMTi, as compared to a cocktail comprising two of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMTi, wherein the first HDACi is valproic, or (iii) a supplement comprising the first HDACi, or a functional equivalent thereof,
  • higher expansion of primitive HSPC subsets e.g. CD34 + CD45RA CD90 + , CD34 + CD45RA CD90 + EPCR + , and CD34 + CD45RA' CD90 + EPCR + CD133 + CD49f
  • higher expansion of primitive HSPC subsets with in vitro multi-lineage differentiation capacity are supported by a supplement comprising the first HDACi and the at least one HMTi, wherein the first HDACi is valproic acid.
  • Methods for culturing and/or expanding target cells such as HSPC (e.g. CD34 + cells).
  • Methods of this disclosure may encompass those steps for culturing and/or expanding target cells, such as HSPCs (e.g. CD34 + cells), in a serum-free culture environment.
  • the methods disclosed herein for culturing and/or expanding cells, such as CD34 + cells - whether normal CD34 + or malignant CD34 + cells - are preferably in vitro methods.
  • the target cells are preferably HSPC, which may express at least CD34.
  • HSPC are preferably mammalian, and more preferably human.
  • HSPC may be directly isolated from a primary sample, such as whole blood, cord blood, (mobilized) peripheral blood, bone marrow, thymus, uterus, liver, gut or secondary lymphoid tissues.
  • the CD34 + are isolated from human cord blood.
  • the CD34 + are isolated from (mobilized) peripheral blood.
  • the CD34 + are isolated from human bone marrow.
  • Target cells used in the methods of this disclosure may be CD34 + cells obtained, enriched, and/or isolated from a cancer, malignant, diseased, or infected sample, such as peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the CD34 + cancer or malignant cells are obtained, enriched, and/or isolated from a chronic myeloid leukemia (CML) sample (e.g. PBMCs).
  • CML chronic myeloid leukemia
  • the CD34 + cancer or malignant cells are obtained, enriched, and/or isolated from an acute myeloid leukemia (AML) sample (e.g. PBMCs).
  • AML acute myeloid leukemia
  • Methods for culturing (e.g. expanding) target cells may comprise contacting one or more cells (e.g. HSPC) with one or more epigenetic modifiers in a culture medium. More specifically, methods of the disclosure may comprise contacting one or more cells (e.g. HSPC) with a cocktail of a plurality of epigenetic modifiers in a culture medium.
  • the nature and other details, such as combinations and concentrations, of the epigenetic modifier(s) in a culture medium of this disclosure may be as described hereinabove.
  • culture media may also apply to the disclosed methods.
  • Hematopoietic cells may require one or more cytokine and/or growth factors for efficient expansion; thus, culture media used in methods of this disclosure may be supplemented with one or more of SCF, FLT-3L, and/or TPO.
  • Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise one or more of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise two or more of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise each of: (i) at least one inhibitor of a histone deacetylase (HDACi); (ii) at least one inhibitor of a histone demethylase (HDMi); and (iii) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise one or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise two or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise three or more of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • Supplements (and as applicable a cocktail) in culture media used in methods of this disclosure may comprise each of: (i) at least a first inhibitor of a histone deacetylase (HDACi); (ii) at least a second inhibitor of a histone deacetylase (HDACi); (iii) at least one inhibitor of a histone demethylase (HDMi); and (iv) at least one inhibitor of a histone methyltransferase (HMTi).
  • HDACi histone deacetylase
  • HDACi histone deacetylase
  • HDMi histone demethylase
  • HMTi histone methyltransferase
  • a supplement (and as applicable a cocktail), as may be comprised in a culture media, for use in methods of this disclosure comprises at least a first HDACi (e.g. valproic acid).
  • a supplement (and as applicable a cocktail) comprises at least a first HDACi (e.g. valproic acid) and/or at least one HMTi.
  • a HSPC (e.g. CD34 + cell) expansion supplement (as may be comprised in culture media) used in methods of this disclosure comprises at least one HDACi, and at least one of an HDMi and HMTi.
  • a HSPC (e.g. CD34 + cell) expansion supplement (as may be comprised in culture media) used in methods of this disclosure comprises at least one HDACi, at least one HDMi, and at least one HMTi.
  • a HSPC (e.g. CD34 + cell) expansion supplement (as may be comprised in culture media) used in methods of this disclosure comprises at least two HDACi, and at least one of an HDMi and HMTi.
  • a HSPC (e.g. CD34 + cell) expansion supplement (as may be comprised in culture media) used in methods of this disclosure comprises at least two HDACi, at least one HDMi, and at least one HMTi.
