[go: up one dir, main page]

WO2025096798A2 - Endothelial cell factors and methods thereof - Google Patents

Endothelial cell factors and methods thereof Download PDF

Info

Publication number
WO2025096798A2
WO2025096798A2 PCT/US2024/053916 US2024053916W WO2025096798A2 WO 2025096798 A2 WO2025096798 A2 WO 2025096798A2 US 2024053916 W US2024053916 W US 2024053916W WO 2025096798 A2 WO2025096798 A2 WO 2025096798A2
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
endothelial
niche
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/053916
Other languages
French (fr)
Other versions
WO2025096798A3 (en
Inventor
Leonard I. Zon
Tianxiao HAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boston Childrens Hospital
Original Assignee
Boston Childrens Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boston Childrens Hospital filed Critical Boston Childrens Hospital
Publication of WO2025096798A2 publication Critical patent/WO2025096798A2/en
Publication of WO2025096798A3 publication Critical patent/WO2025096798A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N5/069—Vascular Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • 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
    • C12N2501/60—Transcription factors
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • Haematopoietic stem and progenitor cells are a rare cell population capable of reconstituting the entire blood system after transplantation. As the functional unit of a bone marrow transplant, these cells offer a curative treatment for many blood and immune diseases. Unfortunately, transplantation is not a viable treatment option for many individuals, particularly those lacking an immune-matched donor. A long-term goal of hematological research has been to culture and expand HSPCs in vitro, for use in transplantation and/or genetic modification. While umbilical cord blood- derived HSPCs are somewhat amenable to in vitro expansion, maintaining and inducing self-renewal of adult-derived HSPCs, in the absence of niche signals, has proven challenging.
  • HSPCs can also be supported outside the bone marrow, during embryonic development and under stress conditions that induce 1 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT extramedullary hematopoiesis in tissues such as the liver, spleen and skull. As in the bone marrow, ECs are thought to function as critical, core components of the HSPC niches in these tissues.
  • One aspect provided herein is describes a method for engineering endothelial niche cells, the method comprising expressing in a cell ETV2; and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • the at least two transcription factors are TFEC and MAFB.
  • the cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a placenta stem cell, an adult stem cell, an amniotic stem cell, and an umbilical vein endothelial cell.
  • ESC embryonic stem cell
  • iPSC induced pluripotent stem cell
  • the ESC, iPSC, placenta stem cell, adult stem cell, amniotic stem cell is differentiated to an endothelial cell prior to contact.
  • ETV2 and the at least two transcription factors are expressed from at least one vector.
  • the at least one vector comprises an exogenous nucleic acid sequence(s) encoding the ETV2 and the at least two transcription factors.
  • expression is transient or stable.
  • the exogenous nucleic acid sequence(s) is incorporated into the genome of the endothelial cell.
  • the cell is a mammalian cell.
  • the one embodiment of this or any aspect herein, the cell is a human cell.
  • the one embodiment of this or any aspect herein, the cell is a nonhuman mammalian cell.
  • the engineered endothelial niche cells secrete at least one of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO.
  • the engineered endothelial niche cells express at least one of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, and CD62E.
  • Another aspect provided herein is describes a method for engineering endothelial niche cells, the method comprising expressing in a cell ETV2, TFEC, and MAFB. 2 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0015] Another aspect provided herein is describes an engineered endothelial niche cell obtained by any of the methods described herein. [0016] Another aspect provided herein is describes an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • Another aspect provided herein is describes an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. [0018] Another aspect provided herein is describes a population of cells comprising any of the engineered endothelial niche cells described herein. [0019] Another aspect provided herein is describes a co-culture comprising any of the engineered endothelial niche cells described herein or population described herein, and a population of stem cells. The one embodiment of this or any aspect herein, the second population of stem cells is an HSPCs.
  • Another aspect provided herein is describes a method for increasing stem cell proliferation, the method comprising co-culturing any of the engineered endothelial niche cells described herein or population described herein, and a population of stem cells for a time sufficient in increase stem cell proliferation.
  • proliferation is increased by at least 10% as compared to an appropriate control.
  • the population of stem cells is a population of HSPCs.
  • the method is performed in vitro.
  • the engineered endothelial niche cells secrete a factor that affects the proliferation of the HSPC cells.
  • Another aspect provided herein is describes a method for treating a subject, the method comprising administering any of the engineered endothelial niche cells described herein, population described herein, or co-culture described herein into a subject in need thereof.
  • Another aspect provided herein is describes a method for treating a subject, the method comprising: identifying a subject in need thereof; and administering any of the engineered endothelial niche cells described herein, population described herein, or co-culture described herein into the subject in need thereof.
  • Another aspect provided herein is describes a method for treating a subject, the method comprising administering the co-culture described herein into a subject in need thereof.
  • Another aspect provided herein is describes a method for treating a subject, the method comprising administering a population of HSPCs that have been previously co-cultured any of the engineered endothelial niche cells described herein or population of described herein into a subject in need thereof. 3 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0029]
  • the method further comprises the step of isolating the population of HSPCs prior to administering.
  • the one embodiment of this or any aspect herein, subject in need thereof is human.
  • the one embodiment of this or any aspect herein, the subject in need thereof has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level.
  • the method further comprises the step of identifying a subject in need thereof having a decreased blood cell level or at risk for developing a decreased blood cell level as compared to a control blood cell level prior to administering.
  • the one embodiment of this or any aspect herein, the blood cell level is decreased at least 1% compared to a reference level.
  • the one embodiment of this or any aspect herein, the subject in need thereof has anemia or blood loss.
  • the subject in need thereof is a bone marrow donor.
  • the one embodiment of this or any aspect herein, the subject in need thereof has depleted bone marrow.
  • the one embodiment of this or any aspect herein, the subject in need thereof has anemia, hemolysis, leukemia, multiple myeloma, or a thyroid disorder.
  • the administering occurs at the liver, spleen, or subcutaneously.
  • Another aspect provided herein is describes a method for generating an ectopic vascular niche, the method comprising administering any of the engineered endothelial niche cells described herein, population described herein, or co-culture described herein to a target site in a subject in need thereof.
  • Another aspect provided herein is describes a method for treating extra medullary hematopoiesis, the method comprising administering any of the engineered endothelial niche cells described herein, population described herein, or co-culture described herein into a subject at a location outside of the bone marrow, thereby creating a synthetic niche.
  • administering is systemic or local administration.
  • the one embodiment of this or any aspect herein, local administration is transplantation.
  • the one embodiment of this or any aspect herein, local administration is administration directly to the liver, spleen, or subcutaneously.
  • Another aspect provided herein is describes a vector comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 operably linked to a promoter.
  • Another aspect provided herein is describes a vector comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB.
  • Figs 1A-1F present exemplary data showing single-cell transcriptional profiling of the endothelial cells from the adult zebrafish kidney marrow and from the adult zebrafish liver.
  • FIG.1A Isolation of endothelial cells from transgenic kdrl:mCherry endothelial cell reporter zebrafish;
  • FIG.1B Pan-endothelial cell marker genes cdh5, kdrl, and pecam1 are expressed by endothelial cells from both organs;
  • Fig.1C Sinusoidal endothelial cell marker genes sele, flt4, and gpr182, are expressed by the sinusoidal endothelial cells from both organs;
  • FIG.1D Liver sinusoidal endothelial cell marker genes gata4 and f8 are exclusively expressed by the sinusoidal endothelial cells from the adult zebrafish liver;
  • FIG.1E HSPC niche supportive genes cxcl12a, mrc1a, dab2 and csf1b, are significantly upregulated in the sinusoidal endothelial cells from the adult zebrafish kidney marrow;
  • FIG.1F Differential
  • Figs 2A-2C present exemplary data showing single-cell RNA-sequencing analyses of adult mouse endothelial cells from the bone marrow, the liver, and the kidney.
  • Fig.2A Pan- endothelial marker genes Cdh5, Kdr, and Pecam1 are expressed by endothelial cells from all three mouse organs;
  • Fig.2B Sinusoidal endothelial cell marker genes Stab2, Flt4, and Gpr182 are highly expressed by sinusoidal endothelial cells from mouse bone marrow and mouse liver;
  • Fig.2C Transcription factors Tfec, Mafb, Foxp4, Hoxb8, and Irf8 are highly expressed by the mouse bone marrow endothelial cells.
  • FIGs 3A-3E present exemplary data showing rca2.2 enhancer drives specific expression in the liver endothelial cells.
  • FIG.3A Schematics of cloning putative enhancers into a GFP reporter construct and creating transgenic zebrafish reporter lines;
  • Fig.3B Gene tracks show a unique ATAC-seq open chromatin region upstream of rca2.2 gene in the liver endothelial cells; anti-GFP immunohistochemistry on rca2.2:GFP transgenic fish reveals specific expression in the liver vascular endothelial cells;
  • FIG.3C Whole-mount immunofluorescence imaging of double transgenic reporter zebrafish liver show overlap between rca2.2:GFP and kdrl:BFP signals in the endothelial cells;
  • Fig.3A Schematics of cloning putative enhancers into a GFP reporter construct and creating transgenic zebrafish reporter lines
  • FIG.3B Gene tracks show a unique ATAC-seq open
  • Figs 4A-4G present exemplary data showing transcription factors tfec and mafbb reprogrammed the liver sinusoidal endothelial cells.
  • Fig.4A Immunofluorescence staining of mrc1a:GFP and kdrl:mCherry show specific labeling of the endothelial cells in the embryonic zebrafish CHT HSPC niche and in the adult zebrafish kidney marrow HSPC niche
  • Fig.4B Immunofluorescence staining show ectopic expression of mrc1a:GFP in the reprogrammed liver upon tfec and mafbb overexpression
  • Fig.4C Heatmap of RNA-sequencing analysis show tfec and mafbb 5 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT reprogrammed the HSPC niche supportive genes without perturbing the normal expression of liver sinusoidal endothelial cell genes
  • Figs 5A-AB present exemplary data showing transplant assay of donor liver cells into immunocompromised recipients.
  • FIG.5A Schematics of transplanting donor liver cells into immunocompromised recipient zebrafish, and post-transplant engraftment assay by flow cytometry;
  • Figs 6A-6B present exemplary data showing generation of human iPSC lines with inducible transcription factor overexpression.
  • FIG.6A Vector map for generating tetracycline- inducible transcription factor overexpression hiPSC lines
  • FIG.6B Schematics of generating new hiPSC lines by nucleofection, drug selection, and PCR genotyping.
  • Figs 7A-7B present exemplary data showing directed differentiation of human iPSCs into endothelial cells in vitro.
  • Fig.7A Schematics of directing differentiation from human iPSCs into induced ECs. Brightfield images show the standard morphology of the cells at each stage.
  • Figs 8A-8D present exemplary data showing transcription factor overexpression in hiPSC-derived endothelial cells putatively upregulated HSPC niche supportive genes.
  • FIG.8D Gene ontology enrichment analyses revealed that many of the genes upregulated by TFEC and MAFB in the iECs are involved in cell adhesion, migration, extravasation
  • Figs 9A-9B present exemplary data showing functional assay of primary human HSPCs co-cultured with TFEC and MAFB overexpression endothelial cells.
  • Fig.9A Colony-forming unit (CFU) assay results of post co-cultured HSPCs; HSPCs with ETV2 iECs did not show significant differences compared to HSPCs without EC co-culture; HSPCs with ETV2-TFEC-MAFB ECs showed a significantly greater number of colonies compared to the other two groups;
  • Embodiments of the technology described herein comprise compositions, kits, vectors, and methods related to generating or engineering endothelial niche cells.
  • One aspect comprises a method to generate/engineer endothelial niche cells, comprising expressing in a cell ETV2; and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • the at least two transcription factors are TFEC and MAFB.
  • Another aspect provided herein comprises a method to generate/engineer endothelial niche cells, comprising expressing in a cell ETV2, TFEC, and MAFB.
  • an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • the at least two transcription factors are TFEC and MAFB.
  • an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB.
  • Yet another aspect provided herein is a vector comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 operably linked to a promoter.
  • the at least two transcription factors are TFEC and MAFB.
  • a vector comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB.
  • the at least two transcription factors can be at least three, at least four, or at least five of the transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • ETS variant transcription factor 2 ETV2 refers to a protein known to enable sequence-specific double-stranded DNA binding activity. It is involved in cell differentiation and regulation of transcription by RNA polymerase II and acts upstream of or within several processes, including cell surface receptor signaling pathway; positive regulation of endothelial cell differentiation; and positive regulation of macromolecule metabolic process.
  • ETV2 sequences are known for a number of species, e.g., human ETV2 (NCBI Gene ID: 2116) polypeptide (e.g., NCBI Ref Seq XP_005258709.1) and mRNA (e.g., NCBI Ref Seq XM_005258652.3).
  • ETV2 can refer to human ETV2, including naturally occurring variants, molecules, and alleles thereof.
  • ETV2 refers to the mammalian ETV2 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like.
  • the nucleic sequence of SEQ ID NO: 1 comprises the nucleic sequence which encodes ETV2.
  • TFEC transcription factor EC
  • MiT microphthalmia
  • the encoded protein is a transcriptional activator of the nonmuscle myosin II heavy chain-A gene and may also co-regulate target genes in osteoclasts as a heterodimer with microphthalmia-associated transcription factor.
  • TFEC sequences are known for a number of species, e.g., human TFEC (NCBI Gene ID: 22797) polypeptide (e.g., NCBI Ref Seq NP_001018068.1) and mRNA (e.g., NCBI Ref Seq NM_001018058.3).
  • TFEC can refer to human TFEC, including naturally occurring variants, molecules, and alleles thereof.
  • TFEC refers to the mammalian TFEC of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like.
  • the nucleic sequence of SEQ ID NO: 3 comprises the nucleic sequence which encodes TFEC.
  • the human polypeptide sequence of SEQ ID NO: 4 comprises the polypeptide sequence of TFEC.
  • MAF bZIP transcription factor B refers to a protein known to encoded by this gene is a basic leucine zipper (bZIP) transcription factor that plays an important role in the regulation of lineage-specific hematopoiesis. The encoded nuclear protein represses ETS1- mediated transcription of erythroid-specific genes in myeloid cells.
  • MAFB sequences are known for a number of species, e.g., human MAFB (NCBI Gene ID: 9935) polypeptide (e.g., NCBI Ref Seq NP_005452.2) and mRNA (e.g., NCBI Ref Seq NM_005461.5).
  • MAFB can refer to human MAFB, including naturally occurring variants, molecules, and alleles thereof.
  • MAFB refers to the mammalian MAFB of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like.
  • the nucleic 8 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT sequence of SEQ ID NO: 5 comprises the nucleic sequence which encodes MAFB.
  • the human polypeptide sequence of SEQ ID NO: 6 comprises the polypeptide sequence of MAFB.
  • FOXP4 forkhead box P4
  • FOXP4 refers to a protein belongs to subfamily P of the forkhead box (FOX) transcription factor family. Forkhead box transcription factors play important roles in the regulation of tissue- and cell type-specific gene transcription during both development and adulthood.
  • FOXP4 sequences are known for a number of species, e.g., human FOXP4 (NCBI Gene ID: 116113) polypeptide (e.g., NCBI Ref Seq NP_ NP_001012426.1) and mRNA (e.g., NCBI Ref Seq NM_ NM_001012426.2).
  • FOXP4 can refer to human FOXP4, including naturally occurring variants, molecules, and alleles thereof.
  • FOXP4 refers to the mammalian FOXP4 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like.
  • the nucleic sequence of SEQ ID NO: 7 comprises the nucleic sequence which encodes FOXP4.
  • the human polypeptide sequence of SEQ ID NO: 8 comprises the polypeptide sequence of FOXP4.
  • “homeobox B8 (HOXB8)” refers to a member of the Antp homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain.
  • HOXB8 sequences are known for a number of species, e.g., human HOXB8 (NCBI Gene ID: 3218) polypeptide (e.g., NCBI Ref Seq NP_076921.1) and mRNA (e.g., NCBI Ref Seq NM_024016.4).
  • HOXB8 can refer to human HOXB8, including naturally occurring variants, molecules, and alleles thereof.
  • HOXB8 refers to the mammalian HOXB8 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like.
  • the nucleic sequence of SEQ ID NO: 9 comprises the nucleic sequence which encodes HOXB8.
  • the human polypeptide sequence of SEQ ID NO: 10 comprises the polypeptide sequence of HOXB8.
  • IRF8 interferon regulatory factor 8
  • IRF8 refers to a transcription factor of the interferon (IFN) regulatory factor (IRF) family. Proteins of this family are composed of a conserved DNA-binding domain in the N-terminal region and a divergent C-terminal region that serves as the regulatory domain.
  • IRF8 sequences are known for a number of species, e.g., human IRF8 (NCBI Gene ID: 3394) polypeptide (e.g., NCBI Ref Seq NP_001350836.1) and mRNA (e.g., NCBI Ref Seq NM_001363907.1).
  • IRF8 can refer to human IRF8, including naturally occurring variants, molecules, and alleles thereof.
  • IRF8 refers to the mammalian IRF8 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like.
  • the nucleic sequence of SEQ ID NO: 11 comprises the nucleic sequence which encodes IRF8.
  • the human polypeptide sequence of SEQ ID NO: 12 comprises the polypeptide sequence of IRF8.
  • Hematopoietic System Development [0062] The development of the haematopoietic system, including the cell populations and molecular pathways, is highly conserved between fish and mammals. HSPCs are born in the aorta-gonad- 9 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT mesonephros (AGM) region and then migrate to a transient fetal niche, the fetal liver in mammals or a vascular plexus in the tail of the fish called the caudal haematopoietic tissue (CHT).
  • AGM aorta-gonad- 9 4867-3387-1598.2
  • HSPCs reside and expand in these developmental sites for several days before migrating to the adult niche – the bone marrow in mammals or the kidney marrow in fish.
  • the CHT is comprised primarily of low-flow sinusoids surrounded by mesenchymal stromal cells. HSPCs initially colonize the CHT niche by lodging within the vascular plexus and interacting directly with cxcl12a + stromal cells.
  • endothelial cells reorganize to form a supportive pocket around the HSPCs, which together with stromal cells and possibly other cell types, forms a niche for the stem cells (the endothelial cells surrounding the HSPCs can be referred to herein as endothelial niche cells).
  • endothelial niche cells In mammals and zebrafish, specific signaling molecules, adhesion proteins and transcription factors have been implicated in mediating communication and physical interaction between stem cells and ECs in the niche.
  • endothelial niche cells are endothelial cells that provide an instructive niche for the differentiation of HSPCs. Endothelial niche cells are typically found in the bone marrow.
  • exogenous expression of specific transcription factors can cause endothelial niche cells to be found in non-bone marrow tissues, thus providing for extramedullary hematopoiesis.
  • endothelial niche cells comprise cells that express ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • the cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a placenta stem cell, an adult stem cell, an amniotic stem cell, and an umbilical vein endothelial cell.
  • ESC embryonic stem cell
  • iPSC induced pluripotent stem cell
  • the ESC, iPSC, placenta stem cell, adult stem cell, amniotic stem cell is differentiated to an endothelial cell prior to contact.
  • One skilled in the art will understand how to differentiate to an ESC, iPSC, placenta stem cell, adult stem cell, or amniotic stem cell to an endothelial cell, e.g., using protocols known in the art.
  • the endothelial cells are human. 10 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0069] In some embodiments, the endothelial cells are mammalian. [0070] In some embodiments, the endothelial cells are nonhuman mammalian. [0071] In some embodiments of any of the aspects, the endothelial niche cells are generated or engineered to express transcription factors, comprising ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. [0072] In some embodiments of any of the aspects, ETV2 and the at least two transcription factors are expressed from at least one vector.
  • the vector comprises an exogenous nucleic acid sequence or sequences encoding ETV2 and the at least two transcription factors.
  • the exogenous nucleic acid sequences are transiently expressed in the endothelial cell.
  • the exogenous nucleic acid sequences are stably expressed in the endothelial cell.
  • the exogenous nucleic acid sequences are incorporated into the genome of the endothelial cell.
  • the exogenous nucleic acid sequences can be incorporated into the genome using viral vectors (e.g., AAV, lentivirus) or CRISPR technologies.
  • One aspect provides for an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • Another aspect provides for a population of engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • compositions comprising an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • the composition can comprise engineered endothelial niche cells.
  • the composition is a therapeutic agent, or the composition further comprises a pharmaceutically acceptable carrier.
  • the composition further comprises a culture dish, 3D cell system, or suspension system.
  • the composition comprises a scaffold.
  • Engineered endothelial niche cells described herein can be identified by assessing if they secrete or present certain growth facts.
  • the engineered endothelial niche cells secrete or presents on the cell surface at least one growth factor.
  • Exemplary growth factors that identify an engineered endothelial niche cells include, but is not limited to SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO.
  • the engineered endothelial niche cells secrete or present on the cell surface at least one of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO.
  • the engineered endothelial niche cells secrete or present on the cell surface, at least two, at least three, at least four, at least five, at least 11 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT six, at least seven, at least eight, at least nine, at least ten, or at least eleven of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO.
  • a skilled person can identify whether the engineered endothelial niche cells secrete or presents the at least one growth factors, e.g., using standard assays in the art.
  • immunofluorescence assays using antibodies to the growth factors can be used to assess whether the engineered endothelial niche cells presents the at least one growth factors on the cell, and assays that detect protein or mRNA expression (e.g., western blot analysis or PCR analysis, respectively) can be used to determine if the engineered endothelial niche cells secretes the at least one growth factors on the cell.
  • Engineered endothelial niche cells described herein can also be identified by assessing if they expression certain cell surface proteins.
  • the engineered endothelial niche cells express at least one cell surface protein.
  • Exemplary cell surface proteins that identify engineered endothelial niche cells include, but are not limited to MRC1, ICAM1, STAB2, VCAM1, and CD62E.
  • the engineered endothelial niche cells express at least one of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, and CD62E.
  • the engineered endothelial niche cells express at least two, at least three, at least four, or at least five of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, CD62E.
  • a skilled person can identify whether the engineered endothelial niche cells express the at least one cell surface protein, e.g., using standard assays in the art.
  • immunofluorescence assays using antibodies to the cell surface proteins can be used to assess whether the engineered endothelial niche cells expresses the at least one cell surface proteins on the cell, and assays that detect protein or mRNA expression (e.g., western blot analysis or PCR analysis, respectively) can be used to determine if the engineered endothelial niche cells expresses the at least one the cell surface proteins.
  • Another aspect provides a method for culturing stem cells, the method comprising culturing a population of stem cells in the presence of a population of engineered endothelial niche cells. In some embodiments of any of the aspects, the method is performed in vitro.
  • the engineered endothelial niche cells secrete a factor (e.g., growth factors) that affects the growth and/or expansion of the population of stem cells.
  • a factor e.g., growth factors
  • the population of stem cells is a population of HSPCs.
  • Another aspect provides a method for culturing HSPCs, the method comprising culturing a population of HSPCs in the presence of a population of engineered endothelial niche cells. In some embodiments of any of the aspects, the method is performed in vitro.
  • the stem cell e.g., HSPCs
  • the engineered endothelial niche cells have at least 10% increased proliferation than stem cells that are cultured in the absence of such engineered endothelial niche cells.
  • the stem cell (e.g., HSPCs) cultured in the presence of the engineered endothelial niche cells have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least
  • the HSPCs cultured in the presence of the engineered endothelial niche cells have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%,
  • the stem cell (e.g., HSPCs) cultured in the presence of the engineered endothelial niche cells can be cultured for at least 3 days longer than stem cells that are cultured in the absence of such engineered endothelial niche cells.
  • the HSPCs cultured in the presence of the engineered endothelial niche cells can be cultured for at least 3 days longer than HSPCs that are cultured in the absence of such engineered endothelial niche cells.
  • the stem cell (e.g., HSPCs) cultured in the presence of the engineered endothelial niche cells can be cultured for at 1 day longer, at least 2 days longer, at least 3 days longer, at least 4 days longer, at least 5 days longer, at least 6 days longer, at least 7 days longer, at least 8 days longer, at least 9 days longer, at least 10 days longer, at least 11 days longer, at least 12 days longer, at least 13 days longer, or at least 14 days longer than stem cell (e.g., HSPCs) that are cultured in the absence of such engineered endothelial niche cells.
  • stem cell e.g., HSPCs
  • the HSPCs cultured in the presence of the engineered endothelial niche cells can be cultured for at 1 day longer, at least 2 days longer, at least 3 days longer, at least 4 days longer, at least 5 days longer, at least 6 days longer, at least 7 days longer, at least 8 days longer, at least 9 days longer, at least 10 days longer, at least 11 days longer, at least 12 days longer, at least 13 days longer, or at least 14 days longer than HSPCs that are cultured in the absence of such engineered endothelial niche cells.
  • the cells are cultured on a biologically compatible scaffold.
  • Non- limiting examples of a biologically compatible scaffold comprise: a hydrogel, biopolymers, or another biomaterial with the ability to grow cells in vitro in preparation for transplantation.
  • the HSPCs cultured in the presence of the engineered endothelial niche cells have increased engraftment when administered to a subject compared to the engraftment of substantially similar HSPCs that were not cultured with engineered endothelial niche cells.
  • engraftment refers to the process wherein transplanted HSPCs begin to grow and produce healthy blood cells. Engraftment is a critical milestone in recovery from an HSPC transplant.
  • Another aspect provides a method of treating a subject, the method comprising, transplanting a composition comprising a population of engineered endothelial niche-cells into the subject.
  • the method can be used to treat myelofibrosis or other hematopoietic diseases where the endogenous bone marrow niche is compromised, non-limiting examples of which are disclosed herein.
  • the method can comprise transplanting a composition comprising a population of HSPCs into the subject.
  • the method can comprise transplanting a 14 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT composition comprising a population of HSPCs and engineered endothelial niche-cells into the subject.
  • Another aspect provides a method for enhancing engraftment of HSPCs, the method comprising administering a composition comprising HSPCs and a population of engineered endothelial niche cells to a subject in need thereof.
  • engraftment of the HSPCs is increased by at least 10% compared to the engraftment of substantially similar HSPCs in the absence of engineered endothelial niche cells.
  • engraftment of the HSPCs is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to the engraftment of substantially similar HSPCs in the absence of engineered endothelial niche cells.
  • Another aspect provides a co-culture comprising engineered endothelial niche cells and HSPCs.
  • the endothelial cells are made by a method described herein.
  • Another aspect provides a method for extra medullary hematopoiesis, the method comprising transplanting engineered-niche endothelial cells into a subject at a location outside of the bone marrow (e.g., the forearm), thereby creating a synthetic niche.
  • extra medullary hematopoiesis refers to hematopoiesis occurring in organs outside of the bone marrow.
  • the endothelial cells are made by a method described herein.
  • Treatment Methods [0093] Another aspect provides a method for treating Extramedullary hematopoiesis (EMH), the method comprising transplanting engineered-niche endothelial cells into a subject at a location outside of the bone marrow (e.g., subcutaneously, e.g., in the forearm), thereby creating a synthetic niche.
  • EMH Extramedullary hematopoiesis
  • the endothelial cells are made by a method described herein.
  • EMH Extramedullary hematopoiesis
  • EMH can arise from, for example, a response to severe anemia, chronic hypoxia, or other conditions that increase the body's demand for blood cells.
  • 15 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT
  • EMH can occur in various tissues and organs, e.g., the liver, spleen, lymph nodes, and, in rare cases, other sites such as the adrenal glands and the skin. The location of EMH can be associated with an additional related disease or disorder.
  • EMH localized in the liver can be associated with myelofibrosis; EMH localized to the spleen can by associated with splenomegaly (enlarged spleen); and EMH localized lymph nodes can be associated with hematological malignancies.
  • EMH localized lymph nodes can be associated with hematological malignancies.
  • One skilled in the art can determine if EMH is present in a subject, for example, by determining if there is a presence of hematopoietic cells, including erythroid, myeloid, and megakaryocytic lineages, outside the bone marrow.
  • the hematopoietic cells outside of the bone marrow can form clusters or nodules within the affected tissues.
  • Another aspect provides a method for treating myelofibrosis or myeloproliferative disorders, the method comprising transplanting engineered-niche endothelial cells into a subject at a location outside of the bone marrow (e.g., subcutaneously, e.g., in the forearm), thereby creating a synthetic niche.
  • the endothelial cells are made by a method described herein.
  • Myelofibrosis belongs to a group of diseases called myeloproliferative disorders, often of a chronic form.
  • Chronic myeloproliferative disorders are a group of slow-growing blood cancers in which the bone marrow makes too many abnormal red blood cells, white blood cells, or platelets, which accumulate in the blood.
  • Non-limiting examples of chronic myeloproliferative neoplasms comprise: Chronic myelogenous leukemia, Polycythemia vera, Primary myelofibrosis (also called chronic idiopathic myelofibrosis), Essential thrombocythemia, Chronic neutrophilic leukemia, and Chronic eosinophilic leukemia.
  • Myelofibrosis is a serious bone marrow disorder that disrupts the body's normal production of blood cells. The result is extensive scarring in bone marrow, leading to severe anemia, weakness, fatigue and often an enlarged spleen. Many subjects or patients with myelofibrosis get progressively worse, and some subjects or patients may eventually develop a more serious form of leukemia.
  • Myelofibrosis can occur when blood stem cells (e.g., HSPCs) develop a genetic mutation.
  • HSPCs blood stem cells
  • Several specific gene mutations have been identified in people with myelofibrosis. The most common is the Janus kinase 2 (JAK2) gene.
  • myelofibrosis Although the cause of myelofibrosis often isn't known, certain factors are known to increase risk. Increased age can be associated with the development of myelofibrosis. Myelofibrosis can affect anyone, but it's most often diagnosed in people older than 50. Patients with another blood cell disorder are at higher risk for developing myelofibrosis. A small portion of people with myelofibrosis develop the condition as a complication of essential thrombocythemia or polycythemia vera. Exposure to certain chemicals can increase the risk for myelofibrosis.
  • Myelofibrosis has been linked to exposure to 16 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT industrial chemicals such as toluene and benzene. Exposure to radiation can increase the risk for myelofibrosis. People exposed to high levels of radiation, such as survivors of atomic bomb attacks, have an increased risk of myelofibrosis. Some people who received a radioactive contrast material called Thorotrast, used until the 1950s, have developed myelofibrosis. [00101] Multiple complications can result from myelofibrosis. A complication of myelofibrosis can include increased pressure on blood flowing into a patient’s liver.
  • Increased blood flow from an enlarged spleen can lead to high blood pressure in the portal vein (e.g., portal hypertension). This in turn can force excess blood into smaller veins in the stomach and esophagus, potentially causing these veins to rupture and bleed. Pain can be another complication of myelofibrosis.
  • a severely enlarged spleen can cause abdominal pain and back pain.
  • Myelofibrosis can lead to growths in other areas of the body. Myelofibrosis can be associated with bleeding complications. As the disease progresses, platelet count tends to drop below normal (thrombocytopenia), and platelet function becomes impaired.
  • Myelofibrosis can also be associated with painful bones and joints. Myelofibrosis can lead to hardening of bone marrow and inflammation of the connective tissue that is found around the bones. This may cause bone and joint pain. Myelofibrosis can also be associated with development of acute leukemia. Some patients with myelofibrosis develop acute myelogenous leukemia, a type of blood and bone marrow cancer that progresses rapidly. [00102] Bone marrow transplantation is currently the only approved treatment for myelofibrosis. Additional treatments can only ameliorate the symptoms of myelofibrosis (e.g., anemia, enlarged spleen).
  • myelofibrosis e.g., anemia, enlarged spleen
  • Ruxolitinib a JAK inhibitor which targets the gene mutation found in most cases of myelofibrosis, can be used to reduce symptoms of an enlarged spleen.
  • levels of functional hematopoiesis can be decreased in myelofibrosis and/or in subjects with myelofibrosis.
  • functional hematopoiesis refers to hematopoiesis that produces normal levels and proportions of blood cells (e.g., red blood cells, white blood cells, platelets).
  • the level of hematopoiesis can be decreased in myelofibrosis or a myeloproliferative disorder and/or in subjects with myelofibrosis or a myeloproliferative disorder.
  • a method of treating myelofibrosis or a myeloproliferative disorder in a subject in need thereof comprising administering HSPCs, engineered endothelial cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) to a subject determined to have a level of functional hematopoiesis that is decreased relative to a reference.
  • transcription factors e.g., ETV2
  • TFEC e.g., MAFB, FOXP4, HOXB8, or IRF8
  • the step of determining if the subject has a functional level of functional hematopoiesis can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level 17 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT of functional hematopoiesis in the subject.
  • the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results.
  • the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results.
  • a method of treating a subject in need thereof comprising administering any of the engineered endothelial cells or co- cultured cells, or a composition or population thereof.
  • a method of treating a subject in need thereof comprising: a) identifying a subject in need thereof; and b) administering any of the engineered endothelial cells or co-cultured cells, or a composition or population thereof.
  • a method of treating a subject having EMH comprising administering any of the engineered endothelial cells or co- cultured cells, or a composition or population thereof.
  • a method of treating a subject in need thereof comprising: a) identifying a subject having or at risk of having EMH; and b) administering any of the engineered endothelial cells or co-cultured cells, or a composition or population thereof.
  • a method of treating a subject having myelofibrosis comprising administering any of the engineered endothelial cells or co-cultured cells, or a composition or population thereof.
  • a method of treating a subject in need thereof comprising: a) identifying a subject having or at risk of having myelofibrosis; and b) administering any of the engineered endothelial cells or co-cultured cells, or a composition or population thereof.
  • a method of treating a subject in need thereof comprising administering stem cells (e.g., HSPCs) that were previously co- cultured cells with any of the engineered endothelial cells disclosed herein.
  • stem cells e.g., HSPCs
  • described herein is a method of treating a subject in need thereof, the method comprising: a) identifying a subject in need thereof; and b) administering stem cells (e.g., HSPCs) that were previously co-cultured cells with any of the engineered endothelial cells disclosed herein.
  • a method of treating a subject having EMH comprising administering stem cells (e.g., HSPCs) that were previously co- cultured cells with any of the engineered endothelial cells disclosed herein. 18 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00113]
  • a method of treating a subject in need thereof comprising: a) identifying a subject having or at risk of having EMH; and b) administering stem cells (e.g., HSPCs) that were previously co-cultured cells with any of the engineered endothelial cells disclosed herein.
  • a method of treating a subject having myelofibrosis comprising administering stem cells (e.g., HSPCs) that were previously co-cultured cells with any of the engineered endothelial cells disclosed herein.
  • stem cells e.g., HSPCs
  • described herein is a method of treating a subject in need thereof, the method comprising: a) identifying a subject having or at risk of having myelofibrosis; and b) administering stem cells (e.g., HSPCs) that were previously co-cultured cells with any of the engineered endothelial cells disclosed herein.
  • the stem cells are isolated prior to administering.
  • method comprises the step of, prior to administering, isolating the stem cells (e.g., HSPCs) prior to administering.
  • the subject in need thereof has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level.
  • the method further comprises the step of, prior to administering, identifying a subject in need thereof having a decreased blood cell level or at risk for developing a decreased blood cell level as compared to a control blood cell level.
  • the blood cell level is decreased at least 1% as compared to a reference level.
  • the blood cell level is decreased at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least
  • a “reference level” refers to an otherwise healthy, identical cell, tissue, organ, or subject.
  • a subject has decreased blood cell levels using standard techniques, e.g., a blood test. 19 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT
  • the subject in need thereof has anemia or blood loss.
  • the subject in need thereof is a bone marrow donor.
  • the subject in need thereof has depleted bone marrow.
  • the subject in need thereof has anemia, hemolysis, leukemia, multiple myeloma, or a thyroid disorder.
  • treating a subject having or at risk of having EMH occurs at a location outside of the bone marrow.
  • Exemplary locations include, but are not limited to, liver, spleen, and subcutaneous.
  • treating a subject having or at risk of having EMH administering is a transplantation.
  • Exemplary transplantation sites include, but are not limited to, liver, spleen, and subcutaneous.
  • administering occurs at a location outside of the bone marrow.
  • Exemplary locations include, but are not limited to, liver, spleen, and subcutaneous.
  • administering is a transplantation.
  • Exemplary transplantation sites include, but are not limited to, liver, spleen, and subcutaneous.
  • administering is systemic.
  • administering is local.
  • local administering is administration to the liver, spleen, or subcutaneous.
  • Administration [00129]
  • the compositions and methods described herein can be administered to a subject having or diagnosed as having EMH.
  • the compositions and methods described herein can be administered to a subject having or diagnosed as having myelofibrosis or a myeloproliferative disorder.
  • the methods described herein comprise administering an effective amount of compositions described herein, e.g.
  • HSPCs engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) to a subject in order to alleviate a symptom of, e.g., EMH, myelofibrosis or a myeloproliferative disorder.
  • transcription factors e.g., ETV2
  • IRF8 transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8
  • Alleviating a symptom is ameliorating any condition or symptom associated with the disease or disorder, e.g., EMH, myelofibrosis or a myeloproliferative disorder.
  • compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
  • the term “effective amount” as used herein refers to the amount of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • transcription factors e.g., ETV2
  • TFEC e.g., MAFB, FOXP4, HOXB8, or IRF8
  • terapéuticaally effective amount therefore refers to an amount of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) that is sufficient to provide a particular anti- disease or disorder effect (e.g., EMH) when administered to a typical subject.
  • An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) which achieves a half- maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model.
  • IC50 i.e., the concentration of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) which achieves a half- maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by
  • the technology described herein relates to a pharmaceutical composition
  • a pharmaceutical composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein, and optionally a pharmaceutically acceptable carrier.
  • the active ingredients of the pharmaceutical composition comprise HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two 21 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein.
