A method to generate endothelial cells

Abstract

Methods are provided for the step-wise generation of endothelial cells. Also provided are methods for generating pure populations of such cell types and derivatives thereof. The instant disclosure also provides methods of screening for cellular responses of the generated cell types and derivatives thereof. Treatment methods making use of the generated cell types and derivatives thereof are also provided. The instant disclosure also provides systems, compositions, and kits for practicing the methods of the disclosure.

Description

A METHOD TO GENERATE ENDOTHELIAL CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 560,495, filed March 1 , 2024 the disclosure of which application is herein incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under contracts HL141851 , HL166693, and HL171102 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Endothelial cells (ECs) lining the inner layer of blood vessels play pivotal roles in transporting oxygen and nutrients, regulating blood flow, modulating immune cell trafficking, and maintaining tissue homeostasis. Vascular dysfunction, resulting from genetic mutations or environmental risk factors, is central to the development of numerous chronic conditions. These include ischemic heart disease and stroke, which are the top two global causes of mortality.

[0004] Human pluripotent stem cells (hPSCs), with their unlimited expansion capacity and multilineage differentiation potential, have found extensive applications in addressing developmental questions, modeling disease pathogenesis, and advancing therapeutic development. Consequently, ECs derived from hPSCs present opportunities to comprehend vascular development defects, evaluate the potential vascular toxicity of drugs, and facilitate vasculogenesis in ischemic tissues. However, a persistent challenge lies in generating hPSC- derived ECs that fully resemble their primary counterparts to achieve maximized cellular functions.

[0005] To use hPSC-derived ECs as a medical composition or a material for research, a method for efficiently generation of ECs with high quality is desired. The term “ECs with high quality” as used herein refers to hPSC-derived ECs that closely resemble their primary counterpart at the functional level and exhibit high differentiation efficiency (high positive ratio of EC markers, e.g., >95% CD144+ / CD31+ cells). Moreover, these cells should demonstrate excellent cell proliferation capacity (e.g., >4 passages), be less susceptible to damage by freezing and thawing, and exhibit homogeneity between iterative differentiations.

[0006] ECs are prone to undergoing the Endothelial-to-Mesenchymal Transition (EndoMT) process when cultured in vitro, particularly in the presence of non-EC populations. Thisproblem is commonly encountered when utilizing extant EC differentiation methods, mainly attributable to their low differentiation efficiency. The presence of non-ECs in the differentiation system can activate the EndoMT program in ECs through a paracrine effect.

[0007] Further, the utilization of animal components, such as Matrigel and fetal bovine serum, in existing EC differentiation methods limits the application of hPSC-derived ECs in the clinical setting, e.g., in vascular regeneration treatment methods.

[0008] Current EC differentiation methods fall short in replicating an environment that closely resembles the in vivo condition by adequately modulating nutrients, pH, and osmotic pressure. Consequently, the microscopic modification or alteration of the shape, size, and characteristics of hPSC-derived ECs may occur, potentially compromising the differentiation, proliferation, and regenerative capacities of these cells.

[0009] A significant obstacle in generating highly functional endothelial cells (ECs) from human pluripotent stem cells (hPSCs) is the inability of existing differentiation methods to accurately recapitulate the developmental trajectory of ECs in vivo. Consequently, these methods frequently fail to attain high cell quality and purity. Furthermore, the inclusion of animal components in existing EC differentiation approaches poses challenges for the application of these cells in regenerative medicine. Therefore, the present invention addresses the development of a method for efficiently generating functional hPSC-derived ECs in a xeno- free and purification-free condition.SUMMARY

[0010] Compositions and methods are provided for the in vitro generation of substantially pure populations of endothelial cells. This is achieved by a controlled sequential induction of homogeneous mesodermal-lineage cells from pluripotent stem cells; of endothelial progenitor cells from mesodermal-lineage cells; and of endothelial cells from endothelial progenitors. Following the induction stages, the endothelial cells are optionally expanded in vitro. The entire differentiation process can be carried out in a xeno-free, feeder-free condition and is accomplished solely by using combinations of differentiation stage-specific extracellular signals. The invention also provides methods for screening optimal extracellular signal cocktails to achieve homogeneous mesodermal-lineage cells, endothelial progenitor cells, and endothelial cells. These methods allow for the mass production of high-purity vascular endothelial cells in large quantities, within a short time frame.

[0011] A benefit of the methods of the disclosure is the high differentiation efficiency, where the percentage of endothelial cells in the final culture is at least greater than 85%, and can be greater than about 95%, greater than about 97%, greater than 98% or more. The complete differentiation protocol from PSC to endothelial cells can be completed within about 5 days, within about 6 days, within about 7 days, within about 8 days, within about 9 days, or more.

[0012] The endothelial cells thus derived closely recapitulate primary vascular ECs at the molecular, cellular, and functional levels; maintain high viability and proliferation rates after repeated cryopreservation; and can be expanded for at least 6 passages without detectable markers indicative of endothelial to mesenchymal transition (EndoMT), for example SOX9, SLUG, and TWIST1 .

[0013] The hPSC-derived ECs derived by the methods of the disclosure find use as a cellular model of diseases relating to endothelial cells, for safety, or toxicity evaluation for clinical use of drugs that may affect endothelial cell viability and / or functions, and in transplantation, e.g. in ischemic tissues to enhance vasculogenesis, thereby facilitating the restoration of blood flow and tissue homeostasis.

[0014] In some embodiments the ECs derived by the methods of the disclosure are used to generate vessel organoids. Vessel organoids can form in the medium disclosed for endothelial cell induction and expansion. The period of time for generating a vessel organoid may be from about 6, 7, 8 or more days following endothelial cell induction. The medium for vessel organoid induction may further comprise an effective dose of a VEGF agonist and an FGF agonist.

[0015] In some embodiments a panel of genetically distinct human ECs are provided, which may be referred to as an “endothelial cell village”. A panel may comprise 5, 10, 15, 20, 25, 30 or more genetically distinct cells. In addition to endothelial cells, a panel may comprise earlier stage cells, for example, iPSCs, mesodermal lineage cells and endothelial progenitor cells, which can be generated according to the methods of the disclosure. In some embodiments a genetic difference comprises a targeted change in a gene of interest, e.g. a naturally occurring mutation, an introduced mutation, and the like. In some embodiments a genetic difference comprises a different ancestry, a different sex, a different region of origin, and the like.

[0016] Aspects of the disclosure relate to screening progenitors and / or differentiated cell types derived or produced according to the methods described herein for a cellular response. In some embodiments a panel of cells is screened, for example to determine the influence of genetic background on drug-induced cellular phenotypes. In certain aspects, a method of screening endothelial progenitors and / or differentiated endothelial cells for a cellular response may include contacting a population of endothelial progenitors and / or differentiated endothelial cells with a pharmacological agent and evaluating the population of cells for a cellular response induced by the pharmacological agent. In certain aspects, the screening may be in vitro screening and the contacting may be performed in vitro. Screening may be high throughput screening, e.g. of drug candidates. In certain aspects, the screening may be in vivo screening and the contacting may be performed by administering the pharmacological agent to a host animal that contains the population of cells.