  • the one or more target cells may be comprised in an initial population of cells, and the initial population of cells may either be an isolated or enriched population of target cells, or a bulk population comprising target cells and off-target cells.
  • methods of this disclosure may comprise isolating or enriching the one or more target cells (e.g. HSPC, or CD34 + cells) from a sample before contacting the population with the cocktail.
  • Approaches to isolating or enriching target cells as may be comprised in an initial population comprising target cells (e.g. HSPC, or CD34 + cells), are known and commercially available, such as by FACS or by immunomagnetic cell separation, as commercialized under the EasySepTM brand (STEMCELL Technologies).
  • the methods may further comprise culturing the one or more target cells (e.g. HSPC, or CD34 + cells) in the presence of the one or more epigenetic modifiers for a time sufficient to produce an expanded population of target cells.
  • target cells e.g. HSPC, or CD34 + cells
  • Culturing duration is not particularly limited other than by build-up of metabolites, pH, etc., by nutrient availabilities, and over-crowding, all of which may be rectified by medium exchanges and/or scaling up vessel size and media volume. Notwithstanding, culturing may be for between about 1 and 35 days, between about 2 and 32 days, between about 3 and 28 days, between about 5 and 21 days, or between about 7 days to about 14 days. In some embodiments, culturing is for 7 days ⁇ 1 day. In some embodiments, culturing is for 10 days ⁇ 1 day. In some embodiments, culturing is for 14 days ⁇ 1 day.
  • Yielding/producing an expanded population of target cells (e.g. HSPC, or CD34 + cells) after culturing for a sufficient time may result in more target cells (e.g. HSPC, or CD34 + cells) compared to when the one or more target cells (as may comprised in an initial population) are not cultured in the presence of the one or more epigenetic modifiers (e.g. the cocktail, or the supplement) for the time sufficient to produce the expanded population of target cells (e.g. HSPCs or CD34 + cells).
  • the one or more epigenetic modifiers e.g. the cocktail, or the supplement
  • Increased expansion of target cells by practicing the methods of this disclosure may also be quantified relative to expansion of target cells (e.g. HSPC, or CD34 + cells) when contacted with and/or cultured in the presence of a reference standard small molecule in the culture medium.
  • target cells e.g. HSPC, or CD34 + cells
  • various such reference molecules/compounds are known including engineered cytokines (e.g. IL-6), pyrimidoindole compounds, or aryl hydrocarbon antagonists.
  • Increased expansion of target cells by practicing the methods of this disclosure may also be quantified relative to expansion of target cells (e.g. HSPC, or CD34 + cells) when contacted with and/or cultured in the presence of fewer than the more than one epigenetic modifier comprised in a cocktail or in a culture medium.
  • target cells e.g. HSPC, or CD34 + cells
  • higher expansion of target cells e.g. HSPC, or CD34 + cells, or primitive subsets thereof: e.g.
  • CD34 + CD45RA _ CD90 + ; CD34 + CD45RA CD90 + EPCR + ; or CD34 + CD45RA CD90 + EPCR + CD133 + CD49f + ) is supported (i) in the presence of four epigenetic modifiers (in an appropriate medium) relative to the presence of three, two or one epigenetic modifiers (in an appropriate medium), (ii) in the presence of three epigenetic modifiers (in an appropriate medium) relative to the presence of two or one epigenetic modifiers (in an appropriate medium), or (iii) in the presence of two epigenetic modifiers (in an appropriate medium) relative to the presence of one epigenetic modifiers (in an appropriate medium).
  • methods of this disclosure may comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34 + cells) when one or more target cells is contacted with and/or cultured in the presence of a cocktail comprising each of a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising only one, two or three of the foregoing.
  • target cells e.g. HSPCs, or CD34 + cells
  • Methods of this disclosure may comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34 + cells) when one or more target cells is contacted with and cultured in the presence of a cocktail comprising a first HDACi, or a functional equivalent thereof, and two of a second HDACi, at least one HDMi, and at least one HMTi, as compared to a cocktail comprising two of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMT, wherein the first HDACi is valproic acid
  • Methods of this disclosure may comprise yielding a more highly expanded population of target cells (e.g. HSPCs, or CD34 + cells) when one or more target cells is contacted with and/or cultured in the presence of a cocktail comprising a first HDACi, or a functional equivalent thereof, and one of either a second HDACi, at least one HDMi, or at least one HMTi, as compared to a cocktail comprising one of the first HDACi, the second HDACi, the at least one HDMi, and the at least one HMTi, wherein the first HDACi is valproic acid.