  • the active ingredients of the pharmaceutical composition consist essentially of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein.
  • the active ingredients of the pharmaceutical composition consist of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as e
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
  • the carrier inhibits the degradation of the active agent, e.g. HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein.
  • the pharmaceutical composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient.
  • parenteral dosage forms 22 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
  • controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS ® -type dosage forms and dose-dumping.
  • Suitable vehicles that can be used to provide parenteral dosage forms of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as disclosed within are well known to those skilled in the art.
  • transcription factors e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 as disclosed within are well known to those skilled in the art.
  • Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection
  • water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene
  • the HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) described herein is administered as a monotherapy, e.g., a second treatment for the disease or disorder (e.g., EMH, myelofibrosis or a myeloproliferative disorder) is not administered to the subject.
  • the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy.
  • Non-limiting examples of a second agent and/or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN ⁇ cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camp
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN ⁇ doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin
  • irinotecan Camptosar, CPT-11 (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva ⁇ )) 24 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT and VEGF-A that reduce
  • the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments. [00139]
  • the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy.
  • a second agent and/or treatment can also be administered a second agent and/or treatment known to be beneficial for subjects suffering from pain or inflammation.
  • the second agent is an anti-inflammation agent.
  • NSAIDs non-steroidal anti- inflammatory drugs
  • corticosteroids including glucocorticoids (e.g. cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone); methotrexate; sulfasalazine; leflunomide; anti- TNF medications; cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g., non-steroidal anti- inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g. cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone); methotrexate;
  • an effective dose of a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein can be administered to a patient once.
  • transcription factors e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 as described herein can be administered to a patient once.
  • an effective dose of a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) can be administered to a patient repeatedly.
  • transcription factors e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8
  • subjects can be administered a therapeutic amount of a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8), such as, e.g.0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
  • the treatments can be administered on a less frequent basis.
  • treatment can be repeated once per month, for six months or a year or longer.
  • Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
  • a marker or symptom of a condition e.g. HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8
  • the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8).
  • the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months.
  • Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.
  • a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
  • the dosage ranges for the administration of HSPCs, engineered endothelial niche cells, and/or transcription factors e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8), according to the methods described herein depend upon, for example, its form, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the extent to which, for example, myelofibrosis or a myeloproliferative disorder is desired to be reduced functional hematopoiesis is desired to be induced.
  • transcription factors e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8
  • the dosage should not be so large as to cause adverse side effects, such as excessive hematopoiesis or excessive extramedullary hematopoiesis. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
  • the efficacy of HSPCs, engineered endothelial niche cells, and/or transcription factors e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 in, e.g.
  • a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms 26 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g.
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms.
  • An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of EMH, myelofibrosis or a myeloproliferative disorder. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.
  • Kits [00145] One aspect described herein provides a kit for culturing HSPCs, the kit comprising: a population of engineered endothelial niche cells, reagents and instructions for use thereof. Another aspect provides for a kit for generating engineered endothelial niche cells comprising: a vector(s) comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 and instructions for use thereof. Described herein are kit components that can be included in one or more of the kits described herein.
  • the kit comprises an effective number of reagents for culturing HSPCs and/or endothelial niche cells.
  • reagents can be supplied in a lyophilized form or a concentrated form that can diluted prior to use with cultured cells. Preferred formulations include those that are non-toxic to the cells and/or does not affect growth rate or viability etc. reagents can be supplied in aliquots or in unit doses.
  • the kit further comprises a vector comprising a nucleic acid encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • kits includes the components described herein, e.g., a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8), a composition(s) that includes a vector comprising e.g., a gene to ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • transcription factors e.g., ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • the kit optionally comprises informational material.
  • the kit can also contain culture dishes and/or a substrate for coating culture dishes, such as laminin, fibronectin, Poly-L-Lysine, or methylcellulose.
  • culture dishes and/or a substrate for coating culture dishes such as laminin, fibronectin, Poly-L-Lysine, or methylcellulose.
  • the compositions in the kit can be provided in a watertight or gas tight container which in some embodiments is substantially free of other components of the kit.
  • a HSPCs, engineered endothelial niche cells, and/or transcription factors e.g., ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 composition
  • ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 composition
  • TFEC e.g., a predetermined number of experiments, e.g., 1, 2, 3 or greater.
  • One or more components as described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the components described herein are substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of endothelial niche cells and/or HSPCs, concentration, date of expiration, batch or production site information, and so forth.
  • the informational material relates to methods for using or administering the components of the kit.
  • the kit can include a component for the detection of a marker for HSPC differentiation and/or endothelial niche cell differentiation.
  • the kit can include one or more antibodies that bind a cell marker, or primers for an RT-PCR or PCR reaction, e.g., a semi-quantitative or quantitative RT-PCR or PCR reaction.
  • a cell marker e.g., a cell marker that binds to an RT-PCR or PCR reaction.
  • primers for an RT-PCR or PCR reaction e.g., a semi-quantitative or quantitative RT-PCR or PCR reaction.
  • Such components can be used to assess the activation of maturation markers or the loss of immature cell markers of endothelial niche cells and/or HSPCs.
  • the detection reagent is an antibody, it can be supplied in dry preparation, e.g., lyophilized, or in a solution.
  • the antibody or other detection reagent can be linked to a label, e.g., a radiological, fluorescent (e.g., GFP) or colorimetric label for use in detection.
  • the detection reagent is a primer
  • it can be supplied in dry preparation, e.g., lyophilized, or in a solution.
  • the kit will typically be provided with its various elements included in one package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a Styrofoam box.
  • the enclosure can be configured 28 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT so as to maintain a temperature differential between the interior and the exterior, e.g., it can provide insulating properties to keep the reagents at a preselected temperature for a preselected time.
  • vectors In some embodiments, one or more of the factors described herein is expressed in a recombinant expression vector or plasmid.
  • the term “vector” refers to a polynucleotide sequence suitable for transferring transgenes into a host cell.
  • the term “vector” includes plasmids, mini-chromosomes, phage, naked DNA and the like. See, for example, U.S. Pat. Nos.4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).
  • vectors refers to a circular double stranded DNA loop into which additional DNA segments are ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments are ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” is used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • a cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence can be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as an RNA transcript.
  • Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase, luciferase or 29 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • the vectors used herein are capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
  • a variety of transcription control sequences e.g., promoter/enhancer sequences
  • the promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene.
  • the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene.
  • conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule.
  • the precise nature of the regulatory sequences needed for gene expression can vary between species or cell types, but in general can include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5′ non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5′ leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. [00159] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA).
  • RNA heterologous DNA
  • the vector is pME Gateway vector (InvitrogenTM). In some embodiments, the vector is p5E GatewayTM vector. In some other embodiments, the vector is pGEX2TKTM vector. In some other embodiments, the vector is TOPO-TATM vector. [00161] Without limitations, the genes described herein can be included in one vector or separate vectors.
  • ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 can be included in the same vector.
  • at least of ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 can be included in a second vector.
  • ETV2 is included in a first vector and the at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 are be included in a second vector.
  • ETV2 is included in a first vector, and the at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 are included in a second vector and third vector.
  • the promoter operably linked to the gene(s) can be zebrafish ubi promoter.
  • one or more of the recombinantly expressed gene can be integrated into the genome of the cell.
  • a nucleic acid molecule that encodes the enzyme of the claimed invention can be introduced into a cell or cells using methods and techniques that are standard in the art.
  • nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule encoding the enzymes of the claimed invention also may be accomplished by integrating the nucleic acid molecule into the genome.
  • “reduce,” “reduction” or “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 or agent) 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. [00171] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean 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, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a “increase” is a statistically significant increase in such level.
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “individual,” “patient” and “subject” are used interchangeably herein. [00173]
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than 32 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT humans can be advantageously used as subjects that represent animal models of myelofibrosis or a myeloproliferative disorder. A subject can be male or female.
  • EMH Extramedullary hematopoiesis
  • EMH is typically a compensatory mechanism that arises in response to certain pathological conditions where the bone marrow's ability to produce blood cells is impaired or insufficient.
  • EMH can be associated with diseases that affect the bone marrow, such as myelofibrosis, leukemia, and other myeloproliferative disorders.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. EMH) or one or more complications related to such a condition, and optionally, have already undergone treatment for EMH or the one or more complications related to EMH.
  • a subject can also be one who has not been previously diagnosed as having EMH or one or more complications related to EMH.
  • a subject can be one who exhibits one or more risk factors for EMH, or one or more complications related to EMH or a subject who does not exhibit risk factors.
  • a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein refers to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and polypeptide are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • protein and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. [00178] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and/or conservative substitution variants of any of the particular polypeptides described are encompassed.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide.
  • conservatively modified variants are in 33 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.
  • a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn).
  • Other such conservative substitutions e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. transcription factor activity and specificity of a native or reference polypeptide is retained.
  • Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).
  • Naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.
  • the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein.
  • a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein.
  • a functional fragment can comprise conservative substitutions of the sequences disclosed herein.
  • the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example.
  • a “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions.
  • Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or 34 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity.
  • a wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.
  • a variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).
  • Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art.
  • Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al.
  • cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single-stranded or double-stranded.
  • a single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA.
  • expression refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or 35 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.
  • the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are tissue specific. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are global. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is systemic. [00188] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences.
  • the gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • "Marker” in the context of the present invention refers to an expression product, e.g., nucleic acid or polypeptide which is differentially present in a sample taken from subjects having myelofibrosis or a myeloproliferative disorder, as compared to a comparable sample taken from control subjects (e.g., a healthy subject).
  • the methods described herein relate to measuring, detecting, or determining the level of at least one marker.
  • detecting or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.
  • a polypeptide, nucleic acid, or cell as described herein can be engineered.
  • engineered refers to the aspect of having been manipulated by the hand of man.
  • a polypeptide is considered to be “engineered” when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature.
  • progeny of an engineered cell is typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • the HSPCs, engineered endothelial niche cells, and/or transcription factors e.g., ETV2, and at least two of TFEC, MAFB, FOXP4, HOXB8, or IRF8 described herein is exogenous.
  • the HSPCs, engineered endothelial niche cells, and/or transcription factors e.g., ETV2, and at least two of TFEC, MAFB, FOXP4, HOXB8, or IRF8 described herein is ectopic.
  • 36 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT the HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two of TFEC, MAFB, FOXP4, HOXB8, or IRF8) described herein is not endogenous.
  • exogenous refers to a substance present in a cell other than its native source.
  • exogenous when used herein can refer to a nucleic acid (e.g.
  • nucleic acid encoding a polypeptide or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism.
  • exogenous can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.
  • ectopic refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment. [00194] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g.
  • a ETV2, TFEC, MAFB, FOXP4, HOXB8, or IRF8 polypeptide is comprised by a vector.
  • a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof is operably linked to a vector.
  • the term "vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
  • a vector can be viral or non-viral.
  • the term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells.
  • a vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
  • the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species.
  • the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).
  • a non-native genetic control element e.g., a promoter, suppressor, activator, enhancer, response element, or the like.
  • the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or 37 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT translated at an improved efficiency in a desired expression system.
  • the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism).
  • the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell.
  • the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.
  • expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. EMH.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with EMH. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced.
  • treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are 38 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
  • “contacting” refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid.
  • Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.
  • the singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.
  • compositions 41 4867-3387-1598.2
  • Attorney Docket No: 701039-000138WOPT functions and concepts of the above references and application to provide yet further embodiments of the disclosure.
  • some changes can be made in protein structure without affecting the biological or chemical action in kind or amount.
  • the at least two transcription factors are TFEC and MAFB.
  • the cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a placenta stem cell, an adult stem cell, an amniotic stem cell, and an umbilical vein endothelial cell.
  • ESC embryonic stem cell
  • iPSC induced pluripotent stem cell
  • a placenta stem cell an adult stem cell
  • amniotic stem cell an umbilical vein endothelial cell.
  • ETV2 and the at least two transcription factors are expressed from at least one vector. 6.
  • the at least one vector comprises an exogenous nucleic acid sequence(s) encoding the ETV2 and the at least two transcription factors. 7. The method of any of the preceding paragraphs, wherein expression is transient or stable. 8. The method of any of the preceding paragraphs, wherein the exogenous nucleic acid sequence(s) is incorporated into the genome of the endothelial cell. 9. The method of any of the preceding paragraphs, wherein the cell is a mammalian cell. 10. The method of any of the preceding paragraphs, wherein the cell is a human cell. 42 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 11.
  • the cell is a nonhuman mammalian cell.
  • the engineered endothelial niche cells secrete at least one of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO.
  • the engineered endothelial niche cells express at least one of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, and CD62E. 14.
  • a method for engineering endothelial niche cells comprising expressing in a cell ETV2, TFEC, and MAFB. 15.
  • An engineered endothelial niche cell obtained by any of the preceding paragraphs.
  • An engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8.
  • An engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB.
  • a population of cells comprising any of the engineered endothelial niche cells of any of the preceding paragraphs. 19.
  • a co-culture comprising any of the engineered endothelial niche cells of any of the preceding paragraphs or population of any of the preceding paragraphs and a population of stem cells.
  • 21. A method for increasing stem cell proliferation, the method comprising co-culturing any of the engineered endothelial niche cells of any of the preceding paragraphs or population of any of the preceding paragraphs and a population of stem cells for a time sufficient in increase stem cell proliferation. 22. The method of any of the preceding paragraphs, wherein proliferation is increased by at least 10% as compared to an appropriate control. 23.
  • the population of stem cells is a population of HSPCs. 24. The method of any of the preceding paragraphs, wherein the method is performed in vitro. 25. The method of any of the preceding paragraphs, wherein the engineered endothelial niche cells secrete a factor that affects the proliferation of the HSPC cells. 43 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 26.
  • a method for treating a subject comprising administering any of the engineered endothelial niche cells of any of the preceding paragraphs, population of any of the preceding paragraphs, or co-culture of any of the preceding paragraphs into a subject in need thereof. 27.
  • a method for treating a subject comprising: a. identifying a subject in need thereof; and b. administering any of the engineered endothelial niche cells of any of the preceding paragraphs, population of any of the preceding paragraphs, or co-culture of any of the preceding paragraphs into the subject in need thereof.
  • 28. A method for treating a subject, the method comprising administering the co-culture of any of the preceding paragraphs into a subject in need thereof.
  • 29. A method for treating a subject, the method comprising administering a population of HSPCs that have been previously co-cultured any of the engineered endothelial niche cells of any of the preceding paragraphs or population of any of the preceding paragraphs into a subject in need thereof.
  • the method of any of the preceding paragraphs comprising the step of isolating the population of HSPCs prior to administering. 31.
  • the method of any of the preceding paragraphs, wherein the subject in need thereof is human.
  • the method of any of the preceding paragraphs, wherein the subject in need thereof has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level.
  • 33. The method of any of the preceding paragraphs, further comprising the step of identifying a subject in need thereof having a decreased blood cell level or at risk for developing a decreased blood cell level as compared to a control blood cell level prior to administering. 34.
  • the method of any of the preceding paragraphs wherein the blood cell level is decreased at least 1% compared to a reference level. 35. The method of any of the preceding paragraphs, wherein the subject in need thereof has anemia or blood loss. 36. The method of any of the preceding paragraphs, wherein the subject in need thereof is a bone marrow donor. 37. The method of any of the preceding paragraphs, wherein the subject in need thereof has depleted bone marrow. 38. The method of any of the preceding paragraphs, the subject in need thereof has anemia, hemolysis, leukemia, multiple myeloma, or a thyroid disorder. 44 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 39.
  • any of the preceding paragraphs wherein the administering occurs at the liver, spleen, or subcutaneously.
  • a method for generating an ectopic vascular niche comprising administering any of the engineered endothelial niche cells of any of the preceding paragraphs, population of any of the preceding paragraphs, or co-culture of any of the preceding paragraphs to a target site in a subject in need thereof. 41.
  • a method for treating extra medullary hematopoiesis comprising administering any of the engineered endothelial niche cells of any of the preceding paragraphs, population of any of the preceding paragraphs, or co-culture of any of the preceding paragraphs into a subject at a location outside of the bone marrow, thereby creating a synthetic niche.
  • administering is systemic or local administration.
  • a vector comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 operably linked to a promoter.
  • a vector comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB.
  • EXAMPLES EXAMPLE 1 [00219] Specialized vascular endothelial cells called venous sinusoids support the growth and maintenance of hematopoietic stem and progenitor cells (HSPCs).
  • HSPCs hematopoietic stem and progenitor cells
  • the transcriptional program of these endothelial cells in determining their HSPC niche supportive programs remains unclear.
  • the inventors performed differential gene expression analyses using sinusoidal endothelial cells from the adult zebrafish kidney marrow HSPC niche and from the adult mouse bone marrow HSPC niche and compared these to adult liver sinusoidal endothelial cells that do not support hematopoiesis.
  • the inventors identified transcription factors that are uniquely upregulated in the endothelial cells within the HSPC niches.
  • liver endothelial cell-specific enhancer to drive the overexpression of 45 4867-3387-1598.2
  • Attorney Docket No: 701039-000138WOPT transcription factors tfec and mafbb they achieved the transcriptional reprogramming of the liver endothelial cells to adopt HSPC niche supportive fate.
  • These reprogrammed liver vascular endothelial cells enabled the homing and maintenance of primary zebrafish HSPCs with long-term repopulating stem-cell potential.
  • the findings uncovered new knowledge about the transcription regulation of the endothelial cells within the HSPC niche and may lead to novel approaches to create synthetic HSPC niches through in vivo reprogramming.
  • Hematopoietic stem and progenitor cells contribute to the multilineage regeneration of blood and immune cells in the blood system (Orkin and Zon, 2008; Sawai et al., 2016).
  • HSPCs reside in specialized vascular microenvironments known as the HSPC niches, and the endothelial cells in the sinusoidal vasculature are the most abundant and one of the most important components of the HSPC niches (Morrison and Scadden, 2014; Wei and Frenette, 2018; Pinho and Frenette, 2019; Comazzetto et al., 2021).
  • vascular endothelial cells and other perivascular cells provide cell surface adhesion proteins and secreted factors that support the maintenance and homeostasis of the HSPCs occupying such vascular niche.
  • HSCs haematopoietic stem cells
  • SCF Stem cell factor
  • HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, nestin-cre- or nestin-creER-expressing cells.
  • HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or leptin receptor (Lepr)- expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells.
  • HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance.
  • the self-renewal of hematopoietic stem cells is regulated by the bone marrow microenvironment.
  • Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression.
  • cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.
  • Bone marrow (BM) perivascular stromal cells and vascular endothelial cells (ECs) are essential for hematopoietic stem cell (HSC) maintenance, but the roles of distinct niche compartments during HSC regeneration are less understood.
  • HSC hematopoietic stem cell
  • LepR+ Leptin receptor-expressing BM stromal cells and ECs dichotomously regulate HSC maintenance and regeneration via secretion of pleiotrophin (PTN).
  • PTN pleiotrophin
  • HSPCs transplanted hematopoietic stem and progenitor cells
  • Monocyte–dendritic cell progenitors map with nonclassical monocytes and conventional dendritic cells; these localize to a subset of blood vessels expressing a major regulator of myelopoiesis, colony-stimulating factor 1 (CSF1, also known as M-CSF)1.
  • CSF1 colony-stimulating factor 1
  • the vascular HSPC niches in the fetal liver and in the adult bone marrow are the primary HSPC niches in the mammalian systems.
  • the liver maintains hematopoietic stem cells (HSCs) during development.
  • HSCs hematopoietic stem cells
  • SCF stem cell factor
  • the zebrafish liver is also rich in sinusoidal vessels, but the endothelial cells in the zebrafish liver do not support hematopoiesis under normal physiological conditions.
  • Prior studies of the vascular HSPC niche in the zebrafish embryonic caudal hematopoietic tissue (CHT) suggested that transcription factors control the fate of these vascular endothelial cells to support HSPCs (Mahony et al., 2016; Xue et al., 2017; Hagedorn et al., 48 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 2023).
  • the transcription factor control of vascular endothelial cells in the adult HSPC niches has not been fully elucidated.
  • the inventors study the transcriptional programs of the sinusoidal vascular endothelial cells in the zebrafish kidney marrow HSPC niche and in the mammalian bone marrow HSPC niche and identify unique transcription factors that control the fate of these endothelial cells to support HSPCs.
  • the inventors study the venous sinusoidal endothelial cells in the adult livers, which have the same venous sinusoidal programs but lack the HSPC niche supportive programs.
  • adult zebrafish as a model, they design synthetic gene expression circuits to drive the overexpression of transcription factors tfec and mafbb in the liver endothelial cells.
  • the transcription factor overexpression is sufficient to induce the fate of adult zebrafish liver endothelial cells to adopt transcriptional programs that modulate HSPC niche recruitment and function.
  • the data presented herein provide functional insights into the transcription factor control of the vascular endothelial cells in the HSPC niches.
  • pan-endothelial cell genes cdh5, kdrl, and pecam1 are expressed by endothelial cells from both organs (Figure 1B), and sinusoidal endothelial cell genes sele, flt4, and gpr182 are expressed by the sinusoidal endothelial cells from both organs ( Figure 1C).
  • Previously identified liver sinusoidal endothelial cell genes gata4 and f8 are exclusively expressed in the liver sinusoidal endothelial cells in the data presented herein ( Figure 1D).
  • Transcription factors are known to regulate transcriptional programs and thus govern cell fate, and prior studies in the field have revealed that transcription factors regulate the endothelial cells in the zebrafish embryonic CHT niche (Mahony et al., 2016; Xue et al., 2017; Hagedorn et al., 2023).
  • the putative HSPC niche supportive genes in the adult zebrafish kidney marrow endothelial cells are consistent with the inventors’ previous study identifying the HSPC niche supportive genes in the embryonic zebrafish CHT endothelial cells (Hagedorn et al., 2023).
  • the inventors aimed to validate that these transcription factor candidates are indeed regulating the endothelial cell fate to function as a HSPC niche. Therefore, the inventors pursued differential gene expression analysis between the adult mouse endothelial cells isolated from the bone marrow, the liver, and the kidney, respectively, in public datasets from the literature (Kalucka et al., 2020; Fang et al., 2020).
  • pan-endothelial cell marker genes Cdh5, Kdr, and Pecam1 are expressed by endothelial cells from all three organs (Figure 2A); and sinusoidal endothelial cell marker genes Stab2, Flt4, and Gpr182 are expressed highly by the sinusoidal endothelial cells in the bone marrow and in the liver ( Figure 2B).
  • Organ-specific endothelial cell enhancer elements in the adult zebrafish liver [00232] To identify cis-regulatory enhancer elements that are specific to the adult zebrafish liver endothelial cells, the inventors pursued Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on the endothelial cells and non-endothelial cells from the adult zebrafish kidney marrow and the adult zebrafish liver. The inventors identified putative enhancer peaks that are uniquely accessible to the adult zebrafish liver endothelial cells, in contrast to other liver cells or the kidney marrow endothelial cells.
  • the inventors aimed to overexpress these transcription factor candidates in the zebrafish liver endothelial cells, which do not normally support HSPCs.
  • the rca2.2 enhancer allowed the inventors to achieve specific overexpression of these transcription factor candidates in the adult zebrafish liver endothelial cells. They generated stable transgenic zebrafish lines that utilized the rca2.2 enhancer to drive the transcription factor candidates in the background of the inventors recently published mrc1a:GFP transgenic reporter line.
  • the mrc1a:GFP reporter specifically labels the endothelial cells in the HSPC niches, including the embryonic zebrafish CHT (Hagedorn et al., 2023) and the adult zebrafish kidney marrow niche (Figure 4A).
  • the inventors found that the overexpression of transcription factors tfec and mafbb can induce a robust ectopic expression of the mrc1a:GFP reporter in the liver endothelial cells (Figure 4B), indicating that these endothelial cells might be reprogrammed into a transcriptional state similar to that of the endothelial cells in the HSPC niches.
  • RNA-sequencing on the endothelial cells of the reprogrammed liver (ectopic mrc1a:GFP+ cells), the wildtype liver (rca2.2:GFP+ or cdh5:GFP+ cells), and the wildtype kidney marrow (mrc1a:GFP+ or cdh5:GFP+ cells) from adult zebrafish.
  • liver endothelial cells with tfec and mafbb overexpression maintained their identity as liver sinusoidal endothelial cells, confirmed by their expression of pan-endothelial (cdh5, kdrl, pecam1, and etv2), venous-sinusoidal (sele, flt4, and gpr182), and liver endothelial signature genes (gata4 and rca2.2) ( Figure 4C).
  • pan-endothelial cdh5, kdrl, pecam1, and etv2
  • venous-sinusoidal sele, flt4, and gpr182
  • liver endothelial signature genes gata4 and rca2.2
  • the inventors confirm that the tfec and mafbb overexpression reprogrammed liver endothelial cells to express HSPC niche supportive genes at the transcriptional level. [00236] To further examine the sufficiency of either transcription factors tfec or mafbb in inducing the changes in the HSPC niche supportive fate in the reprogrammed liver endothelial cells, the inventors conducted experiments on each individual transcription factor overexpression transgenic zebrafish.
  • tfec and mafbb are both required to induce an efficient reprogramming of the liver endothelial cells to a HSPC niche supportive state at the transcriptional level.
  • HSPCs home to the reprogrammed liver vascular endothelial cell niche
  • the inventors aimed to functionally validate HSPC homing and maintenance in the reprogrammed liver vascular HSPC niche.
  • the inventors sought to confirm that the inventors’ reprogramming strategy conferred functional support for the HSPCs.
  • Differential gene expression analyses between the endothelial cells from hematopoietic organs and from the non-hematopoietic organs across different species enable the inventors to identify the conserved set of transcription factor candidates that are upregulated specifically in the endothelial cells that constitute HSPC niches.
  • the inventors are able to drive adult zebrafish liver endothelial-cell specific expression using validated enhancer elements. This strategy is useful to achieve tissue-specific expression of genes of interest in the field.
  • liver endothelial cell-specific enhancer By utilizing a liver endothelial cell-specific enhancer to overexpress the transcription factor candidates tfec and mafbb, the inventors are able to transcriptionally reprogram the liver endothelial cells to express HSPC niche supportive genes.
  • the reprogrammed liver vascular endothelial cells generated a synthetic ectopic HSPC niche that is able to recruit and maintain primary HSPCs. Therefore, the inventors’ work provides a novel understanding of the transcriptional control of the HSPC niche supportive programs of the endothelial cells from hematopoietic organs and establishes a novel approach to create synthetic HSPC niches through in vivo reprogramming.
  • Endothelial cells are known to have complex heterogeneity and plasticity across different tissues of an organism.
  • transcriptional analyses of the endothelial cells from the adult zebrafish kidney marrow HSPC niche and from the adult mouse bone marrow HSPC niche the inventors are able to generate an evolutionarily conserved list of 103 significantly upregulated genes, including previously validated genes such as Sele, Cxcl12, Vcam1, and Osmr.
  • a potential alternative approach to generate human bone marrow-like endothelial cells could be human induced pluripotent stem cells (iPSCs). Human iPSCs hold great potential in the scalable production of desired somatic cell types (Rowe and Daley, 2019; Ng et al., 2021).
  • the transcription factor candidates were identified from differential gene expression analyses between the endothelial cells from hematopoietic organs and the endothelial cells from non-hematopoietic organs across different species, previously described in Chapter Two.
  • the inventors engineered stable piggyBac transgenic human iPSC lines with doxycycline-inducible overexpression of selective transcription factor candidates, allowing us to temporally modulate the activation of these transcription factor expression.
  • This inducible overexpression approach was effective in modulating the transcription factor expression without significantly perturbing the endothelial cell differentiation, and the strategy could be useful to achieve temporally controlled expression of genes of interest in the hiPSC-derived endothelial cells or other somatic cell types in the field.
  • TFEC and MAFB By overexpressing the transcription factors TFEC and MAFB, the inventors were able to induce a transcriptional program in the human iPSC-derived endothelial cells that resembles primary human bone marrow endothelial cells and upregulated previously reported HSPC niche supportive genes. Overexpression of TFEC and MAFB is also sufficient in inducing a functional program in the human iPSC-derived endothelial cells, enabling them to better support the differentiation capacities and engraftment potential of the primary human cord blood CD34+ HSPCs in the endothelial cell co-culture.
  • the scientific rationale for the design is to better focus on the potential downstream targets of each of the single transcription factor candidate.
  • the inventors were also challenged technically by the piggyBac transgenic 60 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT human iPSC generation, during which the inventors used a different drug selection for each of the transgene vectors.
  • ETV2, TFEC, and MAFB are independently delivered by three separate transgene vectors, requiring three different drug resistance genes to fully select for successfully integrated human iPSCs with all three overexpression vectors.
  • the inventors are establishing new transgenic human iPSC vectors with integrated expression systems for multiple transcription factors, such as multicistronic expression vectors using IRES elements or 2A self- cleavage peptides. This approach would allow them to screen for additional combinations of transcription factors, in addition to TFEC and MAFB, that may further improve the induction of HSPC niche supportive genes in the human iPSC-derived endothelial cells and stimulate better functional support of the primary human HSPCs in the endothelial cell co-culture.
  • the inventors’ current endothelial cell differentiation protocol involves doxycycline-inducible expression of the pan-endothelial master transcription factor ETV2 and the transcription factor candidates that upregulate HSPC niche supportive programs in the endothelial cells.
  • the doxycycline-inducible overexpression of these transcription factors was activated during mesodermal progenitor to endothelial differentiation steps and was maintained throughout the period of the endothelial cell and HSPC co-culture time window for continued expression of the transcriptional programs in the endothelial cells.
  • the inventors performed preliminary experiments on the long-term culture of these hiPSC-derived endothelial cells in commercially available endothelial cell growth media, and the endothelial cells maintained their endothelial cell identity without continued doxycycline-inducible expression of the pan-endothelial master transcription factor ETV2.
  • ETV2 pan-endothelial master transcription factor
  • Perivascular mesenchymal stromal cells have also been reported to support primary HSPCs in in vitro co-culture systems (Pinho et al., 2013; Nakahara et al., 2019; Chou et al., 2020).
  • the dynamics of the cell-cell interactions between HSPCs, vascular endothelial cells, and perivascular mesenchymal stromal cells will be an area of interest for further investigation. It is specifically contemplated herein that the HSPC co-culture system with both vascular endothelial cells and perivascular mesenchymal stromal cells would further improve the maintenance and expansion of the primary human HSPCs in vitro.
  • the human iPSCs were passaged to colonies with ReLeSR (STEMCELL Technologies) regularly to prevent overgrowth and spontaneous differentiation.
  • the human iPSCs were maintained in a humidified atmosphere at 37 °C temperature level and 5% CO 2 level.
  • Generation of new transgenic cell lines [00279] Human iPSCs were dissociated into single-cell suspension using TrypLE Select Enzyme (Gibco). The dissociated human iPSCs were mixed with the transcription factor overexpression vectors and the hyperactive piggyBac transposase vector, followed by nucleofection using Cell Line Nucleofector Kit V (Lonza).
  • the nucleofected cells were cultured in StemFlex pluripotent stem cell maintenance media (Gibco) supplemented with CloneR2 (STEMCELL Technologies) or ROCK Inhibitor Y-27632 (STEMCELL Technologies) to improve the viability of single-cell dissociated human iPSCs.
  • Blasticidin, Neomycin, and/or Zeocin were added as previously described in the literature (Holmqvist et al., 2015; Libby et al., 2018; Blanch-Asensio et al., 2022) to select for human iPSCs that are successfully transfected with the transcription factor overexpression vectors with drug resistance genes.
  • Endothelial cell differentiation from human iPSCs [00281] The endothelial cell differentiation protocol was adapted from a previously published protocol (K. Wang et al., 2020), with a modified doxycycline-inducible transcription factor induction method. On day 0, The human iPSCs were dissociated into single-cell suspension using StemPro Accutase Cell Dissociation Reagent (Gibco) and seeded onto Matrigel-coated tissue culture plate at a density of 30,000 cells/cm 2 in StemFlex pluripotent stem cell maintenance media (Gibco) supplemented with CloneR2 (STEMCELL Technologies) or ROCK Inhibitor Y-27632 (STEMCELL Technologies).
  • StemPro Accutase Cell Dissociation Reagent StemPro Accutase Cell Dissociation Reagent (Gibco) and seeded onto Matrigel-coated tissue culture plate at a density of 30,000 cells/cm 2 in Stem
  • the cell culture media was replenished daily with mesoderm induction media.
  • Mesoderm induction media was made with Advanced DMEM/F-12 (Thermo Fisher), 1x GlutaMax supplement (Thermo Fisher), 60 ⁇ g/ml L-Ascorbic Acid (Sigma-Aldrich), and 6 ⁇ M CHIR99021 (Sigma-Aldrich).
  • the mesoderm progenitor cells are dissociated into single-cell suspension using StemPro Accutase Cell Dissociation Reagent (Gibco) and seeded onto Matrigel-coated tissue culture plate at a density of 30,000 cells/cm2 in endothelial cell differentiation media.
  • Endothelial cell differentiation media was made with Advanced DMEM/F-12 (Thermo Fisher), 1x GlutaMax supplement (Thermo Fisher), 60 ⁇ g/mL L-Ascorbic Acid (Sigma-Aldrich), 50 ng/mL FGF-2 (PeproTech), 10 ng/uL EGF (PeproTech), 50 ng/mL VEGF-A (PeproTech), 10 ⁇ M SB431542 (Selleckchem), and 1 ⁇ g/ml Doxycycline (Sigma-Aldrich). On day 4, The cell culture was replenished with fresh endothelial cell differentiation media.
  • Endothelial cells are collected on day 5 of the endothelial cell differentiation protocol. The cells were dissociated into single-cell suspension using StemPro Accutase Cell Dissociation Reagent (Gibco). For flow analyses and fluorescence-activated cell sorting (FACS), the dissociated cells were incubated with BD Pharmingen Alexa Fluor 647 Mouse Anti-Human CD144 (BD Biosciences) at 1:100 dilution for 15 minutes at room temperature in the dark.
  • StemPro Accutase Cell Dissociation Reagent Gibco
  • FACS fluorescence-activated cell sorting