[0017] Aspects of the disclosure relate to screening an animal for a phenotype wherein the host animal has been administered a genetically modified population of endothelialprogenitors, organoid, and / or differentiated endothelial cells derived or produced according to the methods described herein. In certain aspects, the genetically modified population of endothelial progenitors and / or differentiated endothelial cells derived or produced according to the methods described herein may include a genetic modification in at least one genetic locus. In certain aspects, the genetically modified population may include a genetic modification in at least one genetic locus resulting in disruption or deletion of at least one gene. In certain aspects, the host animal may be evaluated, or a detectable phenotype induced by the administered population of cells may be evaluated.

[0018] Aspects of the disclosure relate to methods of treating a subject for a condition through the administration of endothelial progenitors, differentiated endothelial cells, and / or organoids derived or produced according to the methods described herein. In certain aspects, the method of treating a subject for a condition through administration of cells derived according to the methods as described herein may further include co-administration with at least one pro-survival or pro-engraftment factor. In certain aspects, the cells administered to a subject may be genetically modified at least one genetic locus.

[0019] Aspects of the disclosure include kits for the production, derivation, purification, and use of endothelial progenitors and / or differentiated endothelial cells that include one or more induction compositions and / or one or more specific binding agents and / or combinations thereof. In certain aspects, such kits may or may not include one or more cell types described herein.

[0020] Aspects of the disclosure include systems for the production, derivation, purification, and use of endothelial progenitors and / or differentiated endothelial cells that include one or more components configured to administer one or more induction compositions and / or one or more specific inducing agents and / or one or more specific binding agents and / or combinations thereof. In certain aspects, such systems are configured to administer such compositions and / or agents at specific amounts or for specific periods of time according to the methods described herein.

[0021] In some embodiments, human platelet lysate (hPL) is included as a component in the medium for induction of endothelial progenitor cells, and can substitute for the fetal bovine serum in the medium. The inclusion of hPL in EC differentiation medium is highly effective in generating homogenous endothelial progenitor cells and promoting the expansion and maturation of endothelial cells.

[0022] In some embodiments, medium for expansion of the endothelial cells generated by the methods of the disclosure comprises a repressor of EndoMT. In some embodiments the repressor is one or more of simvastatin, GGTi298, chlorpromazine, and cyclosporin A. In some embodiments the repressor is present in the medium at a concentration of from about 0.5 to about 5 jiM, e.g. from around 1 ,uM to around 2 pM.

[0023] In some embodiments, the medium for induction of endothelial cells from endothelial cell progenitors, and medium for expansion of endothelial cells, comprises an effective concentration of a notch inhibitor. Induction in such medium favors the differentiation of vein endothelial cells. Medium lacking a notch inhibitor favors the differentiation of arterial endothelial cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 illustrates a strategy to generate a CDH5-eGFP reporter iPSC line, and flow cytometry data showing linear correlations between eGFP signal and CD31+ / CD34+ / CD144+endothelial cells.

[0025] FIG. 2 illustrates the schematic of the version 1 differentiation method of endothelial cells from human pluripotent stem cells.

[0026] FIG.3 shows the flow cytometry data of endothelial cell purity (CDH5-eGFP+cell ratio) using different basal media and signal inducing cocktails with different concentrations at the endothelial progenitor cell stage. The CDH5-eGFP reporter iPSC line information is shown in FIG.1 and the baseline differentiation method is shown in FIG. 2.

[0027] FIG. 4 shows the single-cell transcriptome data of endothelial progenitor cells derived using the method described in FIG. 2. UMAP plots show a non-endothelial cell cluster (C3) that are negative for endothelial cell markers.

[0028] FIG. 5 illustrates the schematic of the version 2 differentiation method of endothelial cells from human pluripotent stem cells. Two more small molecules, ML7 and Y27632, are added to the endothelial progenitor cell induction medium.

[0029] FIG. 6 shows the single-cell transcriptome data of endothelial progenitor cells derived using the method described in FIG. 5. UMAP plots show that the non-endothelial cell cluster (C3) is significantly reduced compared to that in FIG. 4.

[0030] FIG. 7 shows the receptor-ligand crosstalk between different cell clusters of the endothelial progenitor cells derived using methods from FIG. 2 and FIG. 5. Single-cell data analysis shows that upregulated Wnt activity specifically contributes to the emergence of the non-endothelial cell cluster (C3).

[0031] FIG. 8 illustrates the schematic of the version 3 differentiation method of endothelial cells from human pluripotent stem cells. One more small molecule, Wnt-059, a Wnt inhibitor, is added to the endothelial progenitor cell induction medium.

[0032] FIG. 9 shows the flow cytometry data of endothelial cell purity (CDH5-eGFP+cell ratio) using the differentiation method described in FIG. 8. The seeding density of iPSCs plays an important role in the differentiation efficiency of endothelial cells. The CDH5-eGFP reporter iPSC line information is shown in FIG.1 .

[0033] FIG. 10 shows the bright field images of highly pure endothelial cells differentiated from four iPSC lines using the method described in FIG. 8. Immunofluorescence images show the expression of CD144 (also known as VE-Cadherin) in these endothelial cells.

[0034] FIG. 11 shows the bright field images of endothelial cells with varying purities differentiated from another six iPSC lines using the method described in FIG. 8. These data suggest that the method described in FIG. 8 is not robust enough to give rise to pure endothelial cells across different iPSC lines.

[0035] FIG. 12 shows a novel method denoted as “Baseline Wnt activation” to modulate the overall Wnt signal during mesodermal-lineage cell differentiation. Immunofluorescence images of p-catenin show that 8 pM of CHIR99021 and 1 pM of Wnt-C59 can uniformly activate Wnt activity (nuclear p-catenin signal).

[0036] FIG. 13 shows that the addition of 0.25 pM of a CDK8 inhibitor, BI1347, to the mesoderm induction medium described in FIG. 12 further improve the homogeneity of mesodermal-lineage cells.

[0037] FIG. 14 shows a heatmap of the global gene expression patterns of mesodermal- lineage cells derived using different small molecule cocktails described in FIG. 12 and FIG. 13. The transcriptome profile of mesodermal-lineage cells is drastically different after the GiWiCi cocktail (CHIR99021+Wnt-C59+BI1347) treatment when compared with those from Gi (CHIR99021 ) or GiWi (CHIR99021 + Wnt-C59) groups.

[0038] FIG. 15 shows a correlation map of the transcriptome profile similarities of mesodermal-lineage cells treated with Gi, GiWi, and GiWiCi.