  • target cells e.g. HSPCs, or CD34 + cells
  • Yielding/producing an expanded population of target cells (e.g. HSPC, or CD34 + cells) after culturing for a sufficient time may result in between 2-fold to 100-fold more target cells compared to when the one or more target cells (e.g. HSPC, or CD34 + cells) are not cultured in the presence of the one or more epigenetic modifies (e.g. the cocktail, or the supplement) for the time sufficient to produce the expanded population of target cells.
  • the one or more epigenetic modifies e.g. the cocktail, or the supplement
  • culturing for a sufficient time may yield/produce between 2-fold to more than 100-fold more target cells in an expanded population compared to when the target cells (e.g. HSPC, or CD34 + cells) of the initial population are not cultured in the presence of the one or more epigenetic modifies (e.g. the cocktail, or the supplement) for the time sufficient to produce the expanded population of target cells.
  • an expanded population of target cells may result in about 2- fold more target cells, about 3-fold more target cells, about 4-fold more target cells, about 5- fold more target cells, about 10-fold more target cells, about 15-fold more target cells, about 20-fold more target cells, about 25-fold-more target cells, about 30-fold more target cells, about 40-fold more target cells, about 50-fold more target cells, about 60-fold-more target cells, about 70-fold more target cells, about 80-fold more target cells, about 90-fold more target cells, about 100-fold-more target cells, or more.
  • Methods of this disclosure may yield expanded populations of CD34 + cells which may be further characterized as distinct subsets of CD34-expressing cells.
  • the one or more target cells e.g. CD34 + cells, or HSPC
  • Such phenotypes may correspond with more primitive subsets of CD34-expressing cells and/or HSPC.
  • expanded populations may comprise expanded populations of target cells comprising a CD34 + CD45RA CD90 + phenotype, a CD34 + CD45RA CD90 + EPCR + phenotype, and/or a CD34 + CD45RA _ CD90 + EPCR + CD133 + CD49f + phenotype.
  • an expanded population of target cells may comprise between about 2 to 50-fold, or between about 3 to 40-fold, or between about 4 to 30-fold, or between 5 to 25-fold more of a primitive subset of HSPC or CD34 + cells (e.g CD34 + CD45RA CD90 + ) after culturing one or more such target cells (as may be comprised in an initial population of cells, such as CD34 + cells) in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.
  • a primitive subset of HSPC or CD34 + cells e.g CD34 + CD45RA CD90 +
  • epigenetic modifiers e.g. cocktail or supplement
  • an expanded population of target cells may comprise between about 2 to 50-fold, or between about 3 to 40-fold, or between about 4 to 30-fold, or between 5 to 25-fold more of a primitive subset of HSPC or CD34 + cells (e.g. CD34 + CD45RA CD90 + ) per input CD34 + cells following 7 days or 14 days of expansion culture in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.
  • a primitive subset of HSPC or CD34 + cells e.g. CD34 + CD45RA CD90 +
  • epigenetic modifiers e.g. cocktail or supplement
  • an expanded population of target cells may comprise between about 2 to 25-fold, or between about 3 to 20-fold, or between about 4 to 15-fold, or between 5 to 10-fold more of a primitive subset of HSPC or CD34 + cells (e.g CD34 + CD45RA CD90 + EPCR + ) after culturing one or more such target cells (as may be comprised in an initial population of cells, such as CD34 + cells) in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.
  • a primitive subset of HSPC or CD34 + cells e.g CD34 + CD45RA CD90 + EPCR +
  • epigenetic modifiers e.g. cocktail or supplement
  • an expanded population of target cells may comprise about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or more of a primitive subset of HSPC or CD34 + cells (e.g. CD34 + CD45RA CD90 + EPCR + ) per input CD34 + cells following 7 days or 14 days of expansion culture in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.
  • a primitive subset of HSPC or CD34 + cells e.g. CD34 + CD45RA CD90 + EPCR +
  • epigenetic modifiers e.g. cocktail or supplement
  • an expanded population of target cells may comprise between about 2 to 25-fold, or between about 3 to 20-fold, or between about 4 to 15-fold, or between 5 to 10-fold more of a primitive subset of HSPC or CD34 + cells (e.g. CD34 + CD45RA CD90 + EPCR + CD133 + CD49f) after culturing one or more such target cells (as may be comprised in an initial population of cells, such as CD34 + cells) in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.
  • a primitive subset of HSPC or CD34 + cells e.g. CD34 + CD45RA CD90 + EPCR + CD133 + CD49f
  • an expanded population of target cells may comprise about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6- fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or more of a primitive subset of HSPC or CD34 + cells (e.g. CD34 + CD45RA CD90 + EPCR + CD133 + CD49f + ) per input CD34 + cells following 7 days or 14 days of expansion culture in the presence of one or more epigenetic modifiers (e.g. cocktail or supplement) in a culture medium, relative to when culturing such cells in the absence of the one or more epigenetic modifiers in the medium.