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Vascular Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Developmental Biology & Embryology (AREA)
  • Materials For Medical Uses (AREA)
  • Display Devices Of Pinball Game Machines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The technology described herein relates to compositions and methods of generating endothelial niche cells. Embodiments of the technology described herein comprise compositions, kits, vectors, and methods related to generating or engineering endothelial niche cells.

Description

Attorney Docket No: 701039-000138WOPT ENDOTHELIAL CELL FACTORS AND METHODS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/546,653 filed October 31, 2023, the contents of which are incorporated herein by reference in their entirety. GOVERNMENT SUPPORT [0002] This invention was made with government support under Grant No. DK120535 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] The technology described herein relates to compositions and methods of generating endothelial niche cells. BACKGROUND [0004] Haematopoietic stem and progenitor cells (HSPCs) are a rare cell population capable of reconstituting the entire blood system after transplantation. As the functional unit of a bone marrow transplant, these cells offer a curative treatment for many blood and immune diseases. Unfortunately, transplantation is not a viable treatment option for many individuals, particularly those lacking an immune-matched donor. A long-term goal of hematological research has been to culture and expand HSPCs in vitro, for use in transplantation and/or genetic modification. While umbilical cord blood- derived HSPCs are somewhat amenable to in vitro expansion, maintaining and inducing self-renewal of adult-derived HSPCs, in the absence of niche signals, has proven challenging. [0005] Strategies aimed at in vitro expansion have co-cultured HSPCs with supportive cells in an effort to recapitulate aspects of the microenvironment or ‘niche’ that supports HSPCs in vivo. In the adult bone marrow, multiple cell types are thought to collectively comprise the HSPC niche, with primary contributors being endothelial cells (ECs) and perivascular mesenchymal stromal cells. Different endothelial cell subtypes in the bone marrow can differentially regulate HSPC homeostasis. Arterial ECs (AECs) are less permeable and are believed to promote HSPC quiescence, while sinusoidal ECs (SECs) are leaky and support the differentiation and mobilization of HSPCs. In addition, during haematopoietic recovery after myelosuppression, ECs play a critical role in niche reconstruction and reconstitution of multi-lineage hematopoiesis. HSPCs can also be supported outside the bone marrow, during embryonic development and under stress conditions that induce 1 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT extramedullary hematopoiesis in tissues such as the liver, spleen and skull. As in the bone marrow, ECs are thought to function as critical, core components of the HSPC niches in these tissues. [0006] Researchers have focused on the development of in vitro cultures where HSPCs can be grown in the lab with other cell types that support the maintenance or expansion of the HSPCs for subsequent use in transplantation. To date, however, these in vitro cultures have been only modestly successful. SUMMARY [0007] One aspect provided herein is describes a method for engineering endothelial niche cells, the method comprising expressing in a cell ETV2; and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. The one embodiment of this or any aspect herein, the at least two transcription factors are TFEC and MAFB. [0008] The one embodiment of this or any aspect herein, the cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a placenta stem cell, an adult stem cell, an amniotic stem cell, and an umbilical vein endothelial cell. The one embodiment of this or any aspect herein, the ESC, iPSC, placenta stem cell, adult stem cell, amniotic stem cell is differentiated to an endothelial cell prior to contact. [0009] The one embodiment of this or any aspect herein, ETV2 and the at least two transcription factors are expressed from at least one vector. The one embodiment of this or any aspect herein, the at least one vector comprises an exogenous nucleic acid sequence(s) encoding the ETV2 and the at least two transcription factors. The one embodiment of this or any aspect herein, expression is transient or stable. [0010] The one embodiment of this or any aspect herein, the exogenous nucleic acid sequence(s) is incorporated into the genome of the endothelial cell. [0011] The one embodiment of this or any aspect herein, the cell is a mammalian cell. The one embodiment of this or any aspect herein, the cell is a human cell. The one embodiment of this or any aspect herein, the cell is a nonhuman mammalian cell. [0012] The one embodiment of this or any aspect herein, the engineered endothelial niche cells secrete at least one of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO. [0013] The one embodiment of this or any aspect herein, the engineered endothelial niche cells express at least one of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, and CD62E. [0014] Another aspect provided herein is describes a method for engineering endothelial niche cells, the method comprising expressing in a cell ETV2, TFEC, and MAFB. 2 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0015] Another aspect provided herein is describes an engineered endothelial niche cell obtained by any of the methods described herein. [0016] Another aspect provided herein is describes an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. [0017] Another aspect provided herein is describes an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. [0018] Another aspect provided herein is describes a population of cells comprising any of the engineered endothelial niche cells described herein. [0019] Another aspect provided herein is describes a co-culture comprising any of the engineered endothelial niche cells described herein or population described herein, and a population of stem cells. The one embodiment of this or any aspect herein, the second population of stem cells is an HSPCs. [0020] Another aspect provided herein is describes a method for increasing stem cell proliferation, the method comprising co-culturing any of the engineered endothelial niche cells described herein or population described herein, and a population of stem cells for a time sufficient in increase stem cell proliferation. [0021] The one embodiment of this or any aspect herein, proliferation is increased by at least 10% as compared to an appropriate control. [0022] The one embodiment of this or any aspect herein, the population of stem cells is a population of HSPCs. [0023] The one embodiment of this or any aspect herein, the method is performed in vitro. [0024] The one embodiment of this or any aspect herein, the engineered endothelial niche cells secrete a factor that affects the proliferation of the HSPC cells. [0025] Another aspect provided herein is describes a method for treating a subject, the method comprising administering any of the engineered endothelial niche cells described herein, population described herein, or co-culture described herein into a subject in need thereof. [0026] Another aspect provided herein is describes a method for treating a subject, the method comprising: identifying a subject in need thereof; and administering any of the engineered endothelial niche cells described herein, population described herein, or co-culture described herein into the subject in need thereof. [0027] Another aspect provided herein is describes a method for treating a subject, the method comprising administering the co-culture described herein into a subject in need thereof. [0028] Another aspect provided herein is describes a method for treating a subject, the method comprising administering a population of HSPCs that have been previously co-cultured any of the engineered endothelial niche cells described herein or population of described herein into a subject in need thereof. 3 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0029] The one embodiment of this or any aspect herein, the method further comprises the step of isolating the population of HSPCs prior to administering. [0030] The one embodiment of this or any aspect herein, subject in need thereof is human. The one embodiment of this or any aspect herein, the subject in need thereof has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level. [0031] The one embodiment of this or any aspect herein, the method further comprises the step of identifying a subject in need thereof having a decreased blood cell level or at risk for developing a decreased blood cell level as compared to a control blood cell level prior to administering. The one embodiment of this or any aspect herein, the blood cell level is decreased at least 1% compared to a reference level. [0032] The one embodiment of this or any aspect herein, the subject in need thereof has anemia or blood loss. The one embodiment of this or any aspect herein, the subject in need thereof is a bone marrow donor. The one embodiment of this or any aspect herein, the subject in need thereof has depleted bone marrow. The one embodiment of this or any aspect herein, the subject in need thereof has anemia, hemolysis, leukemia, multiple myeloma, or a thyroid disorder. [0033] The one embodiment of this or any aspect herein, the administering occurs at the liver, spleen, or subcutaneously. [0034] Another aspect provided herein is describes a method for generating an ectopic vascular niche, the method comprising administering any of the engineered endothelial niche cells described herein, population described herein, or co-culture described herein to a target site in a subject in need thereof. [0035] Another aspect provided herein is describes a method for treating extra medullary hematopoiesis, the method comprising administering any of the engineered endothelial niche cells described herein, population described herein, or co-culture described herein into a subject at a location outside of the bone marrow, thereby creating a synthetic niche. [0036] The one embodiment of this or any aspect herein, administering is systemic or local administration. [0037] The one embodiment of this or any aspect herein, local administration is transplantation. The one embodiment of this or any aspect herein, local administration is administration directly to the liver, spleen, or subcutaneously. [0038] Another aspect provided herein is describes a vector comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 operably linked to a promoter. [0039] Another aspect provided herein is describes a vector comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. BRIEF DESCRIPTION OF THE DRAWINGS 4 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0040] Figs 1A-1F present exemplary data showing single-cell transcriptional profiling of the endothelial cells from the adult zebrafish kidney marrow and from the adult zebrafish liver. (Fig.1A) Isolation of endothelial cells from transgenic kdrl:mCherry endothelial cell reporter zebrafish; (Fig.1B) Pan-endothelial cell marker genes cdh5, kdrl, and pecam1 are expressed by endothelial cells from both organs; (Fig.1C) Sinusoidal endothelial cell marker genes sele, flt4, and gpr182, are expressed by the sinusoidal endothelial cells from both organs; (Fig.1D) Liver sinusoidal endothelial cell marker genes gata4 and f8 are exclusively expressed by the sinusoidal endothelial cells from the adult zebrafish liver; (Fig.1E) HSPC niche supportive genes cxcl12a, mrc1a, dab2 and csf1b, are significantly upregulated in the sinusoidal endothelial cells from the adult zebrafish kidney marrow; (Fig.1F) Differential gene expression analysis revealed transcription factor candidates that are uniquely upregulated in the sinusoidal endothelial cells from the adult zebrafish kidney marrow. [0041] Figs 2A-2C present exemplary data showing single-cell RNA-sequencing analyses of adult mouse endothelial cells from the bone marrow, the liver, and the kidney. (Fig.2A) Pan- endothelial marker genes Cdh5, Kdr, and Pecam1 are expressed by endothelial cells from all three mouse organs; (Fig.2B) Sinusoidal endothelial cell marker genes Stab2, Flt4, and Gpr182 are highly expressed by sinusoidal endothelial cells from mouse bone marrow and mouse liver; (Fig.2C) Transcription factors Tfec, Mafb, Foxp4, Hoxb8, and Irf8 are highly expressed by the mouse bone marrow endothelial cells. [0042] Figs 3A-3E present exemplary data showing rca2.2 enhancer drives specific expression in the liver endothelial cells. (Fig.3A) Schematics of cloning putative enhancers into a GFP reporter construct and creating transgenic zebrafish reporter lines; (Fig.3B) Gene tracks show a unique ATAC-seq open chromatin region upstream of rca2.2 gene in the liver endothelial cells; anti-GFP immunohistochemistry on rca2.2:GFP transgenic fish reveals specific expression in the liver vascular endothelial cells; (Fig.3C) Whole-mount immunofluorescence imaging of double transgenic reporter zebrafish liver show overlap between rca2.2:GFP and kdrl:BFP signals in the endothelial cells; (Fig. 3D) Flow analysis shows rca2.2:GFP+ cells are a subset of kdrl:BFP+ endothelial cells from double transgenic zebrafish liver; (Fig.3E) Transcriptional analyses of the kidney marrow endothelial cells isolated from mrc1a:GFP and cdh5:GFP transgenic reporter zebrafish and the liver endothelial cells isolated from rca2.2:GFP and cdh5:GFP transgenic reporter zebrafish. Scale = log2 expression level. Each of the four RNA-sequencing samples is pooled from n=3 animals. [0043] Figs 4A-4G present exemplary data showing transcription factors tfec and mafbb reprogrammed the liver sinusoidal endothelial cells. (Fig.4A) Immunofluorescence staining of mrc1a:GFP and kdrl:mCherry show specific labeling of the endothelial cells in the embryonic zebrafish CHT HSPC niche and in the adult zebrafish kidney marrow HSPC niche; (Fig.4B) Immunofluorescence staining show ectopic expression of mrc1a:GFP in the reprogrammed liver upon tfec and mafbb overexpression; (Fig.4C) Heatmap of RNA-sequencing analysis show tfec and mafbb 5 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT reprogrammed the HSPC niche supportive genes without perturbing the normal expression of liver sinusoidal endothelial cell genes; Scale = log2 expression level; (Fig.4D) Heatmap of RNA- sequencing analysis show tfec and mafbb upregulate putative HSPC niche supportive genes and shifted transcriptional programs toward marrow-like states; Scale = z-score of the expression level (Fig.4E) Volcano plots of RNA-sequencing results show significant upregulation of HSPC niche supportive genes in the reprogrammed liver endothelial cells; (Fig.4F) Flow analyses of the reprogrammed liver with tfec and mafbb co-overexpression show higher percentage of mrc1a:GFP+ cells within the cdh5:mCherry+ endothelial cell population, compared to the reprogrammed liver with mafbb single overexpression; (Fig.4G) Transcriptional analyses using single-cell RNA-sequencing reveal the requirement of both tfec and mafbb in inducing efficient transcriptional changes in the reprogrammed liver endothelial cells. [0044] Figs 5A-AB present exemplary data showing transplant assay of donor liver cells into immunocompromised recipients. (Fig.5A) Schematics of transplanting donor liver cells into immunocompromised recipient zebrafish, and post-transplant engraftment assay by flow cytometry; (Fig.5B) ubi:mCherry+ donor liver cells from the reprogram liver donor animals, but not from the control wildtype liver donor animals, are able to reconstitute the recipient hematopoietic system in their kidney marrows; p = 0.0296 for two-tailed t-test. [0045] Figs 6A-6B present exemplary data showing generation of human iPSC lines with inducible transcription factor overexpression. (Fig.6A) Vector map for generating tetracycline- inducible transcription factor overexpression hiPSC lines; (Fig.6B) Schematics of generating new hiPSC lines by nucleofection, drug selection, and PCR genotyping. [0046] Figs 7A-7B present exemplary data showing directed differentiation of human iPSCs into endothelial cells in vitro. (Fig.7A) Schematics of directing differentiation from human iPSCs into induced ECs. Brightfield images show the standard morphology of the cells at each stage. Flow cytometry plots confirmed that the induced ECs express endothelial cell markers CD144 (VE- Cadherin) and CD31 (PECAM1); (Fig.7B) Flow cytometry plots for induced ECs with overexpression of transcription factor candidates. The transcription factor candidates are tagged with ZsGreen, and the induced EC identity is verified by CD144 (VE-Cadherin). [0047] Figs 8A-8D present exemplary data showing transcription factor overexpression in hiPSC-derived endothelial cells putatively upregulated HSPC niche supportive genes. (Fig.8A) Heatmap of RNA-sequencing analysis show that pan-endothelial cell genes and arteriovenous genes are not significantly changed upon transcription factor overexpression in the induced iECs; Scale = log2 expression level; (Fig.8B) Heatmap of RNA-sequencing analysis shows that putative HSPC niche supportive genes are significantly upregulated in the iECs with TFEC and MAFB overexpression; Scale = z-score of the expression level; (Fig.8C) Heatmap of RNA-sequencing analysis show that primary bone marrow endothelial cell genes are significantly upregulated in the 6 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT iECs with TFEC and MAFB overexpression; Scale = z-score of the expression level; (Fig.8D) Gene ontology enrichment analyses revealed that many of the genes upregulated by TFEC and MAFB in the iECs are involved in cell adhesion, migration, extravasation and stem cell regulation. [0048] Figs 9A-9B present exemplary data showing functional assay of primary human HSPCs co-cultured with TFEC and MAFB overexpression endothelial cells. (Fig.9A) Colony-forming unit (CFU) assay results of post co-cultured HSPCs; HSPCs with ETV2 iECs did not show significant differences compared to HSPCs without EC co-culture; HSPCs with ETV2-TFEC-MAFB ECs showed a significantly greater number of colonies compared to the other two groups; (Fig.9B) Engraftment assay results of post co-cultured HSPCs four months post-transplant; HSPCs with ETV2 iECs did not show significant differences in the percentage of human CD45 detected in the recipient mouse peripheral blood; HSPCs with ETV2-TFEC-MAFB iECs showed a significantly greater percentage of human CD45 detected in the recipient mouse peripheral blood; p = 0.0164 for two- tailed t-test. DETAILED DESCRIPTION Transcription factors [0049] Embodiments of the technology described herein comprise compositions, kits, vectors, and methods related to generating or engineering endothelial niche cells. One aspect comprises a method to generate/engineer endothelial niche cells, comprising expressing in a cell ETV2; and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. In one embodiment, the at least two transcription factors are TFEC and MAFB. [0050] Another aspect provided herein comprises a method to generate/engineer endothelial niche cells, comprising expressing in a cell ETV2, TFEC, and MAFB. [0051] Yet another aspect provided herein is an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. In one embodiment, the at least two transcription factors are TFEC and MAFB. [0052] Yet another aspect provided herein is an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. [0053] Yet another aspect provided herein is a vector comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 operably linked to a promoter. In one embodiment, the at least two transcription factors are TFEC and MAFB. [0054] Yet another aspect provided herein is a vector comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. 7 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0055] In one embodiment, the at least two transcription factors can be at least three, at least four, or at least five of the transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. [0056] As used herein, “ETS variant transcription factor 2 (ETV2)” refers to a protein known to enable sequence-specific double-stranded DNA binding activity. It is involved in cell differentiation and regulation of transcription by RNA polymerase II and acts upstream of or within several processes, including cell surface receptor signaling pathway; positive regulation of endothelial cell differentiation; and positive regulation of macromolecule metabolic process. ETV2 sequences are known for a number of species, e.g., human ETV2 (NCBI Gene ID: 2116) polypeptide (e.g., NCBI Ref Seq XP_005258709.1) and mRNA (e.g., NCBI Ref Seq XM_005258652.3). ETV2 can refer to human ETV2, including naturally occurring variants, molecules, and alleles thereof. ETV2 refers to the mammalian ETV2 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 1 comprises the nucleic sequence which encodes ETV2. The human polypeptide sequence of SEQ ID NO: 2 comprises the polypeptide sequence of ETV2. [0057] As used herein, “transcription factor EC (TFEC)” refers to a protein known as a member of the microphthalmia (MiT) family of basic helix-loop-helix leucine zipper transcription factors. MiT transcription factors regulate the expression of target genes by binding to E-box recognition sequences as homo- or heterodimers, and play roles in multiple cellular processes including survival, growth and differentiation. The encoded protein is a transcriptional activator of the nonmuscle myosin II heavy chain-A gene and may also co-regulate target genes in osteoclasts as a heterodimer with microphthalmia-associated transcription factor. TFEC sequences are known for a number of species, e.g., human TFEC (NCBI Gene ID: 22797) polypeptide (e.g., NCBI Ref Seq NP_001018068.1) and mRNA (e.g., NCBI Ref Seq NM_001018058.3). TFEC can refer to human TFEC, including naturally occurring variants, molecules, and alleles thereof. TFEC refers to the mammalian TFEC of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 3 comprises the nucleic sequence which encodes TFEC. The human polypeptide sequence of SEQ ID NO: 4 comprises the polypeptide sequence of TFEC. [0058] As used herein, “MAF bZIP transcription factor B (MAFB)” refers to a protein known to encoded by this gene is a basic leucine zipper (bZIP) transcription factor that plays an important role in the regulation of lineage-specific hematopoiesis. The encoded nuclear protein represses ETS1- mediated transcription of erythroid-specific genes in myeloid cells. MAFB sequences are known for a number of species, e.g., human MAFB (NCBI Gene ID: 9935) polypeptide (e.g., NCBI Ref Seq NP_005452.2) and mRNA (e.g., NCBI Ref Seq NM_005461.5). MAFB can refer to human MAFB, including naturally occurring variants, molecules, and alleles thereof. MAFB refers to the mammalian MAFB of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic 8 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT sequence of SEQ ID NO: 5 comprises the nucleic sequence which encodes MAFB. The human polypeptide sequence of SEQ ID NO: 6 comprises the polypeptide sequence of MAFB. [0059] As used herein, “forkhead box P4 (FOXP4)” refers to a protein belongs to subfamily P of the forkhead box (FOX) transcription factor family. Forkhead box transcription factors play important roles in the regulation of tissue- and cell type-specific gene transcription during both development and adulthood. FOXP4 sequences are known for a number of species, e.g., human FOXP4 (NCBI Gene ID: 116113) polypeptide (e.g., NCBI Ref Seq NP_ NP_001012426.1) and mRNA (e.g., NCBI Ref Seq NM_ NM_001012426.2). FOXP4 can refer to human FOXP4, including naturally occurring variants, molecules, and alleles thereof. FOXP4 refers to the mammalian FOXP4 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 7 comprises the nucleic sequence which encodes FOXP4. The human polypeptide sequence of SEQ ID NO: 8 comprises the polypeptide sequence of FOXP4. [0060] As used herein, “homeobox B8 (HOXB8)” refers to a member of the Antp homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain. It is included in a cluster of homeobox B genes located on chromosome 17. The encoded protein functions as a sequence-specific transcription factor that is involved in development. HOXB8 sequences are known for a number of species, e.g., human HOXB8 (NCBI Gene ID: 3218) polypeptide (e.g., NCBI Ref Seq NP_076921.1) and mRNA (e.g., NCBI Ref Seq NM_024016.4). HOXB8 can refer to human HOXB8, including naturally occurring variants, molecules, and alleles thereof. HOXB8 refers to the mammalian HOXB8 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 9 comprises the nucleic sequence which encodes HOXB8. The human polypeptide sequence of SEQ ID NO: 10 comprises the polypeptide sequence of HOXB8. [0061] As used herein, “interferon regulatory factor 8 (IRF8)” refers to a transcription factor of the interferon (IFN) regulatory factor (IRF) family. Proteins of this family are composed of a conserved DNA-binding domain in the N-terminal region and a divergent C-terminal region that serves as the regulatory domain. IRF8 sequences are known for a number of species, e.g., human IRF8 (NCBI Gene ID: 3394) polypeptide (e.g., NCBI Ref Seq NP_001350836.1) and mRNA (e.g., NCBI Ref Seq NM_001363907.1). IRF8 can refer to human IRF8, including naturally occurring variants, molecules, and alleles thereof. IRF8 refers to the mammalian IRF8 of, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 11 comprises the nucleic sequence which encodes IRF8. The human polypeptide sequence of SEQ ID NO: 12 comprises the polypeptide sequence of IRF8. Hematopoietic System Development [0062] The development of the haematopoietic system, including the cell populations and molecular pathways, is highly conserved between fish and mammals. HSPCs are born in the aorta-gonad- 9 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT mesonephros (AGM) region and then migrate to a transient fetal niche, the fetal liver in mammals or a vascular plexus in the tail of the fish called the caudal haematopoietic tissue (CHT). HSPCs reside and expand in these developmental sites for several days before migrating to the adult niche – the bone marrow in mammals or the kidney marrow in fish. [0063] The CHT is comprised primarily of low-flow sinusoids surrounded by mesenchymal stromal cells. HSPCs initially colonize the CHT niche by lodging within the vascular plexus and interacting directly with cxcl12a+ stromal cells. In a characteristic vascular remodeling step, endothelial cells (ECs) reorganize to form a supportive pocket around the HSPCs, which together with stromal cells and possibly other cell types, forms a niche for the stem cells (the endothelial cells surrounding the HSPCs can be referred to herein as endothelial niche cells). In mammals and zebrafish, specific signaling molecules, adhesion proteins and transcription factors have been implicated in mediating communication and physical interaction between stem cells and ECs in the niche. Collectively, these studies suggest that ECs within the vascular niches of haematopoietic organs express niche-specific gene programs. To date, however, a comprehensive investigation of the transcriptional circuitry that specifies the niche identity of ECs in the HSPC niche has not been undertaken. Understanding this regulation guides new strategies to improve the efficacy and availability of bone marrow transplantation therapies. Endothelial Niche Cells [0064] As described herein, endothelial niche cells are endothelial cells that provide an instructive niche for the differentiation of HSPCs. Endothelial niche cells are typically found in the bone marrow. However, as described herein, exogenous expression of specific transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) can cause endothelial niche cells to be found in non-bone marrow tissues, thus providing for extramedullary hematopoiesis. [0065] In some embodiments of any of the aspects, endothelial niche cells comprise cells that express ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. [0066] In one embodiment, the cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a placenta stem cell, an adult stem cell, an amniotic stem cell, and an umbilical vein endothelial cell. [0067] In one embodiment, the ESC, iPSC, placenta stem cell, adult stem cell, amniotic stem cell is differentiated to an endothelial cell prior to contact. One skilled in the art will understand how to differentiate to an ESC, iPSC, placenta stem cell, adult stem cell, or amniotic stem cell to an endothelial cell, e.g., using protocols known in the art. [0068] In some embodiments, the endothelial cells are human. 