[0039] FIG. 16 shows a volcano plot of differentially expressed genes (DEGs) between mesodermal-lineage cells derived using Gi vs GiWi. Based on the DEGs in each group, the GiWi-mesodermal cells show a higher potential to give rise to endothelial cells.

[0040] FIG. 17 shows a volcano plot of differentially expressed genes (DEGs) between mesodermal-lineage cells derived using Gi vs GiWiCi. Based on the DEGs in each group, the GiWiCi-mesodermal cells show a higher potential to give rise to endothelial cells.

[0041] FIG. 18 shows a volcano plot of differentially expressed genes (DEGs) between mesodermal-lineage cells derived using GiWi vs GiWiCi. Based on the DEGs in each group, the GiWiCi-mesodermal cells show a higher potential to give rise to endothelial cells.

[0042] FIG. 19 shows a Gene Ontology Pathway analysis of the upregulated biological process in the GiWiCi group of mesodermal-lineage cells compared to those in the Gi and GiWi groups. The data suggest that GiWiCi-treated mesodermal-lineage cells exhibit a high potential to form endothelial cells.

[0043] FIG. 20 shows the bright field images of mesodermal-lineage cells derived using the GiWiCi method from three iPSC lines. Replacing bovine serum albumin (BSA) with humanserum albumin (HSA) in the basal chemically defined medium (CDM) further improve the homogeneity and cell viability of mesodermal-lineage cells.

[0044] FIG. 21 shows the immunofluorescence images of |3-catenin signals in the treatment groups described in FIG. 20. Mesodermal-lineage cells derived using CDM-HSA medium but not CDM-BSA medium give rise to more homogenous nuclear |3-catenin across three different iPSC lines.

[0045] FIG. 22 illustrates the schematic of the version 4 differentiation method of endothelial cells from human pluripotent stem cells based on the data from FIG. 20 and FIG. 21 .

[0046] FIG. 23 shows the bright field images of endothelial cells derived using the method described in FIG. 22. The cells can be further expanded for 8 more passages without changing the cell morphology.

[0047] FIG. 24 shows the bright field images of endothelial cells derived using the method described in FIG. 22. However, cells in different groups were treated with different levels (0.05%-0.5%) human platelet lysate (hPL) in the endothelial progenitor cell induction medium. Flow cytometry data show that 0.05%-0.2% hPL can give rise to highly pure (>97%) endothelial cells. Moreover, the addition of hPL can significantly shorten the differentiation time from 11 -14 days to 5-6 days.

[0048] FIG. 25 shows the bright field images of endothelial cells derived using the method described in FIG. 22. However, cells in different groups were treated with different levels (0.05%-0.5%) knockout serum replacement (KSR) in the endothelial progenitor cell induction medium. Flow cytometry data show that 0.05%-0.5% hPL can give rise to highly pure (>99%) endothelial cells. However, the addition of KSR does not shorten the differentiation time of endothelial cells.

[0049] FIG. 26 shows the bright field images of endothelial cells derived using the method described in FIG. 22. However, cells in different groups were treated with different levels (0.05%-0.5%) fetal bovine serum (FBS) in the endothelial progenitor cell induction medium. Flow cytometry data show that 0.05%-0.5% hPL can give rise to highly pure (>98%) endothelial cells. However, the addition of FBS does not shorten the differentiation time of endothelial cells.

[0050] FIG. 27 illustrates the schematic of the version 5 differentiation method of endothelial cells from human pluripotent stem cells based on the data from FIG. 24 to FIG. 26. Moreover, by substituting EGM2 from Lonza (containing FBS) with EndoGO from Sartorius, the entire differentiation system is animal component-free, except the extracellular matrix (Matrigel).

[0051] FIG. 28 shows the bright field images of endothelial cells derived using the method described in FIG. 27. However, culture vessels are coated with animal component-free extracellular matrices, including laminin 521 , vitronectin, and i Matrix-511 . Flow cytometry datashow that endothelial cells derived using the three matrices show similar purity when compared with those derived using Matrigel.

[0052] FIG. 29 shows the flow cytometer data of endothelial cell markers (CD31 and CD144) derived in a xeno-free condition (matrix: vitronectin and method in FIG. 27). These cells do not lose either endothelial maker at least after 5 passages of subcultures.

[0053] FIG. 30 shows the bright field images of endothelial cells at passage 6 from four iPSC lines. The differentiation system is the same as those described in FIG. 29.

[0054] FIG. 31 shows the bright-field images of endothelial cells differentiated using the method described in FIG. 27. However, mesodermal-lineage cells are not dissociated and replated for further endothelial progenitor cell differentiation. Instead, iPSCs are seeded at a low density (104 / cm2) for mesodermal cell differentiation for 48 hours, and endothelial progenitor cell induction medium containing 0%, 0.1%, or 0.2% hPL is added for further differentiation. Flow cytometry data show that the differentiation efficiency of endothelial cells is significantly decreased when mesodermal-lineage cells are not dissociated before endothelial progenitor cells are further differentiated.

[0055] FIG. 32 shows the immunofluorescence images of endothelial makers (CD31 and CD144 / VE-Cadherin) and the ability of the cells to uptake acetylated low density lipoprotein.

[0056] FIG. 33 shows the immunofluorescence images of fibroblast marker (TE-7), smooth muscle cell markers (Sm22a and aSMA), and pericyte marker (PDGFRp), which are negative in endothelial cells derived using the method described in FIG. 27.

[0057] FIG. 34 shows significantly increased monocyte (THP1 ) adhesion on endothelial cells after TNFa treatment. These cells also highly express adhesion molecules ICAM-1 and VCAM-1 .

[0058] FIG. 35 shows significantly upregulated gene expression levels of cell adhesion molecules ICAM1 , VCAM1 , CCL2, and SELE in endothelial cells after TNFa treatment.

[0059] FIG. 36 shows the tube formation capacity of endothelial cells derived using the method described in FIG 27.

[0060] FIG. 37 shows the single-cell data analysis of endothelial cells derived using the method described in FIG. 27. The majority of endothelial cells express arterial endothelial markers and negative for most venous endothelial and endothelial-to-mesenchymal transition (EndoMT) makers.

[0061] FIG. 38 shows the transcriptome profile similarities between endothelial cells derived using the method described in FIG. 27 and primary organotypic endothelial cells. Among all the six organs examined, the endothelial cells predominantly acquire a cardiac phenotype.

[0062] FIG. 39 shows that the transcriptome profile of the endothelial cells derived using the method described in FIG. 27 similar to that of mature primary cardiac endothelial cells.

[0063] FIG 40. The in-house developed CDM-HSA-based xeno-free medium (version 6.0) demonstrates comparable efficacy to the commercially available EndoGO medium in supporting the expansion of iPSC-derived endothelial cells for at least five passages, without compromising cell purity. The endothelial cell (EC) expansion medium recipe includes 2% human platelet lysate, 25 ng / ml VEGF165, 5 ng / ml EGF, 20 ng / ml IGF, 1 pM hydrocortisone, and 5 pM SB431542, all in a CDM-HSA basal medium. Bright-field imaging and flow cytometry data confirm the high purity of iPSC-derived endothelial cells (CD144+ cells) across different passages.