  • a primitive subset of HSPC or CD34 + cells e.g. CD34 + CD45RA CD90 + EPCR + CD133 + CD49f +
  • an expanded population of target cells may comprise more primitive cells having a phenotype selected from one or more of CD34 + CD45RA CD90 + , CD34 + CD45RACD90 + EPCR + , and/or CD34 + CD45RA' CD90 + EPCR + CD133 + CD49f + compared to when the one or more target cells are not cultured in the presence of the one or more epigenetic modifiers (e.g. the cocktail, or supplement) for a time sufficient to produce the expanded population of cells.
  • the one or more epigenetic modifiers e.g. the cocktail, or supplement
  • methods (and supplements and supplemented media) of this disclosure may preferentially support the expansion of primitive subsets of HSPC or CD34 + cells, such as CD34 + CD45RA CD90 + cells, CD34 + CD45RACD90 + EPCR + cells, and CD34 + CD45RA CD90 + EPCR + CD133 + CD49f + cells.
  • the presence of at least one HDACi helps promote expansion of target cells and to yield target cells capable of downstream differentiation, such as may be measured by CFU assay.
  • a cocktail comprising a first HDACi, a second HDACi, and at least one HMTi may outperform a cocktail comprising each of a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi at least in terms of CFU-GEMM, and/or CFU-G/M/GM, and/or BFU-E output.
  • a cocktail comprising a first HDACi, a second HDACi, and at least one HMTi may outperform any other 3-way combination selected from a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi, at least in terms of CFU-GEMM, and/or CFU-G/M/GM, and/or BFU-E output.
  • a cocktail comprising a first HDACi, a second HDACi, and at least one HDMi may underperform a cocktail comprising each of a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi, at least in terms of CFU-GEMM, and/or CFU-G/M/GM, and/or BFU-E output.
  • cocktails comprising a first HDACi, a second HDACi, and at least one HDMi may underperform any other 3-way combination selected from a first HDACi, a second HDACi, at least one HDMi, and at least one HMTi, at least in terms of CFU-GEMM, and/or CFU-G/M/GM, and/or BFU-E output.
  • in vitro expanded cells e.g. HSPC, and/or CD34 + cells or progenitor and/or primitive subsets thereof.
  • expanded cells may be used in any relevant downstream applications, including in gene editing workflows, clinically or therapeutically, to study hematopoietic development in normal or diseased contexts, or downstream differentiation to downstream hematopoietic lineages (e.g. myeloid, erythroid, and/or lymphoid), etc.
  • a functional HSPC should be capable of reconstituting the hematopoietic lineages of a sub-lethally irradiated recipient, and target cells obtained by practicing the methods disclosed herein have been shown to engraft in a subject after introduction and that they produce myeloid cells, lymphoid cells, and erythroid cells.
  • methods of this disclosure may further comprise introducing expanded cells into a subject, wherein i) a similar or a higher level of lymphoid (CD45 + CD19 + B cells) engraftment occurs relative to uncultured cells in the engrafted subject, and/or ii) a similar or a higher level of myeloid (CD45 + CD33 + ) engraftment occurs relative to uncultured cells, and/or iii) a similar or a higher level of lymphoid and/or myeloid engraftment relative to uncultured cells.
  • a similar or a higher level of lymphoid (CD45 + CD19 + B cells) engraftment occurs relative to uncultured cells in the engrafted subject
  • CD45 + CD33 + myeloid
  • Example 1 Procurement, preparation, and culture of cells
  • Cord blood units were procured from commercial suppliers. Human bone marrow samples and mobilized peripheral blood was procured from STEMCELL Technologies. AML and CML samples were procured from Discovery Life Sciences. CD34 + cells were enriched from the foregoing samples using the EasySepTM Human Cord Blood CD34 Positive Selection Kit II (STEMCELL Technologies), and then either frozen in serum with 10% DMSO or used fresh.
  • ⁇ 4000 CD34 + cells were plated per well of a 24-well plate for 7 days in serum-free StemSpanTM SFEM II (STEMCELL Technologies) supplemented with cytokines contained in StemSpanTM CD34 Expansion Supplement (STEMCELL Technologies).
  • cells were cultured either in the presence of a reference, published small molecule pyrimidoindole compound (Ref) available commercially from STEMCELL Technologies, or one or more epigenetic modifiers.