10 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0069] In some embodiments, the endothelial cells are mammalian. [0070] In some embodiments, the endothelial cells are nonhuman mammalian. [0071] In some embodiments of any of the aspects, the endothelial niche cells are generated or engineered to express transcription factors, comprising ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. [0072] In some embodiments of any of the aspects, ETV2 and the at least two transcription factors are expressed from at least one vector. In some embodiments, the vector comprises an exogenous nucleic acid sequence or sequences encoding ETV2 and the at least two transcription factors. In some embodiments, the exogenous nucleic acid sequences are transiently expressed in the endothelial cell. In some embodiments, the exogenous nucleic acid sequences are stably expressed in the endothelial cell. In some embodiments, the exogenous nucleic acid sequences are incorporated into the genome of the endothelial cell. As a non-limiting example, the exogenous nucleic acid sequences can be incorporated into the genome using viral vectors (e.g., AAV, lentivirus) or CRISPR technologies. [0073] One aspect provides for an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. [0074] Another aspect provides for a population of engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. [0075] One aspect provides for a composition comprising an engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. In some embodiments of any of the aspects, the composition can comprise engineered endothelial niche cells. In some embodiments, the composition is a therapeutic agent, or the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a culture dish, 3D cell system, or suspension system. In some embodiments, the composition comprises a scaffold. [0076] Engineered endothelial niche cells described herein can be identified by assessing if they secrete or present certain growth facts. In one embodiment, the engineered endothelial niche cells secrete or presents on the cell surface at least one growth factor. Exemplary growth factors that identify an engineered endothelial niche cells include, but is not limited to SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO. In one embodiment, the engineered endothelial niche cells secrete or present on the cell surface at least one of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO. In one embodiment, the engineered endothelial niche cells secrete or present on the cell surface, at least two, at least three, at least four, at least five, at least 11 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT six, at least seven, at least eight, at least nine, at least ten, or at least eleven of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO. A skilled person can identify whether the engineered endothelial niche cells secrete or presents the at least one growth factors, e.g., using standard assays in the art. For example, immunofluorescence assays using antibodies to the growth factors can be used to assess whether the engineered endothelial niche cells presents the at least one growth factors on the cell, and assays that detect protein or mRNA expression (e.g., western blot analysis or PCR analysis, respectively) can be used to determine if the engineered endothelial niche cells secretes the at least one growth factors on the cell. [0077] Engineered endothelial niche cells described herein can also be identified by assessing if they expression certain cell surface proteins. In one embodiment, the engineered endothelial niche cells express at least one cell surface protein. Exemplary cell surface proteins that identify engineered endothelial niche cells include, but are not limited to MRC1, ICAM1, STAB2, VCAM1, and CD62E. In one embodiment, the engineered endothelial niche cells express at least one of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, and CD62E. In one embodiment, the engineered endothelial niche cells express at least two, at least three, at least four, or at least five of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, CD62E. A skilled person can identify whether the engineered endothelial niche cells express the at least one cell surface protein, e.g., using standard assays in the art. For example, immunofluorescence assays using antibodies to the cell surface proteins can be used to assess whether the engineered endothelial niche cells expresses the at least one cell surface proteins on the cell, and assays that detect protein or mRNA expression (e.g., western blot analysis or PCR analysis, respectively) can be used to determine if the engineered endothelial niche cells expresses the at least one the cell surface proteins. [0078] Another aspect provides a method for culturing stem cells, the method comprising culturing a population of stem cells in the presence of a population of engineered endothelial niche cells. In some embodiments of any of the aspects, the method is performed in vitro. In some embodiments, the engineered endothelial niche cells secrete a factor (e.g., growth factors) that affects the growth and/or expansion of the population of stem cells. [0079] In one embodiment, the population of stem cells is a population of HSPCs. [0080] Another aspect provides a method for culturing HSPCs, the method comprising culturing a population of HSPCs in the presence of a population of engineered endothelial niche cells. In some embodiments of any of the aspects, the method is performed in vitro. [0081] In some embodiments, the stem cell (e.g., HSPCs) cultured in the presence of the engineered endothelial niche cells have at least 10% increased proliferation than stem cells that are cultured in the absence of such engineered endothelial niche cells. 12 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0082] In some embodiments, the stem cell (e.g., HSPCs) cultured in the presence of the engineered endothelial niche cells have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more; or at least at least 5x, at least 10x, at least 15x, at least 20x, at least 25x, at least 30x, at least 35x, at least 40x, at least 45x, at least 50x, at least 55x, at least 60x, at least 65x, at least 70x, at least 75x, at least 80x, at least 85x, at least 90x, at least 95x, at least 100x, or more increased proliferation than stem cells that are cultured in the absence of such engineered endothelial niche cells. [0083] In some embodiments, the HSPCs cultured in the presence of the engineered endothelial niche cells have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or more; or at least at least 5x, at least 10x, at 13 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT least 15x, at least 20x, at least 25x, at least 30x, at least 35x, at least 40x, at least 45x, at least 50x, at least 55x, at least 60x, at least 65x, at least 70x, at least 75x, at least 80x, at least 85x, at least 90x, at least 95x, at least 100x, or more increased proliferation than HSPCs that are cultured in the absence of such engineered endothelial niche cells. [0084] In some embodiments, the stem cell (e.g., HSPCs) cultured in the presence of the engineered endothelial niche cells can be cultured for at least 3 days longer than stem cells that are cultured in the absence of such engineered endothelial niche cells. [0085] In some embodiments, the HSPCs cultured in the presence of the engineered endothelial niche cells can be cultured for at least 3 days longer than HSPCs that are cultured in the absence of such engineered endothelial niche cells. [0086] In, some embodiments, the stem cell (e.g., HSPCs) cultured in the presence of the engineered endothelial niche cells can be cultured for at 1 day longer, at least 2 days longer, at least 3 days longer, at least 4 days longer, at least 5 days longer, at least 6 days longer, at least 7 days longer, at least 8 days longer, at least 9 days longer, at least 10 days longer, at least 11 days longer, at least 12 days longer, at least 13 days longer, or at least 14 days longer than stem cell (e.g., HSPCs) that are cultured in the absence of such engineered endothelial niche cells. [0087] In, some embodiments, the HSPCs cultured in the presence of the engineered endothelial niche cells can be cultured for at 1 day longer, at least 2 days longer, at least 3 days longer, at least 4 days longer, at least 5 days longer, at least 6 days longer, at least 7 days longer, at least 8 days longer, at least 9 days longer, at least 10 days longer, at least 11 days longer, at least 12 days longer, at least 13 days longer, or at least 14 days longer than HSPCs that are cultured in the absence of such engineered endothelial niche cells. [0088] In some embodiments, the cells are cultured on a biologically compatible scaffold. Non- limiting examples of a biologically compatible scaffold comprise: a hydrogel, biopolymers, or another biomaterial with the ability to grow cells in vitro in preparation for transplantation. In some embodiments, the HSPCs cultured in the presence of the engineered endothelial niche cells have increased engraftment when administered to a subject compared to the engraftment of substantially similar HSPCs that were not cultured with engineered endothelial niche cells. As used herein, “engraftment” refers to the process wherein transplanted HSPCs begin to grow and produce healthy blood cells. Engraftment is a critical milestone in recovery from an HSPC transplant. [0089] Another aspect provides a method of treating a subject, the method comprising, transplanting a composition comprising a population of engineered endothelial niche-cells into the subject. As a non- limiting example, the method can be used to treat myelofibrosis or other hematopoietic diseases where the endogenous bone marrow niche is compromised, non-limiting examples of which are disclosed herein. In some embodiments, the method can comprise transplanting a composition comprising a population of HSPCs into the subject. In some embodiments, the method can comprise transplanting a 14 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT composition comprising a population of HSPCs and engineered endothelial niche-cells into the subject. [0090] Another aspect provides a method for enhancing engraftment of HSPCs, the method comprising administering a composition comprising HSPCs and a population of engineered endothelial niche cells to a subject in need thereof. In some embodiments of any of the aspects, engraftment of the HSPCs is increased by at least 10% compared to the engraftment of substantially similar HSPCs in the absence of engineered endothelial niche cells. In some embodiments of any of the aspects, engraftment of the HSPCs is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to the engraftment of substantially similar HSPCs in the absence of engineered endothelial niche cells. [0091] Another aspect provides a co-culture comprising engineered endothelial niche cells and HSPCs. In some embodiments of any of the aspects, the endothelial cells are made by a method described herein. [0092] Another aspect provides a method for extra medullary hematopoiesis, the method comprising transplanting engineered-niche endothelial cells into a subject at a location outside of the bone marrow (e.g., the forearm), thereby creating a synthetic niche. As used herein, “extra medullary hematopoiesis” refers to hematopoiesis occurring in organs outside of the bone marrow. In some embodiments of any of the aspects, the endothelial cells are made by a method described herein. Treatment Methods [0093] Another aspect provides a method for treating Extramedullary hematopoiesis (EMH), the method comprising transplanting engineered-niche endothelial cells into a subject at a location outside of the bone marrow (e.g., subcutaneously, e.g., in the forearm), thereby creating a synthetic niche. In some embodiments of any of the aspects, the endothelial cells are made by a method described herein. [0094] “Extramedullary hematopoiesis (EMH)” refers to the formation and development of blood cells outside the bone marrow, which is the primary site of hematopoiesis in adults. EMH is typically a compensatory mechanism that arises in response to certain pathological conditions where the bone marrow's ability to produce blood cells is impaired or insufficient. EMH can be associated with diseases that affect the bone marrow, such as myelofibrosis, leukemia, and other myeloproliferative disorders. For example, a subject having EMH can also have at least one of myelofibrosis, a blood cancer (e.g., leukemia), or another myeloproliferative disorders. Onset of EMH can arise from, for example, a response to severe anemia, chronic hypoxia, or other conditions that increase the body's demand for blood cells. 15 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [0095] EMH can occur in various tissues and organs, e.g., the liver, spleen, lymph nodes, and, in rare cases, other sites such as the adrenal glands and the skin. The location of EMH can be associated with an additional related disease or disorder. For example, EMH localized in the liver can be associated with myelofibrosis; EMH localized to the spleen can by associated with splenomegaly (enlarged spleen); and EMH localized lymph nodes can be associated with hematological malignancies. [0096] One skilled in the art can determine if EMH is present in a subject, for example, by determining if there is a presence of hematopoietic cells, including erythroid, myeloid, and megakaryocytic lineages, outside the bone marrow. Typically, the hematopoietic cells outside of the bone marrow can form clusters or nodules within the affected tissues. A skilled person can use, for example, imaging techniques such as ultrasound, CT scans, and MRI can help identify sites of EMH, or biopsy techniques to confirm the presence of hematopoietic tissue outside the bone marrow. [0097] Another aspect provides a method for treating myelofibrosis or myeloproliferative disorders, the method comprising transplanting engineered-niche endothelial cells into a subject at a location outside of the bone marrow (e.g., subcutaneously, e.g., in the forearm), thereby creating a synthetic niche. In some embodiments of any of the aspects, the endothelial cells are made by a method described herein. [0098] Myelofibrosis is an uncommon type of chronic leukemia. Myelofibrosis belongs to a group of diseases called myeloproliferative disorders, often of a chronic form. Chronic myeloproliferative disorders are a group of slow-growing blood cancers in which the bone marrow makes too many abnormal red blood cells, white blood cells, or platelets, which accumulate in the blood. Non-limiting examples of chronic myeloproliferative neoplasms comprise: Chronic myelogenous leukemia, Polycythemia vera, Primary myelofibrosis (also called chronic idiopathic myelofibrosis), Essential thrombocythemia, Chronic neutrophilic leukemia, and Chronic eosinophilic leukemia. [0099] Myelofibrosis is a serious bone marrow disorder that disrupts the body's normal production of blood cells. The result is extensive scarring in bone marrow, leading to severe anemia, weakness, fatigue and often an enlarged spleen. Many subjects or patients with myelofibrosis get progressively worse, and some subjects or patients may eventually develop a more serious form of leukemia. Myelofibrosis can occur when blood stem cells (e.g., HSPCs) develop a genetic mutation. Several specific gene mutations have been identified in people with myelofibrosis. The most common is the Janus kinase 2 (JAK2) gene. [00100] Although the cause of myelofibrosis often isn't known, certain factors are known to increase risk. Increased age can be associated with the development of myelofibrosis. Myelofibrosis can affect anyone, but it's most often diagnosed in people older than 50. Patients with another blood cell disorder are at higher risk for developing myelofibrosis. A small portion of people with myelofibrosis develop the condition as a complication of essential thrombocythemia or polycythemia vera. Exposure to certain chemicals can increase the risk for myelofibrosis. Myelofibrosis has been linked to exposure to 16 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT industrial chemicals such as toluene and benzene. Exposure to radiation can increase the risk for myelofibrosis. People exposed to high levels of radiation, such as survivors of atomic bomb attacks, have an increased risk of myelofibrosis. Some people who received a radioactive contrast material called Thorotrast, used until the 1950s, have developed myelofibrosis. [00101] Multiple complications can result from myelofibrosis. A complication of myelofibrosis can include increased pressure on blood flowing into a patient’s liver. Normally, blood flow from the spleen enters the liver through a large blood vessel called the portal vein. Increased blood flow from an enlarged spleen can lead to high blood pressure in the portal vein (e.g., portal hypertension). This in turn can force excess blood into smaller veins in the stomach and esophagus, potentially causing these veins to rupture and bleed. Pain can be another complication of myelofibrosis. A severely enlarged spleen can cause abdominal pain and back pain. Myelofibrosis can lead to growths in other areas of the body. Myelofibrosis can be associated with bleeding complications. As the disease progresses, platelet count tends to drop below normal (thrombocytopenia), and platelet function becomes impaired. An insufficient number of platelets can lead to easy bleeding. Myelofibrosis can also be associated with painful bones and joints. Myelofibrosis can lead to hardening of bone marrow and inflammation of the connective tissue that is found around the bones. This may cause bone and joint pain. Myelofibrosis can also be associated with development of acute leukemia. Some patients with myelofibrosis develop acute myelogenous leukemia, a type of blood and bone marrow cancer that progresses rapidly. [00102] Bone marrow transplantation is currently the only approved treatment for myelofibrosis. Additional treatments can only ameliorate the symptoms of myelofibrosis (e.g., anemia, enlarged spleen). Ruxolitinib, a JAK inhibitor which targets the gene mutation found in most cases of myelofibrosis, can be used to reduce symptoms of an enlarged spleen. [00103] As described herein, levels of functional hematopoiesis can be decreased in myelofibrosis and/or in subjects with myelofibrosis. As used herein, “functional hematopoiesis” refers to hematopoiesis that produces normal levels and proportions of blood cells (e.g., red blood cells, white blood cells, platelets). In some embodiments of any of the aspects, the level of hematopoiesis can be decreased in myelofibrosis or a myeloproliferative disorder and/or in subjects with myelofibrosis or a myeloproliferative disorder. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating myelofibrosis or a myeloproliferative disorder in a subject in need thereof, the method comprising administering HSPCs, engineered endothelial cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) to a subject determined to have a level of functional hematopoiesis that is decreased relative to a reference. In some embodiments of any of the aspects, the step of determining if the subject has a functional level of functional hematopoiesis can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level 17 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT of functional hematopoiesis in the subject. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results. [00104] In one aspect of any of the embodiments, described herein is a method of treating a subject in need thereof, the method comprising administering any of the engineered endothelial cells or co- cultured cells, or a composition or population thereof. [00105] In one aspect of any of the embodiments, described herein is a method of treating a subject in need thereof, the method comprising: a) identifying a subject in need thereof; and b) administering any of the engineered endothelial cells or co-cultured cells, or a composition or population thereof. [00106] In one aspect of any of the embodiments, described herein is a method of treating a subject having EMH, the method comprising administering any of the engineered endothelial cells or co- cultured cells, or a composition or population thereof. [00107] In one aspect of any of the embodiments, described herein is a method of treating a subject in need thereof, the method comprising: a) identifying a subject having or at risk of having EMH; and b) administering any of the engineered endothelial cells or co-cultured cells, or a composition or population thereof. [00108] In one aspect of any of the embodiments, described herein is a method of treating a subject having myelofibrosis, the method comprising administering any of the engineered endothelial cells or co-cultured cells, or a composition or population thereof. [00109] In one aspect of any of the embodiments, described herein is a method of treating a subject in need thereof, the method comprising: a) identifying a subject having or at risk of having myelofibrosis; and b) administering any of the engineered endothelial cells or co-cultured cells, or a composition or population thereof. [00110] In one aspect of any of the embodiments, described herein is a method of treating a subject in need thereof, the method comprising administering stem cells (e.g., HSPCs) that were previously co- cultured cells with any of the engineered endothelial cells disclosed herein. [00111] In one aspect of any of the embodiments, described herein is a method of treating a subject in need thereof, the method comprising: a) identifying a subject in need thereof; and b) administering stem cells (e.g., HSPCs) that were previously co-cultured cells with any of the engineered endothelial cells disclosed herein. [00112] In one aspect of any of the embodiments, described herein is a method of treating a subject having EMH, the method comprising administering stem cells (e.g., HSPCs) that were previously co- cultured cells with any of the engineered endothelial cells disclosed herein. 18 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00113] In one aspect of any of the embodiments, described herein is a method of treating a subject in need thereof, the method comprising: a) identifying a subject having or at risk of having EMH; and b) administering stem cells (e.g., HSPCs) that were previously co-cultured cells with any of the engineered endothelial cells disclosed herein. [00114] In one aspect of any of the embodiments, described herein is a method of treating a subject having myelofibrosis, the method comprising administering stem cells (e.g., HSPCs) that were previously co-cultured cells with any of the engineered endothelial cells disclosed herein. [00115] In one aspect of any of the embodiments, described herein is a method of treating a subject in need thereof, the method comprising: a) identifying a subject having or at risk of having myelofibrosis; and b) administering stem cells (e.g., HSPCs) that were previously co-cultured cells with any of the engineered endothelial cells disclosed herein. [00116] In one embodiment, the stem cells (e.g., HSPCs) are isolated prior to administering. [00117] In one embodiment, method comprises the step of, prior to administering, isolating the stem cells (e.g., HSPCs) prior to administering. [00118] In one embodiment, the subject in need thereof has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level. In one embodiment, the method further comprises the step of, prior to administering, identifying a subject in need thereof having a decreased blood cell level or at risk for developing a decreased blood cell level as compared to a control blood cell level. In one embodiment, the blood cell level is decreased at least 1% as compared to a reference level. In one embodiment, the blood cell level is decreased at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% as compared to a reference level. As used herein, a “reference level” refers to an otherwise healthy, identical cell, tissue, organ, or subject. One skilled in the art will be able to determine is a subject has decreased blood cell levels using standard techniques, e.g., a blood test. 19 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00119] In one embodiment, the subject in need thereof has anemia or blood loss. [00120] In one embodiment, the subject in need thereof is a bone marrow donor. [00121] In one embodiment, the subject in need thereof has depleted bone marrow. [00122] In one embodiment, the subject in need thereof has anemia, hemolysis, leukemia, multiple myeloma, or a thyroid disorder. [00123] In one embodiment, treating a subject having or at risk of having EMH, administering occurs at a location outside of the bone marrow. Exemplary locations include, but are not limited to, liver, spleen, and subcutaneous. [00124] In one embodiment, treating a subject having or at risk of having EMH, administering is a transplantation. Exemplary transplantation sites include, but are not limited to, liver, spleen, and subcutaneous. [00125] In one embodiment, administering occurs at a location outside of the bone marrow. Exemplary locations include, but are not limited to, liver, spleen, and subcutaneous. [00126] In one embodiment, administering is a transplantation. Exemplary transplantation sites include, but are not limited to, liver, spleen, and subcutaneous. [00127] In one embodiment, administering is systemic. [00128] In one embodiment, administering is local. In one embodiment, local administering is administration to the liver, spleen, or subcutaneous. Administration [00129] The compositions and methods described herein can be administered to a subject having or diagnosed as having EMH. The compositions and methods described herein can be administered to a subject having or diagnosed as having myelofibrosis or a myeloproliferative disorder. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) to a subject in order to alleviate a symptom of, e.g., EMH, myelofibrosis or a myeloproliferative disorder. As used herein, "alleviating a symptom" is ameliorating any condition or symptom associated with the disease or disorder, e.g., EMH, myelofibrosis or a myeloproliferative disorder. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic. 20 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00130] The term “effective amount" as used herein refers to the amount of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) that is sufficient to provide a particular anti- disease or disorder effect (e.g., EMH) when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. [00131] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) which achieves a half- maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. [00132] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two 21 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein. [00133] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non- toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein. [00134] In some embodiments, the pharmaceutical composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms 22 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping. [00135] Suitable vehicles that can be used to provide parenteral dosage forms of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. [00136] In some embodiments of any of the aspects, the HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) described herein is administered as a monotherapy, e.g., a second treatment for the disease or disorder (e.g., EMH, myelofibrosis or a myeloproliferative disorder) is not administered to the subject. [00137] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and/or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN ^ cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1- TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, 23 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN ^ doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK ^ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL ^ paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE ^ Cremophor- free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE ^ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR ^ gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva ^)) 24 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. [00138] In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments. [00139] The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. By way of non-limiting example, if a subject is to be treated for pain or inflammation according to the methods described herein, the subject can also be administered a second agent and/or treatment known to be beneficial for subjects suffering from pain or inflammation. In some embodiments, the second agent is an anti-inflammation agent. Examples of such agents and/or treatments include, but are not limited to, non-steroidal anti- inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g. cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone); methotrexate; sulfasalazine; leflunomide; anti- TNF medications; cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g. endorphins, enkephalins, and dynorphin), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like. [00140] In certain embodiments, an effective dose of a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8), such as, e.g.0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more. [00141] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more. 25 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00142] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8). The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. [00143] The dosage ranges for the administration of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8), according to the methods described herein depend upon, for example, its form, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the extent to which, for example, myelofibrosis or a myeloproliferative disorder is desired to be reduced functional hematopoiesis is desired to be induced. The dosage should not be so large as to cause adverse side effects, such as excessive hematopoiesis or excessive extramedullary hematopoiesis. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication. [00144] The efficacy of HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms 26 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. blood cell counts. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of EMH, myelofibrosis or a myeloproliferative disorder. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed. Kits [00145] One aspect described herein provides a kit for culturing HSPCs, the kit comprising: a population of engineered endothelial niche cells, reagents and instructions for use thereof. Another aspect provides for a kit for generating engineered endothelial niche cells comprising: a vector(s) comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 and instructions for use thereof. Described herein are kit components that can be included in one or more of the kits described herein. [00146] In some embodiments, the kit comprises an effective number of reagents for culturing HSPCs and/or endothelial niche cells. As will be appreciated by one of skill in the art, reagents can be supplied in a lyophilized form or a concentrated form that can diluted prior to use with cultured cells. Preferred formulations include those that are non-toxic to the cells and/or does not affect growth rate or viability etc. reagents can be supplied in aliquots or in unit doses. [00147] In some embodiments the kit further comprises a vector comprising a nucleic acid encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. 