[0064] FIG 41 . Our iPSC-EC version 5.0 protocol can be efficiently adapted to generate vessel organoids. Both commercially available endothelial cell growth media (e.g., EndoGO and EGM2) and our in-house developed CDM-HSA-based xeno-free EC expansion medium, supplemented with 100 ng / ml VEGF165 and 100 ng / ml FGF2, support the formation and maturation of vessel organoids between days 4 and 12. Bright-field images illustrate the morphological changes of vessel organoids throughout the differentiation process.

[0065] FIG 42. Both optimal cutting temperature (OCT) sections and whole-mount imaging confirm the presence of an extensive vascular network within the vessel organoid at day 12 of differentiation. Endothelial cells are labeled with antibodies against CD31 and VE-cadherin, while pericytes are labeled with PDGFRp.

[0066] FIG 43. The small molecules simvastatin, GGTi298, chlorpromazine, and cyclosporin A exhibit a potent repressive effect on the endothelial-to-mesenchymal transition (EndoMT) process during the expansion of iPSC-derived endothelial cells. Flow cytometry data indicate that, in the absence of EndoMT repressors, the purity (CD144+ cells) of iPSC-derived endothelial cells is significantly reduced by passage 6 compared to those at passage 1. In contrast, simvastatin, GGTi298, chlorpromazine, and cyclosporin A preserved the purity of iPSC-derived endothelial cells at passage 6.

[0067] FIG 44. iPSC-derived endothelial cells using protocol version 5.0 express endothelial- specific genetic markers (CD31 , VE-cadherin, ZO-1 ) and mature markers (eNOS and vWF), while lacking expression of mesenchymal (aSMA), fibroblast (TE-7), smooth muscle cell (Sm22a), and pericyte (PDGFRp) markers.

[0068] FIG 45. The inclusion of a NOTCH inhibitor, RO4929097, in our original version 5.0 protocol (new protocol referred to as version 5.0’) from the endothelial cell progenitor stage and throughout the remaining expansion stage (day 2 onward) effectively preserves a vein endothelial cell phenotype. Flow cytometry data reveal a dose-dependent effect of the NOTCH inhibitor in promoting the generation of vein endothelial cells.

[0069] FIG 46. The robustness of our iPSC-endothelial cell differentiation protocol is validated across 30 iPSC lines, collectively referred to as “Cell Village”, which are pooled to represent diverse sexes and ancestral backgrounds (European, African, and East Asian). Co-differentiated endothelial cells in the cell village are demultiplexed using each donor's unique single-nucleotide polymorphism profile. Pooled cells at the iPSC, mesoderm (MES), endothelial progenitor cell (EPC), and passage 0 endothelial cell (ECPO) stages are harvested for single-cell RNA sequencing. Additionally, endothelial cells at passage 1 (ECP1 ), with and without carfilzomib (ECP1 CFZ) treatment, are included in the analysis.

[0070] FIG 47. The proportions of each iPSC line within the pool at the iPSC, mesoderm (MES), endothelial progenitor cell (EPC), passage 0 endothelial cell (ECPO), passage 1 endothelial cell (ECP1 ), and carfilzomib-treated passage 1 endothelial cell (ECP1 CFZ) stages are depicted in pie charts.

[0071] FIG 48. UMAPs illustrate the distribution of canonical endothelial cell markers across the cell populations described in FIG 46.

[0072] FIG 49. UMAPs illustrate the distribution of arterial endothelial cell markers across the cell populations described in FIG 46.

[0073] FIG 50. UMAPs illustrate the distribution of vein endothelial cell markers across the cell populations described in FIG 46.

[0074] FIG 51. UMAPs illustrate the distribution of endocardial markers across the cell populations described in FIG 46.

[0075] FIG 52. UMAPs illustrate the distribution of endothelial-to-mesenchymal transition markers across the cell populations described in FIG 46.

[0076] FIG 53. Violin plots demonstrate no differences in total cell counts between sexes or ancestral backgrounds.

[0077] FIG 54. Principal component analysis (PCA) plots reveal that the differentiation capacity of iPSC-derived endothelial cells is largely independent of sex and ancestry, highlighting the robustness of the differentiation program across diverse genetic backgrounds.

[0078] FIG 55. Box plots show no significant difference in carfilzomib (CFZ) response between males and females. However, a one-way ANOVA revealed a significant difference in CFZ responses among ancestry groups (F = 4.01 , p = 0.029). Post-hoc Tukey’s HSD tests identified a significant difference between the African and East Asian groups (mean difference = 42.9, p = 0.0398), with the African ancestry group exhibiting a larger decrease in cell counts post-treatment. No significant differences were observed between the African and European groups (p = 0.0736) or the East Asian and European groups (p = 0.9550). Several outliers were identified, with certain samples displaying extreme positive or negative CFZ responses, likely reflecting individual-specific variability in treatment response. This key feature underscores the potential of our iPSC-endothelial cell village as a scalable platform for studying population-level pharmacogenomics in vitro, a concept referred to as “populationscale clinical trial-in-dishes”.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0079] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0080] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0081] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0082] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0083] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0084] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission thatthe present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Definitions

[0085] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting development of a disease and / or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and / or symptom(s).

[0086] The terms “recipient”, “individual”, “subject”, “host”, and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human.

[0087] The term "pluripotent stem cell," as used herein, denotes a stem cell characterized by its capability to differentiate into various cell types and exhibit self-proliferation ability. Pluripotent stem cells include, but are not limited to, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), germline stem cells (GS cells), embryonic germ cells (EG cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer ES cells; ntES cells), fused stem cells, and similar entities. A preferred pluripotent stem cell is an iPS cell or an ES cell, with an iPS cell being more preferred.

[0088] An iPS cell is an artificial stem cell derived from somatic cells, possessing properties nearly identical to those of an ES cell. These shared characteristics include differentiation pluripotency and the ability to self-replicate and proliferate. The production of iPS cells can be achieved by introducing specific nuclear reprogramming substances, either in the form of nucleic acids or proteins, into a somatic cell. Alternatively, iPS cells can be generated by elevating the expression levels of endogenous mRNAs and / or proteins of the nuclear reprogramming substances through the use of specific agents. (K. Takahashi and S. Yamanaka (2006), Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131 : 861 -872; J. Yu etal. (2007), Science, 318: 1917-1920; and M. Nakagawa et al. (2008), Nat. Biotechnol., 26: 101 -106). The nuclear reprogramming substance may be a gene specifically expressed in an ES cell, or a gene or a gene product thereof playing an important role in maintenance of undifferentiated state of an ES cell. The substance includes, for example, but not particularly limited to, OCT3 / 4, KLF4, KLF1 , KLF2, KLF5, SOX2, SOX1 , SOX3, SOX15, SOX17, SOX18, C-MYC, L-MYC, N-MYC, TERT, SV40 large T antigen, HPV16 E6, HPV16 E7, BMIL, LIN28, LIN28b, NANOG, ESRRB or ESRRG. These nuclear reprogramming substances may be used in combination when establishing iPS cell. For example, the combination includes at least one, two or three, and preferably the combination includes four of the above nuclear reprogramming substances.