  • Example 2 Flow cytometry and determination of cell count and yield
  • the viable cell count is first multiplied by the % CD34 + obtained by flow cytometry. This number is then divided by the number of input cells (in the case input CD34 + cells) to obtain the final value.
  • Input CD34 + cell numbers were obtained by multiplying total cells cultured in one well by frequency of CD34 + cells after cell separation.
  • Example 3 Expanding HSPCs in the presence of one or more epigenetic modifiers
  • Human cord blood HSPC were isolated and cultured essentially as described in Example 1 (e.g. StemSpanTM SFEM II + StemSpanTM CD34 Expansion Supplement). In certain conditions, the cells were also cultured in the presence of either a reference small molecule (Ref) or various epigenetic modifiers, whether individually (1 ; 2; 3; or 4) or in various 2-way (1-3; 1-4; 1-2; 2-3; or 2-4), 3-way (1-2-3; 2-3-4; 1-2-4; or 1-3-4), or 4-way (4i) cocktails ( Figure 1).
  • Ref reference small molecule
  • CFU colony-forming unit
  • Example 4 Expanding HSPC in a supplement comprising epigenetic modifiers and an antioxidant
  • Figures 2E-F show that an absence of an antioxidant in the 4i cocktail (4i-AO) resulted in lower number of colonies compared to the reference compound and the condition that included the antioxidant in the 4i cocktail (4i).
  • presence of an anti-oxidant in the 4i cocktail of epigenetic modifiers does not appear to influence the output number of primitive HSPCs, it does appear to have a marked impact on colony forming potential.
  • Example 5 Expanding HSPC from different sources in the presence of cocktails of epigenetic modifiers
  • Human cord blood, peripheral blood, and bone marrow HSPC were isolated and cultured for 7 or 14 days, essentially as described in Example 1 , except different basal media formulations were used together with a cytokine supplement and cocktail 4i: StemSpanTM SFEM (STEMCELL Technologies), StemSpanTM SFEM II (STEMCELL Technologies), StemSpanTM-XF (STEMCELL Technologies) or StemSpanTM AOF (STEMCELL Technologies).
  • Example 6 Expanding presumptive cancer stem or progenitor cells from leukemia samples
  • CD34 + cells were isolated from AML and CML samples and cultured essentially as described in Example 1 . After 7 days, the cultured cells were stained with fluorescently labeled antibodies and analyzed by flow cytometry as described in Example 2.
  • Human cord blood HSPCs were isolated and cultured for 7 days in the presence of a cytokine supplement + 4i, essentially as described in Example 1. After 7 days of expansion, progeny of 2500 initial CD34 + cells or 2500 uncultured CD34 + cells (control) were transplanted in sub-lethally irradiated NSG mice. At week 20, long-term multilineage engraftment was measured in bone marrow of transplanted NSG mice.
  • Recipients of 4i expanded cells showed similar or higher frequencies of human CD45 + and CD34 + cells in analyzed mouse bone marrow compared to recipients of uncultured cells. Recipients of 4i expanded cells showed higher levels of HSPC (CD34), lymphoid (CD45 + CD19 + ), and myeloid (CD45 + CD33 + ) cell engraftment, relative to uncultured cells (see Table 1).

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Abstract

La présente divulgation concerne des procédés, des milieux et des suppléments pour la culture de cellules cibles de telles cellules souches et progénitrices hématopoïétiques (HSPC, « hematopoietic stem and progenitor cell »). Les procédés, milieux et compléments de la présente divulgation peuvent comprendre un ou plusieurs modificateurs épigénétiques dans une condition de culture pour cultiver et/ou étendre des cellules cibles, telles que des HSPC ou des cellules CD34+, comme cela peut être obtenu, enrichi ou isolé à partir d'échantillons primaires qui sont infectés/malades ou normaux. Des populations de sortie de HSPC expansées en présence d'un ou de plusieurs modificateurs épigénétiques peuvent être modifiées pour être utilisées dans des applications en aval.
PCT/CA2024/051295 2023-09-29 2024-09-27 Procédés, milieux et suppléments pour expansion de cellules hématopoïétiques Pending WO2025065106A1 (fr)

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ASKARI MARYAM HAJ, SHAHABI MAJID, KOJABAD AMIR ASRI, ZARIF MAHIN NIKOUGOFTAR: "Reconstruction of bone marrow microenvironment for expansion of hematopoietic stem cells by a histone deacetylase inhibitor", CYTOTECHNOLOGY, SPRINGER NETHERLANDS, DORDRECHT, vol. 75, no. 3, 1 June 2023 (2023-06-01), Dordrecht, pages 195 - 206, XP093302043, ISSN: 0920-9069, DOI: 10.1007/s10616-022-00564-w *

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