27 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00148] In some embodiments, the components described herein can be provided singularly or in any combination as a kit. The kit includes the components described herein, e.g., a composition comprising HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8), a composition(s) that includes a vector comprising e.g., a gene to ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. In addition, the kit optionally comprises informational material. The kit can also contain culture dishes and/or a substrate for coating culture dishes, such as laminin, fibronectin, Poly-L-Lysine, or methylcellulose. [00149] In some embodiments, the compositions in the kit can be provided in a watertight or gas tight container which in some embodiments is substantially free of other components of the kit. For example, a HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8) composition can be supplied in more than one container, e.g., it can be supplied in a container having sufficient reagent for a predetermined number of experiments, e.g., 1, 2, 3 or greater. One or more components as described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the components described herein are substantially pure and/or sterile. When the components described herein are provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. [00150] The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein. The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of endothelial niche cells and/or HSPCs, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods for using or administering the components of the kit. [00151] The kit can include a component for the detection of a marker for HSPC differentiation and/or endothelial niche cell differentiation. In addition, the kit can include one or more antibodies that bind a cell marker, or primers for an RT-PCR or PCR reaction, e.g., a semi-quantitative or quantitative RT-PCR or PCR reaction. Such components can be used to assess the activation of maturation markers or the loss of immature cell markers of endothelial niche cells and/or HSPCs. If the detection reagent is an antibody, it can be supplied in dry preparation, e.g., lyophilized, or in a solution. The antibody or other detection reagent can be linked to a label, e.g., a radiological, fluorescent (e.g., GFP) or colorimetric label for use in detection. If the detection reagent is a primer, it can be supplied in dry preparation, e.g., lyophilized, or in a solution. [00152] The kit will typically be provided with its various elements included in one package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a Styrofoam box. The enclosure can be configured 28 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT so as to maintain a temperature differential between the interior and the exterior, e.g., it can provide insulating properties to keep the reagents at a preselected temperature for a preselected time. Vectors [00153] In some embodiments, one or more of the factors described herein is expressed in a recombinant expression vector or plasmid. As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring transgenes into a host cell. The term “vector” includes plasmids, mini-chromosomes, phage, naked DNA and the like. See, for example, U.S. Pat. Nos.4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, wherein additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" is used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. [00154] A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence can be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication can occur actively during a lytic phase or passively during a lysogenic phase. [00155] An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as an RNA transcript. Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, luciferase or 29 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined. [00156] As used herein, a coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide. [00157] When the nucleic acid molecule that encodes any of the factors/polypeptides described herein is expressed in a cell, a variety of transcription control sequences (e.g., promoter/enhancer sequences) can be used to direct its expression. The promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene. A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule. [00158] The precise nature of the regulatory sequences needed for gene expression can vary between species or cell types, but in general can include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5′ non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5′ leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. [00159] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA). That 30 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell. [00160] In some embodiments, the vector is pME Gateway vector (Invitrogen™). In some embodiments, the vector is p5E Gateway™ vector. In some other embodiments, the vector is pGEX2TK™ vector. In some other embodiments, the vector is TOPO-TA™ vector. [00161] Without limitations, the genes described herein can be included in one vector or separate vectors. For example, ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 can be included in the same vector. [00162] In some embodiments, at least of ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 can be included in a second vector. [00163] In one embodiment, ETV2 is included in a first vector and the at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 are be included in a second vector. [00164] In one embodiment, ETV2 is included in a first vector, and the at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 are included in a second vector and third vector. [00165] In some embodiments, the promoter operably linked to the gene(s) can be zebrafish ubi promoter. [00166] In some embodiments, one or more of the recombinantly expressed gene can be integrated into the genome of the cell. [00167] A nucleic acid molecule that encodes the enzyme of the claimed invention can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule encoding the enzymes of the claimed invention also may be accomplished by integrating the nucleic acid molecule into the genome. Definitions [00168] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a 31 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT term in the art and its definition provided herein, the definition provided within the specification shall prevail. [00169] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here. [00170] The terms “decrease” “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “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 or agent) 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. [00171] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean 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, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level. [00172] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein. [00173] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than 32 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT humans can be advantageously used as subjects that represent animal models of myelofibrosis or a myeloproliferative disorder. A subject can be male or female. [00174] “Extramedullary hematopoiesis (EMH)” refers to the formation and development of blood cells outside the bone marrow, which is the primary site of hematopoiesis in adults. EMH is typically a compensatory mechanism that arises in response to certain pathological conditions where the bone marrow's ability to produce blood cells is impaired or insufficient. EMH can be associated with diseases that affect the bone marrow, such as myelofibrosis, leukemia, and other myeloproliferative disorders. [00175] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. EMH) or one or more complications related to such a condition, and optionally, have already undergone treatment for EMH or the one or more complications related to EMH. Alternatively, a subject can also be one who has not been previously diagnosed as having EMH or one or more complications related to EMH. For example, a subject can be one who exhibits one or more risk factors for EMH, or one or more complications related to EMH or a subject who does not exhibit risk factors. [00176] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition. [00177] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. [00178] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and/or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in 33 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure. [00179] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. transcription factor activity and specificity of a native or reference polypeptide is retained. [00180] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu. [00181] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein. [00182] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or 34 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan. [00183] A variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings). [00184] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos.4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization. [00185] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA. [00186] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or 35 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide. [00187] In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are tissue specific. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are global. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is systemic. [00188] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons). [00189] "Marker" in the context of the present invention refers to an expression product, e.g., nucleic acid or polypeptide which is differentially present in a sample taken from subjects having myelofibrosis or a myeloproliferative disorder, as compared to a comparable sample taken from control subjects (e.g., a healthy subject). The term "biomarker" is used interchangeably with the term "marker." [00190] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation. [00191] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell is typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity. [00192] In some embodiments of any of the aspects, the HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two of TFEC, MAFB, FOXP4, HOXB8, or IRF8) described herein is exogenous. In some embodiments of any of the aspects, the HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two of TFEC, MAFB, FOXP4, HOXB8, or IRF8) described herein is ectopic. In some embodiments of any of the aspects, 36 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT the HSPCs, engineered endothelial niche cells, and/or transcription factors (e.g., ETV2, and at least two of TFEC, MAFB, FOXP4, HOXB8, or IRF8) described herein is not endogenous. [00193] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment. [00194] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g. a ETV2, TFEC, MAFB, FOXP4, HOXB8, or IRF8 polypeptide) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. [00195] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like). [00196] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or 37 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell. [00197] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. [00198] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non- essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. [00199] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration. [00200] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. EMH. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with EMH. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are 38 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). [00201] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature. [00202] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated. [00203] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine. [00204] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference. [00205] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. 39 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00206] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation. [00207] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. [00208] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. [00209] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST. [00210] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [00211] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [00212] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell 40 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); 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; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); 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); 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), the contents of which are all incorporated by reference herein in their entireties. [00213] Other terms are defined herein within the description of the various aspects of the invention. [00214] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. [00215] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, 41 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims. [00216] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. [00217] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. [00218] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs: 1. A method for engineering endothelial niche cells, the method comprising expressing in a cell ETV2; and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. 2. The method of paragraph 1, wherein the at least two transcription factors are TFEC and MAFB. 3. The method of any of the preceding paragraphs, wherein the cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a placenta stem cell, an adult stem cell, an amniotic stem cell, and an umbilical vein endothelial cell. 4. The method of any of the preceding paragraphs, wherein the ESC, iPSC, placenta stem cell, adult stem cell, amniotic stem cell is differentiated to an endothelial cell prior to contact. 5. The method of any of the preceding paragraphs, wherein ETV2 and the at least two transcription factors are expressed from at least one vector. 6. The method of any of the preceding paragraphs, wherein the at least one vector comprises an exogenous nucleic acid sequence(s) encoding the ETV2 and the at least two transcription factors. 7. The method of any of the preceding paragraphs, wherein expression is transient or stable. 8. The method of any of the preceding paragraphs, wherein the exogenous nucleic acid sequence(s) is incorporated into the genome of the endothelial cell. 9. The method of any of the preceding paragraphs, wherein the cell is a mammalian cell. 10. The method of any of the preceding paragraphs, wherein the cell is a human cell. 42 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 11. The method of any of the preceding paragraphs, wherein the cell is a nonhuman mammalian cell. 12. The method of any of the preceding paragraphs, wherein the engineered endothelial niche cells secrete at least one of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO. 13. The method of any of the preceding paragraphs, wherein the engineered endothelial niche cells express at least one of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, and CD62E. 14. A method for engineering endothelial niche cells, the method comprising expressing in a cell ETV2, TFEC, and MAFB. 15. An engineered endothelial niche cell obtained by any of the preceding paragraphs. 16. An engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. 17. An engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. 18. A population of cells comprising any of the engineered endothelial niche cells of any of the preceding paragraphs. 19. A co-culture comprising any of the engineered endothelial niche cells of any of the preceding paragraphs or population of any of the preceding paragraphs and a population of stem cells. 20. The co-culture of any of the preceding paragraphs, wherein the second population of stem cells is an HSPCs. 21. A method for increasing stem cell proliferation, the method comprising co-culturing any of the engineered endothelial niche cells of any of the preceding paragraphs or population of any of the preceding paragraphs and a population of stem cells for a time sufficient in increase stem cell proliferation. 22. The method of any of the preceding paragraphs, wherein proliferation is increased by at least 10% as compared to an appropriate control. 23. The method of any of the preceding paragraphs, wherein the population of stem cells is a population of HSPCs. 24. The method of any of the preceding paragraphs, wherein the method is performed in vitro. 25. The method of any of the preceding paragraphs, wherein the engineered endothelial niche cells secrete a factor that affects the proliferation of the HSPC cells. 43 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 26. A method for treating a subject, the method comprising administering any of the engineered endothelial niche cells of any of the preceding paragraphs, population of any of the preceding paragraphs, or co-culture of any of the preceding paragraphs into a subject in need thereof. 27. A method for treating a subject, the method comprising: a. identifying a subject in need thereof; and b. administering any of the engineered endothelial niche cells of any of the preceding paragraphs, population of any of the preceding paragraphs, or co-culture of any of the preceding paragraphs into the subject in need thereof. 28. A method for treating a subject, the method comprising administering the co-culture of any of the preceding paragraphs into a subject in need thereof. 29. A method for treating a subject, the method comprising administering a population of HSPCs that have been previously co-cultured any of the engineered endothelial niche cells of any of the preceding paragraphs or population of any of the preceding paragraphs into a subject in need thereof. 30. The method of any of the preceding paragraphs, comprising the step of isolating the population of HSPCs prior to administering. 31. The method of any of the preceding paragraphs, wherein the subject in need thereof is human. 32. The method of any of the preceding paragraphs, wherein the subject in need thereof has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level. 33. The method of any of the preceding paragraphs, further comprising the step of identifying a subject in need thereof having a decreased blood cell level or at risk for developing a decreased blood cell level as compared to a control blood cell level prior to administering. 34. The method of any of the preceding paragraphs, wherein the blood cell level is decreased at least 1% compared to a reference level. 35. The method of any of the preceding paragraphs, wherein the subject in need thereof has anemia or blood loss. 36. The method of any of the preceding paragraphs, wherein the subject in need thereof is a bone marrow donor. 37. The method of any of the preceding paragraphs, wherein the subject in need thereof has depleted bone marrow. 38. The method of any of the preceding paragraphs, the subject in need thereof has anemia, hemolysis, leukemia, multiple myeloma, or a thyroid disorder. 44 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 39. The method of any of the preceding paragraphs, wherein the administering occurs at the liver, spleen, or subcutaneously. 40. A method for generating an ectopic vascular niche, the method comprising administering any of the engineered endothelial niche cells of any of the preceding paragraphs, population of any of the preceding paragraphs, or co-culture of any of the preceding paragraphs to a target site in a subject in need thereof. 41. A method for treating extra medullary hematopoiesis, the method comprising administering any of the engineered endothelial niche cells of any of the preceding paragraphs, population of any of the preceding paragraphs, or co-culture of any of the preceding paragraphs into a subject at a location outside of the bone marrow, thereby creating a synthetic niche. 42. The method any of the preceding paragraphs, wherein the location is the liver, spleen, or subcutaneous. 43. The method of any of the preceding paragraphs, wherein administering is systemic or local administration. 44. The method of any of the preceding paragraphs, wherein local administration is transplantation. 45. The method of any of the preceding paragraphs, wherein local administration is administration directly to the liver, spleen, or subcutaneously. 46. A vector comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 operably linked to a promoter. 47. A vector comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. EXAMPLES EXAMPLE 1 [00219] Specialized vascular endothelial cells called venous sinusoids support the growth and maintenance of hematopoietic stem and progenitor cells (HSPCs). The transcriptional program of these endothelial cells in determining their HSPC niche supportive programs remains unclear. Here, the inventors performed differential gene expression analyses using sinusoidal endothelial cells from the adult zebrafish kidney marrow HSPC niche and from the adult mouse bone marrow HSPC niche and compared these to adult liver sinusoidal endothelial cells that do not support hematopoiesis. The inventors identified transcription factors that are uniquely upregulated in the endothelial cells within the HSPC niches. Using a liver endothelial cell-specific enhancer to drive the overexpression of 45 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT transcription factors tfec and mafbb, they achieved the transcriptional reprogramming of the liver endothelial cells to adopt HSPC niche supportive fate. These reprogrammed liver vascular endothelial cells enabled the homing and maintenance of primary zebrafish HSPCs with long-term repopulating stem-cell potential. The findings uncovered new knowledge about the transcription regulation of the endothelial cells within the HSPC niche and may lead to novel approaches to create synthetic HSPC niches through in vivo reprogramming. Introduction [00220] Hematopoietic stem and progenitor cells (HSPCs) contribute to the multilineage regeneration of blood and immune cells in the blood system (Orkin and Zon, 2008; Sawai et al., 2016). HSPCs reside in specialized vascular microenvironments known as the HSPC niches, and the endothelial cells in the sinusoidal vasculature are the most abundant and one of the most important components of the HSPC niches (Morrison and Scadden, 2014; Wei and Frenette, 2018; Pinho and Frenette, 2019; Comazzetto et al., 2021). These vascular endothelial cells and other perivascular cells provide cell surface adhesion proteins and secreted factors that support the maintenance and homeostasis of the HSPCs occupying such vascular niche. Several cell types have been proposed to create niches for haematopoietic stem cells (HSCs). However, the expression patterns of HSC maintenance factors have not been systematically studied and no such factor has been conditionally deleted from any candidate niche cell. Thus, the cellular sources of these factors are undetermined. Stem cell factor (SCF; also known as KITL) is a key niche component that maintains HSCs. Here, using Scf-gfp knock-in mice, we found that Scf was primarily expressed by perivascular cells throughout the bone marrow. HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, nestin-cre- or nestin-creER-expressing cells. However, HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or leptin receptor (Lepr)- expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells. Thus, HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance. [00221] The self-renewal of hematopoietic stem cells is regulated by the bone marrow microenvironment. Whereas previous studies have focused on the role of osteoblasts, Ingrid Winkler et al. now show that bone marrow endothelial cells in the so-called 'vascular niche' contribute to this regulation by directly inducing HSC proliferation. In mice, deficiency or antagonism of the endothelial-specific adhesion protein E-selectin promotes HSC quiescence and self-renewal. These findings may point to a new treatment strategy for preserving HSC function in patients undergoing chemotherapy. [00222] The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the 46 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT chemokine cxcl8 and its receptor, cxcr1, are expressed by zebrafish endothelial cells, and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC–endothelial cell “cuddling,” HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression. Finally, using parabiotic zebrafish, we show that cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation. [00223] Bone marrow (BM) perivascular stromal cells and vascular endothelial cells (ECs) are essential for hematopoietic stem cell (HSC) maintenance, but the roles of distinct niche compartments during HSC regeneration are less understood. Here we show that Leptin receptor-expressing (LepR+) BM stromal cells and ECs dichotomously regulate HSC maintenance and regeneration via secretion of pleiotrophin (PTN). BM stromal cells are the key source of PTN during steady-state hematopoiesis because its deletion from stromal cells, but not hematopoietic cells, osteoblasts, or ECs, depletes the HSC pool. Following myelosuppressive irradiation, PTN expression is increased in bone marrow endothelial cells (BMECs), and PTN+ ECs are more frequent in the niche. Moreover, deleting Ptn from ECs impairs HSC regeneration whereas Ptn deletion from BM stromal cells does not. These findings reveal dichotomous and complementary regulation of HSC maintenance and regeneration by BM stromal cells and ECs. [00224] Radiotherapy and chemotherapy disrupt bone vasculature, but the underlying causes and mechanisms enabling vessel regeneration after bone marrow (BM) transplantation remain poorly understood. Here, we show that loss of hematopoietic cells per se, in response to irradiation and other treatments, triggers vessel dilation, permeability, and endothelial cell (EC) proliferation. We further identify a small subpopulation of Apelin-expressing (Apln+) ECs, representing 0.003% of BM cells, which is critical for physiological homeostasis and transplant-induced BM regeneration. Genetic ablation of Apln+ ECs or Apln-CreER-mediated deletion of Kitl and Vegfr2 disrupt hematopoietic stem cell (HSC) maintenance and contributions to regeneration. Consistently, the fraction of Apln+ ECs increases substantially after irradiation and promotes normalization of the bone vasculature in response to VEGF-A, which is provided by transplanted hematopoietic stem and progenitor cells (HSPCs). Together, these findings reveal critical functional roles for HSPCs in maintaining vascular integrity and for Apln+ ECs in hematopoiesis, suggesting potential targets for improving BM transplantation. In contrast to nearly all other tissues, the anatomy of cell differentiation in the bone marrow remains unknown. This is owing to a lack of strategies for examining myelopoiesis—the differentiation of myeloid progenitors into a large variety of innate immune cells—in situ in the bone marrow. Such strategies are required to understand differentiation and lineage-commitment decisions, 47 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT and to define how spatial organizing cues inform tissue function. Here we develop approaches for imaging myelopoiesis in mice, and generate atlases showing the differentiation of granulocytes, monocytes and dendritic cells. The generation of granulocytes and dendritic cells–monocytes localize to different blood-vessel structures known as sinusoids and displays lineage-specific spatial and clonal architectures. Acute systemic infection with Listeria monocytogenes induces lineage-specific progenitor clusters to undergo increased self-renewal of progenitors, but the different lineages remain spatially separated. Monocyte–dendritic cell progenitors (MDPs) map with nonclassical monocytes and conventional dendritic cells; these localize to a subset of blood vessels expressing a major regulator of myelopoiesis, colony-stimulating factor 1 (CSF1, also known as M-CSF)1. Specific deletion of Csf1 in endothelium disrupts the architecture around MDPs and their localization to sinusoids. Subsequently, there are fewer MDPs and their ability to differentiate is reduced, leading to a loss of nonclassical monocytes and dendritic cells during both homeostasis and infection. These data indicate that local cues produced by distinct blood vessels are responsible for the spatial organization of definitive blood cell differentiation. [00225] The vascular HSPC niches in the fetal liver and in the adult bone marrow are the primary HSPC niches in the mammalian systems. The liver maintains hematopoietic stem cells (HSCs) during development. However, it is not clear what cells are the components of the developing liver niche in vivo. Here, we genetically dissected the developing liver niche by systematically determining the cellular source of a key HSC niche factor, stem cell factor (SCF). Most HSCs were closely associated with sinusoidal vasculature. Using Scfgfp knockin mice, we found that Scf was primarily expressed by endothelial and perisinusoidal hepatic stellate cells. Conditional deletion of Scf from hepatocytes, hematopoietic cells, Ng2+ cells, or endothelial cells did not affect HSC number or function. Deletion of Scf from hepatic stellate cells depleted HSCs. Nearly all HSCs were lost when Scf was deleted from both endothelial and hepatic stellate cells. The expression of several niche factors was downregulated in stellate cells around birth, when HSCs egress the developing liver. Thus, hepatic stellate and endothelial cells create perisinusoidal vascular HSC niche in the developing liver by producing SCF. Extramedullary hematopoiesis can occur where HSPCs localize to the vascular spaces in the adult liver and spleen (Cardier and Barberá-Guillem, 1997; Mendt and Cardier, 2012; Inra et al., 2015; Cenariu et al., 2021; Rivera-Torruco et al., 2024). In adult zebrafish, kidney marrow is the primary site of hematopoiesis, where the sinusoidal vasculature serves as the HSPC niche (Orkin and Zon, 2008; White et al., 2008; Tang et al., 2017; Hu et al., 2024). The zebrafish liver is also rich in sinusoidal