[0089] Human iPS cell line established as a cell line may be used in the embodiment of the present invention. Patient-specific iPSC lines may be used, for example, to generate ECs for the potential applications. Furthermore, ECs generated using haplotype human iPS cell lines can function as readily available, off-the-shelf products tailored for specific human subpopulations.

[0090] The ES cell is a type of stem cell derived from the inner cell mass of an early embryo, such as a blastocyst, in mammals like humans or mice. It possesses both differentiation pluripotency and the ability to self-replicate and proliferate. The ES cell has been discovered in mice in 1981 (M. J. Evans and M. H. Kaufman (1981 ), Nature 292: 154-156), and subsequently, the ES cell has been established also in primates such as human and monkey.

[0091] The ES cell can be established by extracting an inner cell mass from a blastocyst of a fertilized egg of a subject animal and culturing the inner cell mass on a feeder of fibroblasts. In addition, the maintenance of cells by subculturing can be carried out by using a medium supplemented with a substance such as LIF or bFGF. The method for establishment and maintenance of ES cell of human and monkey is described in, for example, H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; H. Kawasaki et al. (2002), Proc. Nati. Acad. Sci. USA, 99: 1580-1585, and the like.

[0092] The cultivation of pluripotent stem cells is preferably accomplished by subjecting cells, prepared through any suitable methods, to adhesion culture in a suitable culture vessel or on a culture substrate. In this context, adhesion culture involves cultivating cells in a state where they adhere to a culture vessel or substrate, without the formation of embryoid bodies (EB). This adhesion culture is conducted using a culture vessel or substrate coated with a substance capable of promoting cell adhesion. Such substances include various extracellular matrices (such as collagen, gelatin, laminin, fibronectin, vitronectin, entactin, heparan sulfate proteoglycan, etc.), their altered or modified products, polylysine, or a combination thereof. The methods of the disclosure may utilize animal component-free matrices, such as recombinant vitronectin, laminin 521 , or iMatrix 51 1 (TM).

[0093] For this step and other culturing steps in the present invention, culture vessels or substrates with diverse materials or shapes can be employed, as long as they do not impede the maintenance, viability, differentiation, maturation, or self-replication of the cells. The shape of the culture vessels or substrates is not restricted and may include options such as flasks, plates, dishes, bags, and incubators. Various commercially available culture vessels or substrates can be utilized. Additionally, culturing using a culture vessel equipped with a hollow thread or a culture substrate, such as microcarriers, is also feasible.

[0094] As a basal medium for hPSCs, any medium suitable for the culture of animal cells may be employed. Examples include RPMI 1640 medium, DEF-OS medium, Medium 199, MCDB131 medium, IMDM, EMEM, aMEM, DMEM, Ham's F12 medium, Fischer's medium, or a combination thereof. A commercially available medium sold as a medium for culturing pluripotent stem cells, for example, StemFit (registered trademark), mTeSR 1 or mTeSR plus (registered trademark), Essential 8 (registered trademark), StemFlex (registered trademark), StemMACS™ iPS-Brew XF (registered trademark), NutriStem® hPSC XF Medium (registered trademark) or the like may be used. StemMACS™ iPS-Brew XF may be used for hPSC maintenance.

[0095] Cells are seeded onto a culture vessel coated with a substance promoting cell adhesion at a density ranging, for instance, from 0.5x104to 2.5x104cells / cm2, preferably from 1 x104to 1 .6x104cells / cm2. The cells are then cultured in a suitable medium. The culturing period for each stage of intermediate cells may extend for 48 hours or more, with the duration determined based on the confluency of differentiating cells. Additionally, medium exchanges or subculturing may be appropriately performed during the culturing period.

[0096] The method for separating hPSCs that have undergone adhesion culture and other cells in the present invention from a culture vessel encompasses various techniques. These include physical methods, chelator-based methods, and enzymatic methods utilizing a separating solution with protease and / or collagenase activity. In some instances, the dissociation of the pluripotent progenitors is chemical, molecular (e.g., enzyme mediated), or mechanical dissociation. Methods of chemical, molecular, and / or enzyme mediated dissociation will vary and in some instances may include but are not limited to the use of, e.g., trypsin, TrypLE Express™, TrypLE Select™, Accutase®, StemPro® (Life Technologies, Inc., Grand Island, N.Y.), calcium and magnesium free media, low calcium and magnesium medium, or the like, and a combination thereof. After dissociating pluripotent stem cell colonies through an enzymatic approach, a method involving the physical dispersion of cells can be employed. Typically, the separating operation is performed when hPSCs reach approximately 80% confluence in relation to the utilized culture vessel.

[0097] The terms “progenitor cells”, “progenitors”, “pluripotent stem cells”, “multipotent progenitor cells” and the like, as used herein refer to cells that are capable of differentiating into two or more different cell types and proliferating. Non limiting examples of multipotent progenitor cells include but are not limited to ectodermal derived stem cells, endodermal derived stem cells, mesodermal derived stem cells, neural crest cells, amniotic stem cells, cord blood stem cells, adult or somatic stem cells, neural stem cells, bone marrow stem cells, bone marrow stromal stem cells, hematopoietic stem cells, lymphoid progenitor cell, myeloid progenitor cell, mesenchymal stem cells, epithelial stem cells, adipose derived stem cells, skeletal muscle stem cells, muscle satellite cells, side population cells, intestinal stem cells, pancreatic stem cells, liver stem cells, hepatocyte stem cells, endothelial progenitor cells, hemangioblasts, gonadal stem cells, germline stem cells, and the like.

[0098] Of interest herein are pluripotent progenitors having the capacity to generate endothelial cells or derivatives thereof, through a step-wise progression of specific progenitor cells. Induced pluripotent stem cells (iPSC) may find use for this purpose.

[0099] Aspects of the disclosure include methods for deriving endodermal cells from pluripotent progenitor cells. Pluripotent progenitors of the instant disclosure may be acquired from any convenient source, including but not limited to newly derived from a subject of interest or tissue specimen or other cellular sample, obtained from a public repository, obtained from a commercial vendor, and the like. In some instances, pluripotent cells of interest include human cells including but not limited to, e.g., human embryonic stem cells, human induced pluripotent stem cells, human fetal stem cells, and the like.

[0100] In some instances, pluripotent progenitor cells of the subject disclosure may be unmodified such that the cells have not been genetically or otherwise modified from their natural state prior to modification according to the methods described herein. In other instances, pluripotent progenitor cells of the subject disclosure may be unmodified such that the cells have been genetically or otherwise modified from their natural state prior to modification according to the methods described herein. Modification of pluripotent progenitors and derived endodermal cells is described in further detail elsewhere herein.

[0101] The terms “endothelial progenitors” and “endothelial progenitor cells” are used interchangeably herein and generally refer to precursor and / or progenitor cells capable of directly giving rise to one or more endothelial cell types and proliferating.

[0102] The terms “differentiated endothelial cell types” and “differentiated endothelial cells” are used interchangeably herein and refer to cells that are terminally differentiated and are readily identifiable as such. Such differentiated endothelial cells may or may not beproliferative. As described herein, mesodermal cells include those adult cell types and cells of adult tissues derived from endothelium that are well-known to the ordinary skilled artisan.

[0103] Endothelial cells line the inside of all blood and lymphatic vessels, and have key roles in delivering oxygen and nutrients, regulating blood flow, modulating immune cell trafficking and maintaining tissue homeostasis. Endothelial cells may have tissue specific features. Endothelial cells induced by the methods of the disclosure may be characterized by the presence of cell surface and cytoplasmic markers. In some embodiments, the cells are CDH5 (CD144, VE-cadherin)+and PECAM1 (CD31 )+.

[0104] Additional markers typically expressed by endothelial cells that may be used to characterize endothelial cells produced by the methods of the disclosure include, without limitation, Endoglin (CD105), von Willebrand factor (vWF), kinase insert domain receptor (KDR), FOXF1 , BMP4, MOX1 , SDF1 , and similar markers. Endothelial progenitor cells may be characterized by KDR, also known as vascular endothelial growth factor receptor-2 (VEGFR-2) or Flk-1 expression. The cells may also be characterized by the absence of markers such as fibroblast marker (TE-7), smooth muscle cell markers (Calponin, Sm22a and aSMA), and pericyte marker (PDGFRp), which are negative in endothelial cells.

[0105] Human vein endothelial cells (VECs) and arterial endothelial cells (AECs) can be distinguished based on the expression of specific molecular markers related to their distinct physiological functions. Key markers include Ephrin type-B receptor 4 (EPHB4), which is highly expressed in vein endothelial cells, serving as a crucial marker for venous identity. Ephrin-B2 (EFNB2) is selectively expressed in arterial endothelial cells, marking arterial lineage. Notch signaling components, e.g., NOTCH1 , DLL4 are enriched in arterial endothelial cells. The transcription factor NR2F2 is highly expressed in vein endothelial cells, and absent in arterial cells.

[0106] The ECs obtained through the present disclosure typically contain CD31 -positive cells in a ratio of, for example, 85% or more, 90% or more, 95% or more, 98% or more, without limiting the scope of the present invention to these values. The cells maintain high viability and proliferation rates after repeated cryopreservation; and can be expanded for at least 6 passages without detectable markers indicative of endothelial to mesenchymal transition (EndoMT), for example SOX9, SLUG, and TWIST1.

[0107] The term “population”, e.g., “cell population” or “population of cells”, as used herein means a grouping (i.e. , a population) of two or more cells that are separated (i.e., isolated) from other cells and / or cell groupings. For example, a 6-well culture dish can contain 6 cell populations, each population residing in an individual well. The cells of a cell population can be, but need not be, clonal derivatives of one another. A cell population can be derived fromone individual cell. For example, if individual cells are each placed in a single well of a 6-well culture dish and each cell divides one time, then the dish will contain 6 cell populations.

[0108] The cells of a cell population can be, but need not be, derived from more than one cell, i.e. non-clonal. The cells from which a non-clonal cell population may be derived may be related or unrelated and include but are not limited to, e.g., cells of a particular tissue, cells of a particular sample, cells of a particular lineage, cells having a particular morphological, physical, behavioral, or other characteristic, etc. A cell population can be any desired size and contain any number of cells greater than one cell. For example, a cell population can be 2 or more, 10 or more, 100 or more, 1 ,000 or more, 5,000 or more, 104or more, 105or more, 106or more, 107or more, 108or more, 109or more, 1010or more, 1011or more, 1012or more, 1013or more, 1014or more, 1015or more, 1016or more, 1017or more, 1018or more, 1019or more, or 102° or more cells.

[0109] The terms “homogenous population”, as it relates to cell populations, refers to a cell population that is essentially pure and does not consist of a significant amount of undesired or contaminating cell types. By significant amount, in this context, is meant an amount of undesired or contaminating cell types that negatively impacts the use of the isolated desired cell population. As such, the actual amount of undesired or contaminating cells that defines a significant amount will vary and depend on the particular type of undesired or contaminating cells and / or the particular use of the desired cell type. For example, in a population of differentiated endothelial cells used in the treatment of a subject, a significant amount of improperly differentiated contaminating cell types will be small, as such cells may a high capacity to negatively impact the use of the generated desired cell population.

[0110] In some instances, a homogenous population refers to a highly enriched population. Levels of homogeneity will vary, as described, and may, in some instances, be greater than 80% pure, including e.g., more than 85%, more than 90%, more than 95%, more than 98%.

[0111] The term “heterologous”, as it refers to a “heterologous sequence” or “heterologous nucleic acid”, means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.Methods

[0112] The generation of endothelial cell types from pluripotent progenitors as described herein generally involves sequential lineage restriction events in which cultured pluripotent progenitor cells are subjected to treatments causing the cultured cells or a population thereofto take on the features of endothelial cells. In certain instances, lineage restriction events may be performed successively such that a first mesodermal cell type may be achieved by a first linage restriction event and the first cell type may be subjected to a second lineage restriction event to achieve a desired second endothelial progenitor cell, and then differentiated endothelial cells. This is achieved by a controlled sequential induction of homogeneous mesodermal-lineage cells from pluripotent stem cells; of endothelial progenitor cells from mesodermal-lineage cells; and of endothelial cells from endothelial progenitors.

[0113] Homogeneous populations of endothelial cells are produced by contacting cells with various signaling pathway modulators that promote the formation of a desired cell type and various signaling pathway modulators that block the formation of other cell types.

[0114] Lineage restriction events as described herein may be induced by induction compositions wherein an induction composition is a composition that contains one or more induction agents useful in guiding cellular development or lineage restricting a cell along a particular lineage. Induction agents include those agents that activate or inhibit particular developmental signaling pathways that drive development. Such signaling pathways that may be activated or inhibited by induction agents include but are not limited to those signaling pathways that upon activation and inhibition generally promote mesodermal and endothelial cell differentiation. As will be clear from the instant disclosure, whether activation or inhibition of a particular signaling pathway is necessary to generate a particular cell type of interest will depend on a number of factors including but not limited to, e.g., the particular desired cell type, the timing of use of the particular inductive agent and / or induction composition, the starting cell type to be induced, etc.