vessels, but the endothelial cells in the zebrafish liver do not support hematopoiesis under normal physiological conditions. Prior studies of the vascular HSPC niche in the zebrafish embryonic caudal hematopoietic tissue (CHT) suggested that transcription factors control the fate of these vascular endothelial cells to support HSPCs (Mahony et al., 2016; Xue et al., 2017; Hagedorn et al., 48 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 2023). However, the transcription factor control of vascular endothelial cells in the adult HSPC niches has not been fully elucidated. [00226] Here, the inventors study the transcriptional programs of the sinusoidal vascular endothelial cells in the zebrafish kidney marrow HSPC niche and in the mammalian bone marrow HSPC niche and identify unique transcription factors that control the fate of these endothelial cells to support HSPCs. In comparison, the inventors study the venous sinusoidal endothelial cells in the adult livers, which have the same venous sinusoidal programs but lack the HSPC niche supportive programs. Using adult zebrafish as a model, they design synthetic gene expression circuits to drive the overexpression of transcription factors tfec and mafbb in the liver endothelial cells. The transcription factor overexpression is sufficient to induce the fate of adult zebrafish liver endothelial cells to adopt transcriptional programs that modulate HSPC niche recruitment and function. The data presented herein provide functional insights into the transcription factor control of the vascular endothelial cells in the HSPC niches. Results [00227] Transcriptional control of the endothelial cells in the HSPC niches [00228] The inventors hypothesized that the transcriptional programs of the endothelial cells from the hematopoietic and non-hematopoietic organs are different, contributing to their capacity to support HSPCs in the niche. Therefore, they pursued single-cell RNA-sequencing to investigate the gene expression differences in the endothelial cells isolated from the adult zebrafish kidney marrow and from the adult zebrafish liver respectively (Figure 1A). The inventors confirmed that pan-endothelial cell genes cdh5, kdrl, and pecam1 are expressed by endothelial cells from both organs (Figure 1B), and sinusoidal endothelial cell genes sele, flt4, and gpr182 are expressed by the sinusoidal endothelial cells from both organs (Figure 1C). Previously identified liver sinusoidal endothelial cell genes gata4 and f8 are exclusively expressed in the liver sinusoidal endothelial cells in the data presented herein (Figure 1D). Differential gene expression analysis revealed that the adult kidney marrow endothelial cells express previously reported HSPC niche supportive genes such as cxcl12a, mrc1a, dab2, and csf1b (Figure 1E), confirming that the sinusoidal endothelial cells in the adult zebrafish kidney marrow and in the adult zebrafish liver have different transcriptional programs. A HPSC niche endothelial function must support hematopoiesis. The inventors observed that a small subset of the adult zebrafish kidney marrow sinusoidal endothelial cells expresses particularly high levels of the putative HSPC niche supportive genes, and these might be the putative endothelial cell population that actively supports the HSPCs in the vascular endothelial cell niche. [00229] Transcription factors are known to regulate transcriptional programs and thus govern cell fate, and prior studies in the field have revealed that transcription factors regulate the endothelial cells in the zebrafish embryonic CHT niche (Mahony et al., 2016; Xue et al., 2017; Hagedorn et al., 2023). Therefore, the inventors aimed to identify the unique expression of transcription factors in the 49 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT endothelial cells from the adult zebrafish kidney marrow that might control their cell fate to function as a HSPC niche. Differential gene expression analysis revealed that transcription factors tfec, mafba, mafbb, foxp4, hoxb8a, and irf8 are highly expressed in the adult zebrafish kidney marrow endothelial cells but not in the adult liver endothelial cells (Figure 1F). The putative HSPC niche supportive genes in the adult zebrafish kidney marrow endothelial cells are consistent with the inventors’ previous study identifying the HSPC niche supportive genes in the embryonic zebrafish CHT endothelial cells (Hagedorn et al., 2023). [00230] Furthermore, the inventors aimed to validate that these transcription factor candidates are indeed regulating the endothelial cell fate to function as a HSPC niche. Therefore, the inventors pursued differential gene expression analysis between the adult mouse endothelial cells isolated from the bone marrow, the liver, and the kidney, respectively, in public datasets from the literature (Kalucka et al., 2020; Fang et al., 2020). Similar to the inventors zebrafish analyses, they validated the expression of pan-endothelial cell marker genes Cdh5, Kdr, and Pecam1 are expressed by endothelial cells from all three organs (Figure 2A); and sinusoidal endothelial cell marker genes Stab2, Flt4, and Gpr182 are expressed highly by the sinusoidal endothelial cells in the bone marrow and in the liver (Figure 2B). The mammalian homologs of these transcription factors, Tfec, Mafb, Foxp4, Hoxb8, and Irf8, are also highly expressed in the adult mouse bone marrow endothelial cells, confirming that the expression of these transcription factors is correlated with the endothelial cells in the HSPC niches across different species (Figure 2C). [00231] Organ-specific endothelial cell enhancer elements in the adult zebrafish liver [00232] To identify cis-regulatory enhancer elements that are specific to the adult zebrafish liver endothelial cells, the inventors pursued Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on the endothelial cells and non-endothelial cells from the adult zebrafish kidney marrow and the adult zebrafish liver. The inventors identified putative enhancer peaks that are uniquely accessible to the adult zebrafish liver endothelial cells, in contrast to other liver cells or the kidney marrow endothelial cells. They then validated these putative enhancers in vivo by cloning them into a GFP reporter construct and creating transgenic zebrafish reporter lines (Figure 3A). An enhancer element upstream of the rca2.2 gene showed very specific chromatin accessibility in the zebrafish liver endothelial cells, and it robustly drives the GFP expression specifically in the liver sinusoidal endothelial cells (Figure 3B). Double transgenic reporter of rca2.2:GFP and a pan- endothelial marker kdrl:BFP showed that rca2.2:GFP labels a subset of endothelial cells within the adult zebrafish liver (Figure 3C and 3D). Transcriptional analyses of the endothelial cells isolated from either the rca2.2:GFP reporter line or from a previously published pan-endothelial cdh5:GFP reporter line showed similar gene expression profiles in the liver sinusoidal endothelial cell genes including pan-endothelial marker genes (cdh5, kdrl, pecam1, and etv2), sinusoidal endothelial cell genes (sele, flt4, and gpr182), and liver sinusoidal endothelial cell (gata4 and rca2.2), suggesting that 50 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT the rca2.2 upstream enhancer elements can be used to achieve specific expression of genes of interest in the zebrafish liver endothelial cells (Figure 3E) [00233] Transcription factors tfec and mafbb reprogrammed liver endothelial cells to express markers of HSPC niche supportive endothelial cells [00234] The inventors hypothesized that the transcription factor candidates that they identified from the adult zebrafish kidney marrow endothelial cells and from the adult mouse bone marrow endothelial cells play critical roles in regulating the transcriptional programs of the HSPC niche supportive endothelial cells. To evaluate this hypothesis, the inventors aimed to overexpress these transcription factor candidates in the zebrafish liver endothelial cells, which do not normally support HSPCs. The rca2.2 enhancer allowed the inventors to achieve specific overexpression of these transcription factor candidates in the adult zebrafish liver endothelial cells. They generated stable transgenic zebrafish lines that utilized the rca2.2 enhancer to drive the transcription factor candidates in the background of the inventors recently published mrc1a:GFP transgenic reporter line. The mrc1a:GFP reporter specifically labels the endothelial cells in the HSPC niches, including the embryonic zebrafish CHT (Hagedorn et al., 2023) and the adult zebrafish kidney marrow niche (Figure 4A). The inventors found that the overexpression of transcription factors tfec and mafbb can induce a robust ectopic expression of the mrc1a:GFP reporter in the liver endothelial cells (Figure 4B), indicating that these endothelial cells might be reprogrammed into a transcriptional state similar to that of the endothelial cells in the HSPC niches. [00235] To further validate the transcriptional changes, the inventors performed RNA-sequencing on the endothelial cells of the reprogrammed liver (ectopic mrc1a:GFP+ cells), the wildtype liver (rca2.2:GFP+ or cdh5:GFP+ cells), and the wildtype kidney marrow (mrc1a:GFP+ or cdh5:GFP+ cells) from adult zebrafish. The reprogrammed liver endothelial cells with tfec and mafbb overexpression maintained their identity as liver sinusoidal endothelial cells, confirmed by their expression of pan-endothelial (cdh5, kdrl, pecam1, and etv2), venous-sinusoidal (sele, flt4, and gpr182), and liver endothelial signature genes (gata4 and rca2.2) (Figure 4C). Interestingly, many of the genes that have been previously described to contribute to the HSPC niche functions are upregulated (Figure 4D and 4E). Therefore, the inventors confirm that the tfec and mafbb overexpression reprogrammed liver endothelial cells to express HSPC niche supportive genes at the transcriptional level. [00236] To further examine the sufficiency of either transcription factors tfec or mafbb in inducing the changes in the HSPC niche supportive fate in the reprogrammed liver endothelial cells, the inventors conducted experiments on each individual transcription factor overexpression transgenic zebrafish. They found that mafbb single overexpression is sufficient to induce the ectopic expression of mrc1a:GFP in the liver endothelial cells; however, tfec single overexpression is insufficient in activating the ectopic expression of this marker in the reprogrammed liver endothelial cells. 51 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT Consequently, the inventors further evaluated the efficiency of reprogramming between mafbb single overexpression and tfec-mafbb double overexpression. Flow analyses of the reprogrammed livers suggested that tfec-mafbb double overexpression had greater efficiency in inducing the ectopic mrc1a:GFP expression in the liver compared to mafbb single overexpression (Figure 4F). Transcriptional analyses by single-cell RNA-sequencing from cdh5:mCherry+ liver endothelial cells showed that mafbb single overexpression could induce the GFP expression of the mrc1a:GFP transgene. However, the combined tfec and mafbb overexpression is necessary to fully induce the expression of endogenous HSPC niche supportive genes, including mrc1a and dab2 (Figure 4G). Thus, tfec and mafbb are both required to induce an efficient reprogramming of the liver endothelial cells to a HSPC niche supportive state at the transcriptional level. [00237] HSPCs home to the reprogrammed liver vascular endothelial cell niche [00238] Following the validation of the transcriptional programs in the reprogrammed liver endothelial cells induced by tfec and mafbb overexpression, the inventors aimed to functionally validate HSPC homing and maintenance in the reprogrammed liver vascular HSPC niche. Thus, the inventors sought to confirm that the inventors’ reprogramming strategy conferred functional support for the HSPCs. However, a significant challenge in studying adult zebrafish hematopoietic biology is the difficulty in isolating primary HSPCs due to the absence of antibodies that can specifically recognize cell surface markers on HSPCs and the lack of transgenic zebrafish reporter lines that selectively label restricted populations of putative HSPCs (Tamplin et al., 2015; Gansner et al., 2017). These limitations impede direct isolation and visualization of primary adult zebrafish HSPCs and their interactions with the vascular endothelial cells in the HSPC niche in the inventors’ zebrafish model in vivo. [00239] To address the challenges of specific labeling of primary HSPCs, the inventors pursued an alternative strategy of performing transplant assay. Long-term repopulating HSPCs from donor animals are able to transplant and engraft in immunocompromised recipient animals, and reconstitute the hematopoietic system (Traver et al., 2003; White et al., 2008; Li et al., 2015). Therefore, the inventors performed transplant assays using donor liver cells into immunocompromised recipients to assess the presence of long-term repopulating HSPCs in the reprogrammed liver vascular endothelial cell HSPC niche (Figure 5A). Recipient zebrafish were conditioned immunodeficient by irradiation. Whole liver cells from either the reprogram donors or the wildtype control donors were enzymatically dissociated, and 200,000 enriched non-parenchymal cells were transplanted retro-orbitally into irradiated recipient animals. Post-transplant engraftment analyses revealed that long-term repopulating HSPCs are present in the donor liver cells harvested from the animals with liver endothelial cell-specific tfec and mafbb overexpression, giving rise to the presence of donor-derived cells in the recipient hematopoietic system from 7/26 transplanted recipient zebrafish. In contrast, 0/20 recipient animals that received donor liver cells from the wildtype control animals had any 52 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT engraftment in the hematopoietic system (Figure 5B). The engraftment results (p = 0.0296 for two- tailed t-test ) confirmed that the reprogrammed liver vascular endothelial cell HSPC niche has functionally recruited and supported primary HSPCs that have long-term repopulating stem cell potential. [00240] Discussion [00241] In this study, the inventors aimed to elucidate the transcription factor code that specifies the transcriptional programs of vascular endothelial cells in supporting the HSPCs in their niches. Differential gene expression analyses between the endothelial cells from hematopoietic organs and from the non-hematopoietic organs across different species enable the inventors to identify the conserved set of transcription factor candidates that are upregulated specifically in the endothelial cells that constitute HSPC niches. Through investigating the chromatin accessibility assay and validating putative enhancer elements in vivo using zebrafish models, the inventors are able to drive adult zebrafish liver endothelial-cell specific expression using validated enhancer elements. This strategy is useful to achieve tissue-specific expression of genes of interest in the field. By utilizing a liver endothelial cell-specific enhancer to overexpress the transcription factor candidates tfec and mafbb, the inventors are able to transcriptionally reprogram the liver endothelial cells to express HSPC niche supportive genes. The reprogrammed liver vascular endothelial cells generated a synthetic ectopic HSPC niche that is able to recruit and maintain primary HSPCs. Therefore, the inventors’ work provides a novel understanding of the transcriptional control of the HSPC niche supportive programs of the endothelial cells from hematopoietic organs and establishes a novel approach to create synthetic HSPC niches through in vivo reprogramming. [00242] The inventors find that the ability of the endothelial cells to function as HSPC niches is built on their unique gene expression profiles. Endothelial cells are known to have complex heterogeneity and plasticity across different tissues of an organism. By transcriptional analyses of the endothelial cells from the adult zebrafish kidney marrow HSPC niche and from the adult mouse bone marrow HSPC niche, the inventors are able to generate an evolutionarily conserved list of 103 significantly upregulated genes, including previously validated genes such as Sele, Cxcl12, Vcam1, and Osmr. They have also confirmed that many of these genes are shared with the endothelial cells from the embryonic zebrafish CHT niche (Hagedorn et al., 2023). [00243] In conclusion, the inventors’ current model establishes a transcription factor code that allows them to induce the HSPC niche supportive functions in the liver sinusoidal endothelial cells. Their discovery can be used to develop novel therapeutic approaches to generate synthetic extramedullary HSPC niches in the liver in the context of pathological bone marrow failures such as primary myelofibrosis. The transcription factors identified can also be used to generate bone marrow-like HSPC niche supportive endothelial cells in vitro, which will improve cell-based therapies for hematological disorders. 53 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT Materials and Methods [00244] Transgenic animal models [00245] Wildtype zebrafish TU strain, transparent zebrafish Capser-EKK strain, and transgenic zebrafish lines kdrl:mCherry (Chi et al., 2008), kdrl:mTagBFP (Matsuoka et al., 2016), mrc1a:GFP (Hagedorn et al., 2023), cdh5:GFP (Hagedorn et al., 2023), runx:GFP (Tamplin et al., 2015), runx:mCherry (Tamplin et al., 2015), cd41:GFP (Lin et al., 2005), ubi:mCherry (Mosimann et al., 2011) were used in this study. All zebrafish were housed at Boston Children’s Hospital and handled according to the approved Institutional Animal Care and Use Committee (IACUC) of Boston Children's Hospital protocols. [00246] Generation of new transgenic animal models [00247] New transgenic zebrafish lines were generated following a standard Tol2-transposase protocol (Mosimann et al., 2011). The cis-regulatory enhancer elements and the exon sequences for the transcription factors were synthesized as gBlocks from Integrated DNA Technologies (IDT). The cis-regulatory enhancer element gBlocks were cloned into the p5E Gateway vector using pENTR 5- Topo TA cloning kit (Invitrogen), and the open reading frames for the exon sequences for the transcription factors were cloned into the pME Gateway vector using pENTR/D-Topo cloning kit (Invitrogen). Gateway LR Clonase II Plus enzyme mix (Invitrogen) was used to recombine p5E regulatory elements, pME expression vector, p3E polyA tail, and pDest Tol2 integration backbone together into one vector. The vector and Tol2 mRNA are co-injected into one-cell stage zebrafish embryos. The injected embryos were screened for transgenesis markers, raised to adulthood, and bred with wild-type zebrafish to establish new stable transgenic zebrafish animal lines. [00248] Primary zebrafish cell sample preparation for sequencing [00249] Adult zebrafish were euthanized. Kidney marrow and liver were manually dissected into 0.9 x PBS and dissociated in Liberase TM (Roche) for 20 minutes at 37 ˚C. The dissociated cells are filtered through a 40 μm mesh filter, centrifuged at 500 x g for 5 minutes, and resuspended in 2% FBS in PBS for downstream sample processing with flow cytometry and fluorescence-activated cell sorting (FACS). For single-cell RNA-sequencing, cells are collected in 0.04% BSA for downstream sample processing following the Chromium Single Cell 3’ Reagent Kit (10x Genomics). For bulk RNA-sequencing, cells are collected in 2% FBS, centrifuged at 500 x g for 5 minutes, and resuspended in TRI Reagent (Zymo Research). Direct-zol RNA microprep kits (Zymo Research) were used to purify RNA, and NEBNext low input RNA library prep kits (New England Biolabs) were used to prepare the RNA-sequencing libraries. For ATAC-sequencing, primary zebrafish cells are collected in 2% FBS for downstream sample processing following the published ATAC-sequencing protocol (Buenrostro et al., 2013). [00250] Histology and microscopy 54 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00251] Adult zebrafish were euthanized and fixated in 10% buffered formalin (VWR) for 72 hours. The fixated zebrafish were processed with decalcification, paraffin embedding, and sectioning at the Specialized Histopathology Core at Brigham and Women’s Hospital. The histological sections were stained with H&E, anti-GFP immunofluorescence, anti-mCherry immunofluorescence, or anti-GFP immunohistochemistry at the Specialized Histopathology Core at Brigham and Women’s Hospital. Microscopic imaging of the histological sections was performed using a Yokogawa CSU-X1 spinning disk mounted on an inverted Nikon Eclipse Ti microscope equipped with dual Andor iXon EMCCD cameras, and the images were processed using NIS-Elements (Nikon). [00252] Bioinformatic analyses [00253] RNA-sequencing and ATAC-sequencing libraries were sequenced by Next Generation Sequencing (NGS) services at Genewiz. For RNA-sequencing, quality control was performed by Fast QC and Cutadapt to remove adaptor sequences and low-quality regions. High-quality reads were aligned to UCSC build danRer11/GRCz11 reference zebrafish genome using Tophat. The aligned reads were used for differential gene expression analyses using DESeq2 following recommendations from Harvard Chan Bioinformatic Core. For ATAC-sequencing, reads are aligned to UCSC build danRer11/GRCz11 reference zebrafish genome using Bowtie2. Chromatin accessible regions were called using the MACS2 package. Single-cell RNA-sequencing libraries were sequenced with Illumina NovaSeq at the Harvard Bauer Core Facility. The libraries were demultiplexed and aligned to UCSC build danRer11/GRCz11 reference zebrafish genome using the Cell Ranger algorithm from 10x Genomics. The aligned reads were analyzed following the R toolkit Seurat (Hao et al., 2024) following recommendations from Harvard Chan Bioinformatics Core. [00254] Transplant assay [00255] Casper-EKK adult zebrafish that are between three-month-old and six-month-old age were used for irradiation. Female fish were bred the day before irradiation to reduce the number of eggs that they carry for better irradiation efficiency and decreased risk of infections. The Casper-EKK adult fish received split-dose irradiation of 14 Gy each for two consecutive days prior to transplant. Adult zebrafish livers were harvested, dissociated, and processed with Debris Removal Solution (Miltenyi Biotec) for the donor cells. The cells are centrifuged at 50 x g for 2 minutes to pellet the hepatocytes, and the supernatant enriched for non-parenchymal cells is collected. The non-parenchymal supernatant fraction was then centrifuged at 500 x g for 5 minutes and resuspended in 0.9 x FBS, 5% FBS, and 1% penicillin-streptomycin transplant buffer. Resuspended non-parenchymal fraction cells are counted with a hemocytometer, and 200,000 cells are delivered into the recipient circulatory system through retro-orbital injection, as previously described (Li et al., 2015). Recipients that died before six weeks post-transplant due to infections were excluded from downstream analysis. EXAMPLE 2 55 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00256] Hematopoietic stem and progenitor cells (HSPCs) are capable of multilineage reconstitution of the hematopoietic system upon transplant, offering curative therapies for multiple hematological disorders. However, maintaining and expanding primary HSPC ex vivo remains challenging. Endothelial cells are a critical component of the native HSPC microenvironment in vivo, providing essential secreted cytokines and cell surface interactions that actively support the maintenance and homeostasis of resident HSPCs. Previous research indicated beneficial effects of co-culturing HSPCs in vitro with human umbilical vein endothelial cells (HUVECs), but these endothelial cells do not accurately represent native HSPC niche supportive bone marrow sinusoidal endothelial cells. Here, the inventors present a platform to derive bone marrow-like endothelial cells from human induced pluripotent stem cells (hiPSCs). By overexpressing transcription factors TFEC and MAFB, they successfully modulated the expression of HSPC niche supportive genes. Co-culture with the inventors’ engineered endothelial cells significantly enhanced the differentiation potential and engraftment capacity of primary human cord blood-derived CD34+ HSPCs. The inventors’ findings presented herein provide a novel strategy for generating bone marrow-like HSPC niche supportive endothelial cells from hiPSCs. This improves the maintenance and expansion of primary human HSPCs used in ex vivo research studies and in the clinical applications of HSPC-based cell therapies. Introduction [00257] Hematopoietic stem and progenitor cells (HSPCs) can reconstitute the multilineage hematopoietic system upon transplant, offering curative therapies for many hematological disorders (Copelan Edward A., 2006; Brunstein et al., 2010; Chabannon et al., 2018). However, it remains challenging to maintain and expand primary HSPCs ex vivo. A variety of approaches have been proposed to overcome the challenge, including HSPC supportive chemicals and cytokines, overexpression of transcriptional regulators, bioengineered extracellular matrices, and co-culture with HSPC niche supportive cell types (Dahlberg et al., 2011; Kumar and Geiger, 2017; Pineault and Abu- Khader, 2015; Wilkinson et al., 2019, 2020; Sakurai et al., 2023). [00258] Endothelial cells are the most abundant and one of the most important cell types within the natural HSPC microenvironment, and they provide secreted cytokines and cell surface contact interactions with the HSPCs (Morrison and Scadden, 2014; Wei and Frenette, 2018; Pinho and Frenette, 2019; Comazzetto et al., 2021). Previous studies have shown that endothelial cells have the potential to maintain and support primary HSPCs ex vivo in two-dimensional or three-dimensional co- culture systems (Butler et al., 2010; Kobayashi et al., 2010; Butler et al., 2012; Rafii et al., 2016a; Chou et al., 2020; Ingber, 2022). However, due to donor availability, the endothelial cells used in these studies are human umbilical vein endothelial cells, and they do not truly represent the bone marrow sinusoidal endothelial cells in the native HSPC niches. Therefore, the inventors developed a platform for bone marrow-like endothelial cells and their co-culture with primary human HSPCs. 56 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00259] A potential alternative approach to generate human bone marrow-like endothelial cells could be human induced pluripotent stem cells (iPSCs). Human iPSCs hold great potential in the scalable production of desired somatic cell types (Rowe and Daley, 2019; Ng et al., 2021). Endothelial cells have been successfully derived from human iPSCs in vitro; however, the tissue-specific heterogeneity and plasticity of the human iPSC-derived endothelial cells are very limited (Patsch et al., 2015; K. Wang et al., 2020; Gage et al., 2020; Nguyen et al., 2021; Ang et al., 2022). Thus, the inventors optimized the directed differentiation of human iPSCs into endothelial cells with bone marrow-like gene signatures and functions. [00260] Here, the inventors report herein on the derivation of endothelial cells in vitro from human iPSCs with overexpression of selective transcription factor candidates that stimulated the expression of HSPC niche supportive genes. The transcription factor candidates were selected based on differential gene expression analyses of highly upregulated transcription factors in endothelial cells isolated from HSPC niches, including the zebrafish kidney marrow and the mammalian bone marrow. They found that the combination of transcription factors TFEC and MAFB is sufficient in inducing the expression of secreted signaling molecules and cell surface proteins that are highly expressed in the native HSPC niche supportive endothelial cells in the bone marrow. Upon co-culture with the engineered endothelial cells, primary human cord blood-derived HSPCs showed better differentiation potential and improved engraftment capacity. The inventors’ findings presented herein provide a new strategy for acquiring bone marrow-like endothelial cells from hiPSCs and for improving the maintenance and expansion of human primary HSPCs in an optimized endothelial cell co-culture system. Results [00261] Generation of human iPSC lines with inducible transcription factor overexpression [00262] To achieve robust and controllable overexpression of the transcription factor candidates in hiPSCs, the inventors utilized the piggyBac transposase system to achieve high-efficiency transgene integration and stable transgene expression. The overexpression vectors have transcription factor overexpression sequences under the control of a tetracycline-inducible promoter, which allows the inventors precise temporal control of the transcription factor overexpression during endothelial cell differentiation. The overexpression vectors also have integrated tetracycline transactivator and antibiotic resistance genes, which allows the inventors to efficiently select the hiPSC clones with targeted insertion of the overexpression vectors (Figure 6A). They co-transfected the human iPSCs with the overexpression vectors and a piggyBac transposase expression vector. The transfected cells are subjected to antibiotic drug selection to select for successfully transfected clones. Following successful drug selection, PCR genotyping and sequencing were performed to validate the targeted insertion of overexpression vectors in the newly generated hiPSC lines (Figure 6B). Through this approach, the inventors generated hiPSC lines overexpressing the endothelial cell-lineage master 57 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT regulatory transcription factor ETV2, together with the transcription factor candidates TFEC, MAFB, FOXP4, HOXB8, or IRF8. [00263] Directed differentiation of human iPSCs into endothelial cells in vitro [00264] ETV2 is the master transcription factor that directs pluripotent stem cells toward endothelial cell-lineage differentiation (K. Wang et al., 2020; Ng et al., 2021; Gong et al., 2022). Here, the inventors adapted a published protocol that directed the differentiation of human iPSCs into endothelial cells through a mesoderm intermediate stage (K. Wang et al., 2020). Human iPSCs are directed into mesoderm progenitor cells by small molecule modulation, followed by doxycycline- inducible overexpression of ETV2 to differentiate these mesoderm progenitor cells into endothelial cells (Figure 7A). The transcription factor candidates, TFEC, MAFB, FOXP4, HOXB8, or IRF8, are also being overexpressed at the same time as ETV2 under the doxycycline induction. The transcription factor candidates are tagged by a fluorescent marker ZsGreen to confirm their expression. They found that the endothelial cell differentiation efficiency was not significantly changed upon the overexpression of most of the inventors identified transcription factors, with the exception of HOXB8 (Figure 7B). HOXB8 has been implicated in strong myeloid commitment of cell fate (Alharbi et al., 2013; Orosz et al., 2021), which could explain why the overexpression of HOXB8 might interfere with the endothelial cell differentiation from mesoderm progenitor cells in the experiments presented herein. Hence, the inventors excluded HOXB8 overexpression hiPSC-derived cells from the downstream analyses. Nevertheless, the inventors were able to efficiently generate CD144+ endothelial cells from hiPSC differentiation and with overexpression of the transcription factor candidates. [00265] TFEC and MAFB directed the induced endothelial cells to express HSPC niche supportive genes [00266] The inventors purified hiPSC-derived endothelial cells with the transcription factor overexpression by fluorescence-activated cell sorting (FACS). The inventors hypothesized that the transcription factors would upregulate the HSPC niche supportive genes within these engineered endothelial cells. Therefore, the inventors pursued transcriptional analyses of these FACS-isolated endothelial cells by RNA-sequencing. They first validated the expression of pan-endothelial cell marker genes, including CDH5 (CD144), KDR, and PECAM1 (CD31), and confirmed the endothelial cell identity of the transcription factor overexpression hiPSC-derived endothelial cells (Figure 8A). The inventors further analyzed markers of arteriovenous identities, including EFNB2, EPHB4, NR2F2, and PODXL, and ruled out the possibility that these transcription factors are regulating the arterial or venous differentiation of the hiPSC-derived endothelial cells (Figure 8A). Next, the inventors investigated the HSPC niche supportive genes that were previously identified with differential gene expression analyses from HSPC niche supportive endothelial cells from zebrafish kidney marrow endothelial cells and mouse bone marrow endothelial cells. Interestingly, the inventors 58 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT found that TFEC and MAFB seem to upregulate a complementary set of HSPC niche supportive genes, and the combination of TFEC and MAFB co-overexpression most effectively induced the expression of HSPC niche supportive genes (Figure 8B). [00267] The inventors further investigated whether the transcription factor overexpression induced important cell signaling molecules and surface receptors that are highly expressed in primary human bone marrow sinusoidal endothelial cells. They confirmed that the combination of transcription factors TFEC and MAFB co-overexpression indeed upregulated many of these important cytokine signaling genes and cell surface protein genes that are expressed by the primary human bone marrow endothelial cell transcriptional dataset (Bandyopadhyay et al., 2024) (Figure 8C). Furthermore, gene ontology enrichment analyses revealed that many of the genes upregulated by TFEC and MAFB in the iECs are involved in cell adhesion, migration, extravasation, and stem cell regulation (Figure 8D), suggesting that the engineered endothelial cells might upregulate critical pathways that resemble the capacity of bone marrow endothelial cells in HSPC interactions and maintenance. [00268] TFEC and MAFB overexpression in the endothelial cells better support primary human cord blood HSPCs [00269] The inventors hypothesized that the genes upregulated by the transcription factor combination TFEC and MAFB may functionally support primary human HSPCs in vitro. Thus, the inventors performed co-culture experiments using engineered iECs and primary human cord blood CD34+ cells. Equal numbers of primary human cord blood CD34+ cells are cultured in three groups: without endothelial cell co-culture, with ETV2 iECs, and with ETV2-TFEC-MAFB iECs. The co- culture was maintained in the HSPC maintenance media for five days before the HSPCs were collected for functional analyses. FACS-purified CD34+ CD45+ HSPCs were seeded in equal numbers for colony-forming unit (CFU) assay. The CFU results show that the HSPCs co-cultured with ETV2-TFEC-MAFB iECs demonstrated significantly better differentiation potential including greater numbers of CFU-GEMM (23.3±2.4 vs.10.0±2.1, p=0.004), CFU-G (89.3±13.4 vs.46.0±4.2, p=0.027), and CFU-E (42.3±3.3 vs.26.7±0.9, p=0.021) (Figure 9A). [00270] Furthermore, the inventors aimed to interrogate the in vivo engraftment potential of the co- cultured HSPCs. Therefore, the inventors harvested the post co-culture HSPCs and transplanted them into immunodeficient NOD KitW-41J Prkdc scid Il2rg tm1Wjl (NBSGW) mouse models that can support multilineage engraftment of human HSPCs without irradiation conditioning (McIntosh et al., 2015). The post co-culture HSPCs are harvested from the suspension without enzymatic dissociation from the adherent endothelial cell monolayer, followed by intravenous injection into the NBSGW mouse recipients. Four months post-transplant, retro-orbitally collected peripheral blood samples were collected from the mouse recipients, and the collected samples were analyzed with human CD45 antibody staining to detect the presence of human HSPC-derived hematopoietic cells. The inventors found that the experimental group, which received the co-cultured with ETV2-TFEC-MAFB iECs, 59 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT showed a significantly higher percentage of human CD45+ cells compared to the control group, which received human CD34+ HSPCs without co-culture (p = 0.0164 for two-tailed t-test). In contrast, the experimental group that received HSPCs co-cultured with ETV2 iECs did not show statistical significance in human CD45+ percentage (Figure 9B). The engraftment results suggested that the transcription factors TFEC and MAFB directed the HSPC niche supportive fates in the endothelial cells, which enabled these endothelial cells to functionally maintain the stem cell potential of the primary human HSPCs in an in vitro co-culture system. Discussion [00271] This study aimed to decode the transcription factor regulation of the HSPC niche supportive fate in the vascular endothelial cells using an in vitro human iPSC-derived endothelial cell model. The transcription factor candidates were identified from differential gene expression analyses between the endothelial cells from hematopoietic organs and the endothelial cells from non-hematopoietic organs across different species, previously described in Chapter Two. The inventors engineered stable piggyBac transgenic human iPSC lines with doxycycline-inducible overexpression of selective transcription factor candidates, allowing us to temporally modulate the activation of these transcription factor expression. This inducible overexpression approach was effective in modulating the transcription factor expression without significantly perturbing the endothelial cell differentiation, and the strategy could be useful to achieve temporally controlled expression of genes of interest in the hiPSC-derived endothelial cells or other somatic cell types in the field. By overexpressing the transcription factors TFEC and MAFB, the inventors were able to induce a transcriptional program in the human iPSC-derived endothelial cells that resembles primary human bone marrow endothelial cells and upregulated previously reported HSPC niche supportive genes. Overexpression of TFEC and MAFB is also sufficient in inducing a functional program in the human iPSC-derived endothelial cells, enabling them to better support the differentiation capacities and engraftment potential of the primary human cord blood CD34+ HSPCs in the endothelial cell co-culture. Therefore, data presented herein provides a deeper understanding of the transcriptional control of the HSPC niche supportive programs in the endothelial cells and may offer a potential new platform for maintaining and expanding human primary HSPCs in vitro. The findings from the data presented herein provides new insights for the directed differentiation of organ-specific endothelial cells from human iPSCs and has therapeutic potential in improving current HSPC-based cell therapies targeting hematologic disorders and malignancies. [00272] The inventors’ current approach generated stable piggyBac transgenic human iPSCs with single transcription factor overexpression (TFEC, MAFB, FOXP4, HOXB8, and IRF8) or a combination of two transcription factor candidates (TFEC and MAFB). The scientific rationale for the design is to better focus on the potential downstream targets of each of the single transcription factor candidate. In addition, the inventors were also challenged technically by the piggyBac transgenic 60 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT human iPSC generation, during which the inventors used a different drug selection for each of the transgene vectors. For instance, ETV2, TFEC, and MAFB are independently delivered by three separate transgene vectors, requiring three different drug resistance genes to fully select for successfully integrated human iPSCs with all three overexpression vectors. The inventors are establishing new transgenic human iPSC vectors with integrated expression systems for multiple transcription factors, such as multicistronic expression vectors using IRES elements or 2A self- cleavage peptides. This approach would allow them to screen for additional combinations of transcription factors, in addition to TFEC and MAFB, that may further improve the induction of HSPC niche supportive genes in the human iPSC-derived endothelial cells and stimulate better functional support of the primary human HSPCs in the endothelial cell co-culture. [00273] Furthermore, the inventors’ current endothelial cell differentiation protocol involves doxycycline-inducible expression of the pan-endothelial master transcription factor ETV2 and the transcription factor candidates that upregulate HSPC niche supportive programs in the endothelial cells. The doxycycline-inducible overexpression of these transcription factors was activated during mesodermal progenitor to endothelial differentiation steps and was maintained throughout the period of the endothelial cell and HSPC co-culture time window for continued expression of the transcriptional programs in the endothelial cells. The inventors performed preliminary experiments on the long-term culture of these hiPSC-derived endothelial cells in commercially available endothelial cell growth media, and the endothelial cells maintained their endothelial cell identity without continued doxycycline-inducible expression of the pan-endothelial master transcription factor ETV2. [00274] In previously published literature, the most important HSPC niche supportive microenvironmental cell types are the vascular endothelial cells and perivascular mesenchymal stromal cells (Morrison and Scadden, 2014; Wei and Frenette, 2018; Pinho and Frenette, 2019; Comazzetto et al., 2021). Perivascular mesenchymal stromal cells have also been reported to support primary HSPCs in in vitro co-culture systems (Pinho et al., 2013; Nakahara et al., 2019; Chou et al., 2020). The dynamics of the cell-cell interactions between HSPCs, vascular endothelial cells, and perivascular mesenchymal stromal cells will be an area of interest for further investigation. It is specifically contemplated herein that the HSPC co-culture system with both vascular endothelial cells and perivascular mesenchymal stromal cells would further improve the maintenance and expansion of the primary human HSPCs in vitro. [00275] In summary, the inventors’ current results revealed a transcription factor code that regulates the HSPC niche supportive functions in the human iPSC-derived endothelial cells. The findings presented herein will improve the HSPC-based cell therapies for hematologic disorders and malignancies. Materials and Methods [00276] hiPSC culture and maintenance 61 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00277] Human iPSCs were cultured in StemFlex pluripotent stem cell maintenance media (Gibco). The tissue culture plates for hiPSC the tissue culture plate pre-coated with hiPSC-grade Matrigel (Corning) diluted in DMEM/F-12 (Thermo Fisher). The human iPSCs were passaged to colonies with ReLeSR (STEMCELL Technologies) regularly to prevent overgrowth and spontaneous differentiation. The human iPSCs were maintained in a humidified atmosphere at 37 °C temperature level and 5% CO2 level. [00278] Generation of new transgenic cell lines [00279] Human iPSCs were dissociated into single-cell suspension using TrypLE Select Enzyme (Gibco). The dissociated human iPSCs were mixed with the transcription factor overexpression vectors and the hyperactive piggyBac transposase vector, followed by nucleofection using Cell Line Nucleofector Kit V (Lonza). The nucleofected cells were cultured in StemFlex pluripotent stem cell maintenance media (Gibco) supplemented with CloneR2 (STEMCELL Technologies) or ROCK Inhibitor Y-27632 (STEMCELL Technologies) to improve the viability of single-cell dissociated human iPSCs. Blasticidin, Neomycin, and/or Zeocin were added as previously described in the literature (Holmqvist et al., 2015; Libby et al., 2018; Blanch-Asensio et al., 2022) to select for human iPSCs that are successfully transfected with the transcription factor overexpression vectors with drug resistance genes. [00280] Endothelial cell differentiation from human iPSCs [00281] The endothelial cell differentiation protocol was adapted from a previously published protocol (K. Wang et al., 2020), with a modified doxycycline-inducible transcription factor induction method. On day 0, The human iPSCs were dissociated into single-cell suspension using StemPro Accutase Cell Dissociation Reagent (Gibco) and seeded onto Matrigel-coated tissue culture plate at a density of 30,000 cells/cm2 in StemFlex pluripotent stem cell maintenance media (Gibco) supplemented with CloneR2 (STEMCELL Technologies) or ROCK Inhibitor Y-27632 (STEMCELL Technologies). On day 1 and day 2, the cell culture media was replenished daily with mesoderm induction media. Mesoderm induction media was made with Advanced DMEM/F-12 (Thermo Fisher), 1x GlutaMax supplement (Thermo Fisher), 60 μg/ml L-Ascorbic Acid (Sigma-Aldrich), and 6 μM CHIR99021 (Sigma-Aldrich). On day 3, the mesoderm progenitor cells are dissociated into single-cell suspension using StemPro Accutase Cell Dissociation Reagent (Gibco) and seeded onto Matrigel-coated tissue culture plate at a density of 30,000 cells/cm2 in endothelial cell differentiation media. Endothelial cell differentiation media was made with Advanced DMEM/F-12 (Thermo Fisher), 1x GlutaMax supplement (Thermo Fisher), 60 μg/mL L-Ascorbic Acid (Sigma-Aldrich), 50 ng/mL FGF-2 (PeproTech), 10 ng/uL EGF (PeproTech), 50 ng/mL VEGF-A (PeproTech), 10 μM SB431542 (Selleckchem), and 1 μg/ml Doxycycline (Sigma-Aldrich). On day 4, The cell culture was replenished with fresh endothelial cell differentiation media. [00282] Induced endothelial cell sample collection 62 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT [00283] Endothelial cells are collected on day 5 of the endothelial cell differentiation protocol. The cells were dissociated into single-cell suspension using StemPro Accutase Cell Dissociation Reagent (Gibco). For flow analyses and fluorescence-activated cell sorting (FACS), the dissociated cells were incubated with BD Pharmingen Alexa Fluor 647 Mouse Anti-Human CD144 (BD Biosciences) at 1:100 dilution for 15 minutes at room temperature in the dark. DAPI (Thermo Fisher) was added to the sample immediately before FACS for the live-dead staining. FACS-purified endothelial cells were collected in TRI Reagent (Zymo Research). Direct-zol RNA Microprep kits (Zymo Research) were used to purify RNA. NEBNext Ultra II RNA Library Prep Kits (New England Biolabs) were used to prepare the RNA-sequencing libraries. For long-term culture of the human iPSC-derived endothelial cells, the cells were dissociated into single-cell suspension using StemPro Accutase Cell Dissociation Reagent (Gibco) and seeded onto Matrigel-coated tissue culture plate at a density of 30,000 cells/cm2. All endothelial cell cultures from day 5 onwards were in VascuLife Complete Endothelial Medium (Lifeline Cell Technology) supplemented with 10 μM SB431542 (Selleckchem). [00284] RNA-sequencing bioinformatic analyses [00285] RNA-sequencing libraries were sequenced by Next Generation Sequencing (NGS) services at Genewiz. For RNA-sequencing, quality control was performed by Fast QC and Cutadapt to remove adaptor sequences and low-quality regions. High-quality reads were aligned to UCSC build GRCh38/hg38 human reference genome using Tophat. The aligned reads were used for differential gene expression analyses using DESeq2 following recommendations from Harvard Chan Bioinformatic Core. [00286] Endothelial cell and HSPC co-culture [00287] Endothelial cells were dissociated into single-cell suspension using StemPro Accutase Cell Dissociation Reagent (Gibco) and seeded onto Matrigel-coated tissue culture plates at a density of 30,000 cells/cm2. In each co-culture well on a 6-well plate, 300,000 endothelial cells were seeded at least 2 hours before adding the human primary cord blood CD34+ cells.20,000 freshly thawed human cord blood CD34+ cells (STEMCELL Technologies) were added to each co-culture well pre-seeded with 300,000 endothelial cells. The co-culture cells were maintained in StemSpan SFEM II Medium (STEMCELL Technologies). [00288] Colony-forming unit (CFU) assay and transplant assay [00289] For the CFU assay, post co-cultured HSPCs were purified by fluorescence-activated cell sorting (FACS) using BD Pharmingen PE-Cy7 Mouse Anti-Human CD34 (BD Biosciences) and BD Pharmingen APC-Cy7 Mouse Anti-Human CD45 (BD Biosciences).10,000 CD34+ CD45+ HSPCs were seeded in MethoCult SF H4636 methylcellulose medium (STEMCELL Technologies) following the manufacturer's instructions. Colonies were counted after 14 days in the MethoCult culture. For the transplant assay, all cells in the suspension of the tissue culture plates were collected and transplanted into 8-week-old NOD KitW-41J Prkdcscid Il2rgtm1Wjl (NBSGW) mice through tail-vein injection. 63 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT Peripheral blood samples were collected by retro-orbital bleeding of the recipient animals 4 months post-transplant. The samples were stained for BD Pharmingen APC-Cy7 Mouse Anti-Human CD45 (BD Biosciences), and the percentage of human CD45+ cells in each sample was recorded using flow analysis. SEQUENCES SEQ ID NO: 1 a tggacctgtg gaactgggat gaggcatccc cacaggaagt 481 gcctccaggg aacaagctgg cagggcttgg taggctgccg aggctgccac aacgtgtgtg 541 gggagggtgt ccaggtgggg cctctgctga ccctaacccc ttatcgcctg cagaaggagc 601 caaattaggc ttctgtttcc ctgatctggc actccaaggg gacacgccga cagcgacagc 661 agagacatgc tggaaaggta caagctcatc cctggcaagc ttcccacagc tggactgggg 721 ctccgcgtta ctgcacccag aagttccatg gggggcggag cccgactctc aggctcttcc 781 gtggtccggg gactggacag acatggcgtg cacagcctgg gactcttgga gcggcgcctc 841 gcagaccctg ggccccgccc ctctcggccc gggccccatc cccgccgccg gctccgaagg 901 cgccgcgggc cagaactgcg tccccgtggc gggagaggcc acctcgtggt cgcgcgccca 961 ggccgccggg agcaacacca gctgggactg ttctgtgggg cccgacggcg atacctactg 1021 gggcagtggc ctgggcgggg agccgcgcac ggactgtacc atttcgtggg gcgggcccgc 1081 gggcccggac tgtaccacct cctggaaccc ggggctgcat gcgggtggca ccacctcttt 1141 gaagcggtac cagagctcag ctctcaccgt ttgctccgaa ccgagcccgc agtcggaccg 1201 tgccagtttg gctcgatgcc ccaaaactaa ccaccgaggt cccattcagc tgtggcagtt 1261 cctcctggag ctgctccacg acggggcgcg tagcagctgc atccgttgga ctggcaacag 1321 ccgcgagttc cagctgtgcg accccaaaga ggtggctcgg ctgtggggcg agcgcaagag 1381 aaagccgggc atgaattacg agaagctgag ccggggcctt cgctactact atcgccgcga 1441 catcgtgcgc aagagcgggg ggcgaaagta cacgtaccgc ttcgggggcc gcgtgcccag 1501 cctagcctat ccggactgtg cgggaggcgg acggggagca gagacacaat aa SEQ ID NO: 2 MDLWNWDEASPQEVPPGNKLAGLGRLPRLPQRVWGGCPGGASAD PNPLSPAEGAKLGFCFPDLALQGDTPTATAETCWKGTSSSLASFPQLDWGSALLHPEV PWGAEPDSQALPWSGDWTDMACTAWDSWSGASQTLGPAPLGPGPIPAAGSEGAAGQNC VPVAGEATSWSRAQAAGSNTSWDCSVGPDGDTYWGSGLGGEPRTDCTISWGGPAGPDC TTSWNPGLHAGGTTSLKRYQSSALTVCSEPSPQSDRASLARCPKTNHRGPIQLWQFLL ELLHDGARSSCIRWTGNSREFQLCDPKEVARLWGERKRKPGMNYEKLSRGLRYYYRRD IVRKSGGRKYTYRFGGRVPSLAYPDCAGGGRGAETQ SEQ ID NO: 3 atgaccc ttgatcatca gatcatcaat ccaactctta 241 aatggtcaca acctgcagtg ccaagtggtg ggcctcttgt gcagcatgca cacacaactc 301 tggacagtga tgctggcctc acagaaaacc cactcaccaa gttactagct attgggaaag 361 aagatgacaa tgcacaatgg catttatctg gaagtatttt ggatgtgtat agcggtgaac 421 aaggaatttc accaattaac atggggctta caagtgcttc ttgtccaagt agtctaccaa 481 tgaaaagaga aattacagaa actgacacta gagctttagc aaaagagaga caaaaaaagg 541 acaaccacaa cctcattgaa agaagaagaa ggtataatat taattaccga atcaaggagc 601 ttggcactct tattccaaag tctaatgatc ctgatatgcg ctggaacaaa ggaaccattc 661 taaaagcatc agtggagtac atcaagtggc tacaaaaaga acaacagaga gcccgagaat 721 tggaacacag acagaagaaa ttagagcagg ctaacaggcg acttctactt cggattcagg 781 aactagaaat tcaggctcgt actcatggtc tgccaaccct ggcttcactt ggcacggttg 841 atttaggtgc tcatgtcacc aaacagcaga gccatcctga gcagaattca gtagactatt 901 gccaacaact gactgtgtct caggggccaa gccctgagct ctgtgatcaa gctatagcct 961 tttctgatcc tttgtcatac ttcacagatt tatcatttag tgctgcattg aaagaggaac 1021 aaagattgga tggcatgcta ttggatgaca caatctctcc atttggaaca gatcctctgc 1081 tatctgccac ttcccctgca gtttccaaag aaagcagtag gagaagtagc tttagctcag 1141 atgatggtga tgaattataa 64 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT SEQ ID NO: 4 MTLDHQIINPTLKWSQPAVPSGGPLVQHAHTTLDSDAGLTENPL TKLLAIGKEDDNAQWHLSGSILDVYSGEQGISPINMGLTSASCPSSLPMKREITETDT RALAKERQKKDNHNLIERRRRYNINYRIKELGTLIPKSNDPDMRWNKGTILKASVEYI KWLQKEQQRARELEHRQKKLEQANRRLLLRIQELEIQARTHGLPTLASLGTVDLGAHV TKQQSHPEQNSVDYCQQLTVSQGPSPELCDQAIAFSDPLSYFTDLSFSAALKEEQRLD GMLLDDTISPFGTDPLLSATSPAVSKESSRRSSFSSDDGDEL SEQ ID NO: 5 atgg ccgcggagct gagcatgggg ccagagctgc 421 ccaccagccc gctggccatg gagtatgtca acgacttcga cctgctcaag ttcgacgtga 481 agaaggagcc actggggcgc gcggagcgtc cgggcaggcc ctgcacacgc ctgcagccag 541 ccggctcggt gtcctccaca ccgctcagca ctccgtgtag ctccgtgccc tcgtcgccca 601 gcttcagccc gaccgaacag aagacacacc tcgaggatct gtactggatg gcgagcaact 661 accagcagat gaaccccgag gcgctcaacc tgacgcccga ggacgcggtg gaagcgctca 721 tcggctcgca cccagtgcca cagccgctgc aaagcttcga cagctttcgc ggcgctcacc 781 accaccacca tcaccaccac cctcacccgc accacgcgta cccgggcgcc ggcgtggccc 841 acgacgagct gggcccgcac gctcacccgc accatcacca tcatcaccaa gcgtcgccgc 901 cgccgtccag cgccgctagc ccggcgcaac agctgcccac tagccacccc gggcccgggc 961 cgcacgcgac ggcctcggcg acggcggcgg gcggcaacgg cagcgtggag gaccgcttct 1021 ccgacgacca gctcgtgtcc atgtccgtgc gcgagctgaa ccgccacctg cggggcttca 1081 ccaaggacga ggtgatccgc ctgaagcaga agcggcggac cctgaagaac cggggctacg 1141 cccagtcttg caggtataaa cgcgtccagc agaagcacca cctggagaat gagaagacgc 1201 agctcattca gcaggtggag cagcttaagc aggaggtgtc ccggctggcc cgcgagagag 1261 acgcctacaa ggtcaagtgc gagaaactcg ccaactccgg cttcagggag gcgggctcca 1321 ccagcgacag cccctcctct cccgagttct ttctgtga SEQ ID NO: 6 MAAELSMGPELPTSPLAMEYVNDFDLLKFDVKKEPLGRAERPGR PCTRLQPAGSVSSTPLSTPCSSVPSSPSFSPTEQKTHLEDLYWMASNYQQMNPEALNL TPEDAVEALIGSHPVPQPLQSFDSFRGAHHHHHHHHPHPHHAYPGAGVAHDELGPHAH PHHHHHHQASPPPSSAASPAQQLPTSHPGPGPHATASATAAGGNGSVEDRFSDDQLVS MSVRELNRHLRGFTKDEVIRLKQKRRTLKNRGYAQSCRYKRVQQKHHLENEKTQLIQQ VEQLKQEVSRLARERDAYKVKCEKLANSGFREAGSTSDSPSSPEFFL SEQ ID NO: 7 atgatgg tggaatctgc ctcggagaca atcaggtcgg 541 ctccatctgg tcagaatggc gtgggcagcc tctctgggca agccgatggc agcagcggcg 601 gggccacagg gacaactgca agtggcacgg gcagggaagt gaccacgggt gcagacagca 661 atggtgagat gagtcccgca gagctgctgc acttccagca gcaacaggct ctccaagtgg 721 cccggcagtt cctgctgcag caggcctcag gcctgagctc cccagggaac aatgacagca 781 aacagtctgc ctctgctgtg caggtgcctg tgtcggtggc catgatgtcg ccgcagatgc 841 ttaccccgca acagatgcag cagatcctgt cgcccccgca gctgcaggcc ttgctccagc 901 agcagcaagc cctcatgctc cagcagctac aggagtacta caagaagcag caggagcagc 961 tccacctgca gctcctcacc cagcagcagg ctgggaaacc gcagcccaaa gaggcactgg 1021 ggaacaagca gctggccttc cagcagcagc tcctgcaaat gcaacagttg cagcagcagc 1081 acctgctcaa cctgcagagg caggggctgg tcagcctgca gcccaaccaa gcctcggggc 1141 ccctccagac ccttccgcaa gcagctgttt gcccaacaga cctgccccag ctgtggaagg 1201 gcgagggtgc ccccgggcag cctgccgagg acagcgtcaa gcaggagggg ctggacctca 1261 ctggcacggc cgccaccgct acctcgtttg ccgctccccc caaggtctca ccccccctct 1321 cccaccatac cctgcccaac ggacagccta ctgtgctcac atctcggaga gacagctctt 1381 cccacgagga gacccccggc tcccaccccc tgtacggaca cggagagtgc aagtggccag 1441 gctgtgagac cctgtgtgaa gacctgggcc agtttatcaa acacctcaac acagagcacg 1501 ccctggatga ccggagtaca gcccagtgcc gggtacagat gcaggtggtg cagcagctgg 1561 agatccagct cgccaaggag agcgagcggc tgcaggccat gatggcccac ctgcacatgc 1621 ggccctcgga gcccaagccc ttcagccagc cactgaaccc ggtccccggc tcctcctcat 1681 tctccaaggt gaccgtctct gcagcagact cattcccaga tggtctcgtg caccccccga 1741 cctcggccgc agcccctgtc acccctctac ggccccctgg cctgggctct gcctccctgc 1801 atggtggggg cccagcccgt cggagaagca gtgacaagtt ctgctccccc atctcctcag 1861 agctggccca gaatcatgag ttctacaaga acgccgacgt ccggcccccc ttcacctacg 65 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 1921 cctccctcat ccgccaggcc atcctggaaa cccctgacag gcagctgacc ctgaatgaga 1981 tctataactg gttcaccagg atgttcgcct atttccgcag aaacactgcc acctggaaga 2041 acgccgtgcg ccacaacctc agcctgcaca agtgcttcgt ccgcgtggag aacgtcaagg 2101 gtgccgtgtg gactgtggac gagcgggagt atcagaagcg gagaccgcca aagatgacag 2161 ggagccccac cctggtgaag aacatgatct ctggcctcag ctatggagca cttaatgcca 2221 gctaccaggc cgccctggcc gagagcagct tccccctcct caacagccct ggcatgctga 2281 accctggctc cgccagcagc ctgctgcccc tcagccacga tgacgtgggt gcccccgtgg 2341 agccgctgcc cagcaacggc agcagcagcc ctcctcgcct ctccccgccc cagtacagcc 2401 accaggtgca ggtgaaggag gagccagcag aggcagagga agacaggcag cccgggcctc 2461 ccctgggcgc ccctaacccc agcgcctcgg ggcctccgga agacagggac ctggaggagg 2521 agctgccggg agaagaactg tcctaa
Figure imgf000068_0001
EVTTGADSNGEMSPAELLHFQQQQALQVARQFLLQQASGLSSPGNNDSKQSASAVQVP VSVAMMSPQMLTPQQMQQILSPPQLQALLQQQQALMLQQLQEYYKKQQEQLHLQLLTQ QQAGKPQPKEALGNKQLAFQQQLLQMQQLQQQHLLNLQRQGLVSLQPNQASGPLQTLP QAAVCPTDLPQLWKGEGAPGQPAEDSVKQEGLDLTGTAATATSFAAPPKVSPPLSHHT LPNGQPTVLTSRRDSSSHEETPGSHPLYGHGECKWPGCETLCEDLGQFIKHLNTEHAL DDRSTAQCRVQMQVVQQLEIQLAKESERLQAMMAHLHMRPSEPKPFSQPLNPVPGSSS FSKVTVSAADSFPDGLVHPPTSAAAPVTPLRPPGLGSASLHGGGPARRRSSDKFCSPI SSELAQNHEFYKNADVRPPFTYASLIRQAILETPDRQLTLNEIYNWFTRMFAYFRRNT ATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEREYQKRRPPKMTGSPTLVKNMISGLS YGALNASYQAALAESSFPLLNSPGMLNPGSASSLLPLSHDDVGAPVEPLPSNGSSSPP RLSPPQYSHQVQVKEEPAEAEEDRQPGPPLGAPNPSASGPPEDRDLEEELPGEELS SEQ ID NO: 9 at gagctcttat 601 ttcgtcaact cactgttctc caaatacaaa accggggagt ccctgcgccc caattattat 661 gactgcggct tcgcccagga cctgggcggc cgacccaccg tggtgtacgg tcccagcagc 721 ggcggcagct tccagcaccc gtcgcaaatc caggagttct accacgggcc gtcgtcgctg 781 tccacggctc cctaccagca gaacccgtgc gccgtggcgt gccacgggga ccccggcaat 841 ttctacggct acgacccgct gcaacgccag agcctattcg gtgcgcagga tccagacctg 901 gtgcagtacg cagactgcaa gcttgccgcc gccagcggcc tgggcgagga ggccgagggc 961 tccgagcaga gcccgtcgcc cacacagctc ttcccctgga tgcgcccgca agcagccgcc 1021 ggacgcaggc gaggccgaca gacctacagc cgctaccaga ccctggagct ggagaaggag 1081 ttcctattta atccctatct gactcgtaag cggcgaatcg aggtatcgca cgccctggga 1141 ctgacagaga gacaggtcaa aatctggttc cagaaccgga ggatgaagtg gaaaaaagag 1201 aacaacaaag acaagttccc cagcagcaaa tgcgagcagg aggagctgga gaaacagaag 1261 ctggagcggg ccccagaggc ggcggacgag ggcgacgcgc agaagggcga caagaagtag SEQ ID NO: 10 MSSYFVNSLFSKYKTGESLRPNYYDCGFAQDLGGRPTVVYGPSS GGSFQHPSQIQEFYHGPSSLSTAPYQQNPCAVACHGDPGNFYGYDPLQRQSLFGAQDP DLVQYADCKLAAASGLGEEAEGSEQSPSPTQLFPWMRPQAAAGRRRGRQTYSRYQTLE LEKEFLFNPYLTRKRRIEVSHALGLTERQVKIWFQNRRMKWKKENNKDKFPSSKCEQE ELEKQKLERAPEAADEGDAQKGDKK SEQ ID NO: 11 atggcaggt gtcccggagt ccctgaatct 61 gatgtgtgac cggaatggtg gtcggcggct tcgacagtgg ctgatcgagc agattgacag 121 tagcatgtat ccaggactga tttgggagaa tgaggagaag agcatgttcc ggatcccttg 181 gaaacacgct ggcaagcaag attataatca ggaagtggat gcctccattt ttaaggcctg 241 ggcagttttt aaagggaagt ttaaagaagg ggacaaagct gaaccagcca cttggaagac 301 gaggttacgc tgtgctttga ataagagccc agattttgag gaagtgacgg accggtccca 361 actggacatt tccgagccat acaaagttta ccgaattgtt cctgaggaag agcaaaaatg 421 caaactaggc gtggcaactg ctggctgcgt gaatgaagtt acagagatgg agtgcggtcg 481 ctctgaaatc gacgagctga tcaaggagcc ttctgtggac gattacatgg ggatgatcaa 541 aaggagccct tccccgccgg aggcctgtcg gagtcagctc cttccagact ggtgggcgca 601 gcagcccagc acaggcgtgc cgctggtgac ggggtacacc acctacgacg cgcaccattc 66 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 661 agcattctcc cagatggtga tcagcttcta ctatgggggc aagctggtgg gccaggccac 721 caccacctgc cccgagggct gccgcctgtc cctgagccag cctgggctgc ccggcaccaa 781 gctgtatggg cccgagggcc tggagctggt gcgcttcccg ccggccgacg ccatccccag 841 cgagcgacag aggcaggtga cgcggaagct gttcgggcac ctggagcgcg gggtgctgct 901 gcacagcagc cggcagggcg tgttcgtcaa gcggctgtgc cagggccgcg tgttctgcag 961 cggcaacgcc gtggtgtgca aaggcaggcc caacaagctg gagcgtgatg aggtggtcca 1021 ggtcttcgac accagccagt tcttccgaga gctgcagcag ttctataaca gccagggccg 1081 gcttcctgac ggcagggtgg tgctgtgctt tggggaagag tttccggata tggccccctt 1141 gcgctccaaa ctcattctcg tgcagattga gcagctgtat gtccggcaac tggcagaaga 1201 ggctgggaag agctgtggag ccggctctgt gatgcaggcc cccgaggagc cgccgccaga 1261 ccaggtcttc cggatgtttc cagatatttg tgcctcacac cagagatcat ttttcagaga 1321 aaaccaacag atcaccgtct aa SEQ ID NO: 12 MAGVPESLNLMCDRNGGRRLRQWLIEQIDSSMYPGLIWENEEKS MFRIPWKHAGKQDYNQEVDASIFKAWAVFKGKFKEGDKAEPATWKTRLRCALNKSPDF EEVTDRSQLDISEPYKVYRIVPEEEQKCKLGVATAGCVNEVTEMECGRSEIDELIKEP SVDDYMGMIKRSPSPPEACRSQLLPDWWAQQPSTGVPLVTGYTTYDAHHSAFSQMVIS FYYGGKLVGQATTTCPEGCRLSLSQPGLPGTKLYGPEGLELVRFPPADAIPSERQRQV TRKLFGHLERGVLLHSSRQGVFVKRLCQGRVFCSGNAVVCKGRPNKLERDEVVQVFDT SQFFRELQQFYNSQGRLPDGRVVLCFGEEFPDMAPLRSKLILVQIEQLYVRQLAEEAG KSCGAGSVMQAPEEPPPDQVFRMFPDICASHQRSFFRENQQITV 67 4867-3387-1598.2