[0115] In some instances, an agent useful in a particular induction composition may include an inhibitor of the TGF-beta (transforming growth factor |3 (TGF-p)) pathway. Inhibitors of the TGF-beta pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the TGF-beta pathway resulting in a corresponding activation or inhibition in cellular TGF-beta signaling. Components and downstream effectors of the TGF-beta pathway include but are not limited to, e.g., 14-3-3 e (UniProtID P62258), ark (UniProtID Q6ZNA4), axinl (UniProtID 015169), bambi (UniProtID Q13145), beta arrestin 2 (UniProtID P32121 ), beta catenin (UniProtID P35222), beta glycan (UniProtID Q03167), camkiia (UniProtID Q9UQM7), caveolin-1 (UniProtID Q03135), ctgf (UniProtID P29279), dab2 (UniProtID P98082), dapper2 (UniProtID Q5SW24), daxx (UniProtID Q9UER7), eif2a (UniProtID Q9BY44), elf (UniProtID Q01082), endofin (UniProtID Q7Z3T8), fkbpl 2 (UniProtID P62942), gadd34 (UniProtID 075807), grb2 (UniProtID P62993), itch (UniProtID Q96J02), km23-1 (UniProtID Q9NP97), nedd4-2 (UniProtID Q96PU5), ocln(UniProtID Q16625), p70s6k (UniProtID P23443), par6 (UniProtID Q9NPB6), pdk1 (UniProtID 015530), pml (UniProtID P29590), ppplca (UniProtID P62136), ppp2ca (UniProtID P67775), ppp2cb (UniProtID P62714), ppp2r2a (UniProtID P63151), rhoa (UniProtID P61586), sara (UniProtID 095405), she (UniProtID P29353), smad2 (UniProtID Q15796), smad3 (UniProtID P84022), smad4 (UniProtID Q13485), smad7 (UniProtID 015105), smurfl (UniProtID Q9HCE7), smurf2 (UniProtID Q9HAU4), snon (UniProtID P12757), sos1 (UniProtID Q07889), strap (UniProtID Q9Y3F4), tab1 (UniProtID Q15750), tab2 (UniProtID Q9NYJ8), tak1 (UniProtID 043318), TGFB1 (UniProtID P01137), TGFB2 (UniProtID P61812), TGFB3 (UniProtID P10600), tgfbrl (UniProtID P36897), tgfbr2 (UniProtID P37173), trap-1 (UniProtID 060466), wwp1 (UniProtID Q9HOMO), xiap (UniProtID P98170), yap65 (UniProtID P46937), and the like.

[0116] Inhibitors of the TGF-beta pathway include but are not limited to, e.g., A-83-01 (3-(6- Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1 H-pyrazole-1 -carbothioamide), D4476 (4-[4- (2,3-Dihydro-1 ,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1 H-imidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2-Pyridinyl)-1 H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), LY 364947 (4-[3-(2-Pyridinyl)-1 H-pyrazol-4-yl]-quinoline), RepSox (2-(3-(6-Methylpyridine-2- yl)-1 H-pyrazol-4-yl)-1 ,5-naphthyridine), SB431542 (4-[4-(1 ,3-benzodioxol-5-yl)-5-(2- pyridinyl)- 1 H-imidazol-2-yl]benzamide), SB-505124 (2-[4-(1 ,3-Benzodioxol-5-yl)-2-(1 ,1 - dimethylethyl)-1 H-imidazol-5-yl]-6-methyl-pyridine), SB 525334 (6-[2-(1 ,1 -Dimethylethyl)-5- (6-methyl-2-pyridinyl)-1 H-imidazol-4-yl]quinoxaline), SD208 (2-(5-Chloro-2-fluorophenyl)-4- [(4-pyridyl)amino]pteridine), ITD1 (4-[1 ,1 ’-Biphenyl]-4-yl- 1 ,4,5,6,7,8-hexahydro-2,7,7- trimethyl-5-oxo-3-quinolinecarboxylic acid ethyl ester), DAN / Fc, antibodies to TGF-beta and TGF-beta receptors, TGF-beta inhibitory nucleic acids, TGF-beta inhibitory peptides, and the like. In some embodiments the inhibitor is SB431542.

[0117] In some instances, an inducing agent useful in a particular induction composition may include an activator or inhibitor of the Wnt pathway. Activators and inhibitors of the Wnt pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the Wnt pathway resulting in a corresponding activation or inhibition in cellular Wnt signaling. Components and downstream effectors of the Wnt pathway include but are not limited to, e.g., cthrcl (UniProtID Q96CG8), dkk1 (UniProtID 094907), fzd1 (UniProtID Q9UP38), fzd10 (UniProtID Q9ULW2), fzd2 (UniProtID Q14332), fzd4 (UniProtID Q9ULV1 ), fzd5 (UniProtID Q13467), fzd6 (UniProtID 060353), fzd7 (UniProtID 075084), fzd8 (UniProtID Q9H461), fzd9 (UniProtID 000144), igfbp4 (UniProtID P22692), kremen 1 (UniProtID Q96MU8), kremen 2 (UniProtID Q8NCW0), Irp5 (UniProtID 075197), Irp6 (UniProtID 075581), prr (UniProtID 075787), ror2 (UniProtID Q01974), rspol(UniProtID Q2MKA7), ryk (UniProtID P34925), wnt inhibitory 1 (UniProtID Q9Y5W5), wnt1 (UniProtID P04628), wnt2 (UniProtID P09544), wnt3 (UniProtID P56703), wnt3a (UniProtID P56704), wnt5a (UniProtID P41221 ), wnt7a (UniProtID 000755), wnt7b (UniProtID P56706), CTNNB1 (UniProtID P35222), GSK3A (UniProtID P49840), GSK3B (UniProtID P49841 ), TNKS1 (UniProtID 095271 ), TNKS2 (UniProtID Q9H2K2) and the like.

[0118] Activators of the WNT pathway include but are not limited to, e.g., CHIR99021 (6-[[2- [[4-(2,4-Dichlorophenyl)-5-(5-methyl-1 H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3- pyridinecarbonitrile), WNT family ligands (e.g., including but not limited to Wnt-1 , Wnt-2, Wnt- 2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt- 9a, Wnt-9b, Wnt-1 Oa, Wnt-1 Ob, Wnt-1 1 , Wnt-16b, etc.), RSPO co-agonists (e.g., RSPO2), lithium chloride, TDZD8 (4-Benzyl-2-methyl-1 ,2, 4-thiadiazolidine-3, 5-dione), BIO-Acetoxime ((2 ' Z,3 ' E)-6-Bromoindirubin-3 ' -acetoxime), A1070722 (1 -(7-Methoxyquinolin-4-yl)-3-[6- (trifluoromethyl)pyridin-2-yl]urea), HLY78 (4-Ethyl-5,6-Dihydro-5-methyl-[1 ,3]dioxolo[4,5- j]phenanthridine), CID 11210285 hydrochloride (2-Amino-4-(3,4- (methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride), WAY-316606, (hetero)arylpyrimidines, IQ1 , QS1 1 , SB-216763, DCA, and the like. In some instances, activation of the Wnt pathway may be achieved through repression of a Wnt pathway inhibitor, e.g., including but not limited to the use of an inhibitory nucleic acid targeting an inhibitor of the Wnt pathway or an antibody or small molecule directed to a Wnt pathway inhibitor. In some embodiments a Wnt activator is CHIR99021 .