Claims

Attorney Docket No: 701039-000138WOPT CLAIMS What is claimed herein is: 1) A method for engineering endothelial niche cells, the method comprising expressing in a cell ETV2; and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. 2) The method of claim 1, wherein the at least two transcription factors are TFEC and MAFB. 3) The method of claim 1 or 2, wherein the cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a placenta stem cell, an adult stem cell, an amniotic stem cell, and an umbilical vein endothelial cell. 4) The method of claim 3, wherein the ESC, iPSC, placenta stem cell, adult stem cell, amniotic stem cell is differentiated to an endothelial cell prior to contact. 5) The method of any of claims 1-5, wherein ETV2 and the at least two transcription factors are expressed from at least one vector. 6) The method of claim 5, wherein the at least one vector comprises an exogenous nucleic acid sequence(s) encoding the ETV2 and the at least two transcription factors. 7) The method of any of claims 1-6, wherein expression is transient or stable. 8) The method of claim 6, wherein the exogenous nucleic acid sequence(s) is incorporated into the genome of the endothelial cell. 9) The method of any one of claims 1-8, wherein the cell is a mammalian cell. 10) The method of any one of claims 1-8, wherein the cell is a human cell. 11) The method of any one of claims 1-8, wherein the cell is a nonhuman mammalian cell. 12) The method of any of claims 1-12, wherein the engineered endothelial niche cells secrete at least one of the growth factors selected from the group consisting of: SCF/KL, CXCL12, ANGTPL2, ANGPTL4, BMP4, BMP6, FLT3L, JAG1, DLL4, FLT3L, and TPO. 68 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 13) The method of any of claims 1-12, wherein the engineered endothelial niche cells express at least one of the cell surface proteins selected from the group consisting of: MRC1, ICAM1, STAB2, VCAM1, and CD62E. 14) A method for engineering endothelial niche cells, the method comprising expressing in a cell ETV2, TFEC, and MAFB. 15) An engineered endothelial niche cell obtained by any of the claims of 1-14. 16) An engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8. 17) An engineered endothelial niche cell comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. 18) A population of cells comprising any of the engineered endothelial niche cells of claim 15-17. 19) A co-culture comprising any of the engineered endothelial niche cells of claim 15-17 or population of claim 18 and a population of stem cells. 20) The co-culture of claim 19, wherein the second population of stem cells is an HSPCs. 21) A method for increasing stem cell proliferation, the method comprising co-culturing any of the engineered endothelial niche cells of claim 15-17 or population of claim 18 and a population of stem cells for a time sufficient in increase stem cell proliferation. 22) The method of claim 21, wherein proliferation is increased by at least 10% as compared to an appropriate control. 23) The method of claim 21, wherein the population of stem cells is a population of HSPCs. 24) The method of claim 21, wherein the method is performed in vitro. 25) The method of claim 21, wherein the engineered endothelial niche cells secrete a factor that affects the proliferation of the HSPC cells. 26) A method for treating a subject, the method comprising administering any of the engineered endothelial niche cells of claim 15-17, population of claim 18, or co-culture of claims 19 or 20 into a subject in need thereof. 69 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 27) A method for treating a subject, the method comprising: a. identifying a subject in need thereof; and b. administering any of the engineered endothelial niche cells of claim 15-17, population of claim 18, or co-culture of claims 19 or 20 into the subject in need thereof. 28) A method for treating a subject, the method comprising administering the co-culture of claims 19 or 20 into a subject in need thereof. 29) A method for treating a subject, the method comprising administering a population of HSPCs that have been previously co-cultured any of the engineered endothelial niche cells of claim 15-17 or population of claim 18 into a subject in need thereof. 30) The method of claim 29, comprising the step of isolating the population of HSPCs prior to administering. 31) The method of any of claims 26-30, wherein the subject in need thereof is human. 32) The method of any of claims 26-30, wherein the subject in need thereof has a decreased blood cell level or is at risk for developing a decreased blood cell level as compared to a control blood cell level. 33) The method of any of claims 26-30, further comprising the step of identifying a subject in need thereof having a decreased blood cell level or at risk for developing a decreased blood cell level as compared to a control blood cell level prior to administering. 34) The method of claim 32 or 33, wherein the blood cell level is decreased at least 1% compared to a reference level. 35) The method of any of claims 26-30, wherein the subject in need thereof has anemia or blood loss. 36) The method of any of claims 26-30, wherein the subject in need thereof is a bone marrow donor. 37) The method of any of claims 26-30, wherein the subject in need thereof has depleted bone marrow. 38) The method of any of claims 26-30, the subject in need thereof has anemia, hemolysis, leukemia, multiple myeloma, or a thyroid disorder. 70 4867-3387-1598.2 Attorney Docket No: 701039-000138WOPT 39) The method of any of claims 26-30, wherein the administering occurs at the liver, spleen, or subcutaneously. 40) A method for generating an ectopic vascular niche, the method comprising administering any of the engineered endothelial niche cells of claim 15-17, population of claim 18, or co-culture of claims 19 or 20 to a target site in a subject in need thereof. 41) A method for treating extra medullary hematopoiesis, the method comprising administering any of the engineered endothelial niche cells of claim 15-17, population of claim 18, or co- culture of claims 19 or 20 into a subject at a location outside of the bone marrow, thereby creating a synthetic niche. 42) The method of claim 41, wherein the location is the liver, spleen, or subcutaneous. 43) The method of any of claims 21-42, wherein administering is systemic or local administration. 44) The method of claim 43, wherein local administration is transplantation. 45) The method of claim 43, wherein local administration is administration directly to the liver, spleen, or subcutaneously. 46) A vector comprising one or more exogenous nucleic acid sequences encoding ETV2 and at least two transcription factors selected from the group consisting of TFEC, MAFB, FOXP4, HOXB8, or IRF8 operably linked to a promoter. 47) A vector comprising one or more exogenous nucleic acid sequences encoding ETV2, TFEC, and MAFB. 71 4867-3387-1598.2
PCT/US2024/053916 2023-10-31 2024-10-31 Endothelial cell factors and methods thereof Pending WO2025096798A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363546653P 2023-10-31 2023-10-31
US63/546,653 2023-10-31

Publications (2)

Publication Number Publication Date
WO2025096798A2 true WO2025096798A2 (en) 2025-05-08
WO2025096798A3 WO2025096798A3 (en) 2025-07-10

Family

ID=95583326

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/053916 Pending WO2025096798A2 (en) 2023-10-31 2024-10-31 Endothelial cell factors and methods thereof

Country Status (1)

Country Link
WO (1) WO2025096798A2 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105229144A (en) * 2013-02-22 2016-01-06 细胞动力学国际有限公司 By combination genetic engineering and chemical engineering via forward programming produce liver cell
CA3009225A1 (en) * 2015-12-23 2017-06-29 Monash University Cell reprogramming
CA3094837A1 (en) * 2018-03-23 2019-09-26 The Children's Medical Center Corporation Endothelial cell factors and methods thereof

Also Published As

Publication number Publication date
WO2025096798A3 (en) 2025-07-10

Similar Documents

Publication Publication Date Title
Larsson et al. The role of Smad signaling in hematopoiesis
US9381210B2 (en) Induced pluripotent stem cell model of chronic myeloid leukemia revealed olfactomedin 4 as a novel therapeutic target in leukemia stem cells
Lin et al. Angiopoietin-like proteins stimulate HSPC development through interaction with notch receptor signaling
US20240417695A1 (en) Endothelial cell factors and methods thereof
AU2016222587B2 (en) Compositions and methods of treating Fanconi Anemia
Fazio et al. SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance
WO2025096798A2 (en) Endothelial cell factors and methods thereof
US10526402B2 (en) Anti-TGF-beta antibody for the treatment of fanconi anemia
CA3103423A1 (en) Engineered hematopoietic stem cells for the treatment of acute myeloid leukemia
Watt Dissecting the role of TGFβ in pancreatic ductal adenocarcinoma
Abdallah Investigation of the Initiation, Progression and Treatment of Pediatric Gliomas
Warsi Novel Insights into Haematopoietic Stem Cell Regulation and Function
Hao Regulation of Stress Erythropoiesis via Splenic Erythroid Microenvironment Development and Metabolic Programming
Westerlund Novel aspects of neuroblastoma: to hypoxia and beyond
Bonner Developing Novel Models and Therapeutics for ZFTA-RELA Ependymoma
Hazzazi The role of resistin-like gamma expressed by haematopoietic stem cells in controlling their maintenance and commitment to differentiation
Vaníčková Emergency granulopoiesis at the level of hematopoietic stem and progenitor cells
Mendoza-Castrejon Mechanisms of MLL-Rearranged Infant Leukemogenesis
Abbas The Consequences Of Disrupting The Mdm2-P53 Balance In Hematopoiesis
CA3237598A1 (en) Nucleoside modified mrna and uses thereof
Youshani Characterising glioma associated macrophages and microglia in glioblastoma
Almale del Barrio Crosstalk between HGF and TGF-ß signaling pathways in adult liver progenitor cells and hepatocellular carcinoma cells
WO2022269007A1 (en) A molecule capable of inhibiting the integration of calcineurin with a substrate and uses thereof
Rörby Regulation of Hematopoietic Stem Cells
Friend The Role of SAMSN1 in Multiple Myeloma