[0119] Inhibitors of the WNT pathway include but are not limited to, e.g., Wnt-C59 (4-(2- Methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide), DKK1 , IWP-2 (N-(6-Methyl- 2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]- acetamide), Ant1 .4Br, Ant 1 .4CI, Niclosamide, apicularen, bafilomycin, XAV939 (3, 5,7,8- Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), IWR-1 (4- (1 ,3,3a,4,7,7a-Hexahydro-1 ,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl- Benzamide), NSC668036 (N-[(1 ,1 -Dimethylethoxy)carbonyl]-L-alanyl-(2S)-2-hydroxy-3- methylbutanoyl-L-Alanine-(1 S)-1 -carboxy-2-methylpropyl ester hydrate), 2,4-diamino- quinazoline, Quercetin, ICG-001 ((6S,9aS)-Hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1 - naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1 ,2-a]pyrimidine-1 (6H)- carboxamide), PKF1 15-584, BML-284 (2-Amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3- methoxyphenyl)pyrimidine), FH-535, iCRT-14, JW-55, JW-67, antibodies to Wnts and Wnt receptors, Wnt inhibitory nucleic acids, and the like. In some embodiments a Wnt inhibitor is Wnt-C59.

[0120] Glycogen synthase kinase 3 (GSK-3) inhibitors are used in cell culture to regulate signaling pathways involved in cell survival, differentiation, and pluripotency maintenance.GSK-3 is a serine / threonine kinase with two isoforms, GSK-3a and GSK-3p, that play key roles in multiple pathways, including Wnt / p-catenin, PI3K / Akt, and Hedgehog signaling. One of the most commonly used inhibitors is CHIR99021 , a highly selective and potent inhibitor of GSK-3 that stabilizes -catenin, promoting self-renewal in human and mouse embryonic stem cells. Another widely used inhibitor, LiCI (lithium chloride), is a non-specific GSK-3 inhibitor that works by competing with magnesium ions, indirectly increasing p-catenin levels. BIO (6- bromoindirubin-3'-oxime) is another selective inhibitor used in neural and cardiac differentiation studies due to its ability to modulate Wnt signaling. Other inhibitors, such as TWS1 19, are used to promote mesodermal and neural differentiation in stem cell cultures. In some embodiments a GSK3 inhibitor is CHIR99021 .

[0121] Repressors of the endothelial-to-mesenchymal transition (EndoMT) process during the expansion of iPSC-derived endothelial cells include, without limitation, simvastatin, GGTi298, chlorpromazine, and cyclosporin A. One or more of the repressors may be included in culture medium, e.g. at a concentration of from about 0.5 LIM to about 5 y.M, and may be from about 1 uM to about 2 .M.

[0122] Notch signaling inhibitors include DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester), a y-secretase inhibitor that prevents the cleavage and activation of Notch intracellular domain (NICD), thereby blocking downstream transcriptional activation. LY411575 and RO4929097 are other potent y-secretase inhibitors that have been used to suppress Notch signaling. Furfurylamine derivatives, such as MRK-560, selectively inhibit specific y-secretase complexes, providing a more targeted approach. Additionally, DLL4-Fc (a soluble Delta-like ligand 4 fusion protein) can act as a Notch ligand decoy, preventing receptor activation. Jaggedl peptide inhibitors and monoclonal antibodies against Notch receptors (e.g., anti-Notch1 or anti-Notch2 antibodies) offer alternative strategies to disrupt ligandreceptor interactions. In some embodiments a notch inhibitor is RO4929097.

[0123] In some instances, an inducing agent useful in a particular induction composition may include an activator of the FGF pathway, e.g. FGF2 activator. In some instances, an activator of the FGF pathway may also include activators of related signal transduction pathways including but not limited to, e.g., the MAPK / ERK signal transduction pathway. Activators of the FGF pathway include FGF proteins, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the FGF pathway resulting in a corresponding activation or inhibition in cellular FGF signaling. Components anddownstream effectors of the FGF pathway include but are not limited to, e.g., akt1 (UniProtID P31749), beta-klotho (UniProtID Q86Z14), camkiia (UniProtID Q9UQM7), cb1 (UniProtID P22681 ), cortactin (UniProtID Q14247), e-cadherin (UniProtID P12830), erk1 (UniProtID P27361 ), erk2 (UniProtID P28482), FGF1 (UniProtID P05230), FGF16 (UniProtID 060258), FGF17 (UniProtID 060258), FGF18 (UniProtID 076093), FGF19 (UniProtID 095750), FGF2 (UniProtID P09038), fgf23 (UniProtID Q9GZV9), FGF4 (UniProtID P08620), FGF6 (UniProtID P10767), FGF8 (UniProtID P55075), FGF9 (UniProtID P31371 ), fgfrl (UniProtID P1 1362), fgfr2 (UniProtID P21802), fgfr2b (UniProtID P21802-18), FGFR2c (UniProtID P21802-5), FGFR3C (UniProtID P22607-1 ), FGFR4 (UniProtID P22455), fos (UniProtID P01 100), frs2 (UniProtID Q8WU20), gab1 (UniProtID Q13480), grb2 (UniProtID P62993), hgf (UniProtID P14210), jun (UniProtID P05412), klotho (UniProtID Q9UEF7), mapk 14 (UniProtID Q16539), met (UniProtID P08581 ), mkp-3 (UniProtID Q16828), mmp9 (UniProtID P14780), n-cad-ctfl (UniProtID P19022), n-cad-ctf2 (UniProtID P19022), n-cadherin (UniProtID P19022), ncam (UniProtID P13591 ), osteocalcin (UniProtID P02818), osteopontin (UniProtID P10451 ), p11 flalpha (UniProtID P42336), p120ctn (UniProtID 060716), p90-rsk 1 (UniProtID Q15418), pak4 (UniProtID Q8WYL5), pak4 (UniProtID 096013), pdk1 (UniProtID 015530), pik3r1 (UniProtID P27986), plcgammal (UniProtID P19174), pro-e-cadherin (UniProtID P12830), pro-mmp9 (UniProtID P14780), ps1 (UniProtID gamma), pyk2 (UniProtID Q14289), runx2 (UniProtID Q13950), se-cad (UniProtID P12830), secad-ntf2 (UniProtID P12830), sef (UniProtID Q8NFM7), she (UniProtID P29353), shp2 (UniProtID Q06124), sn-cad (UniProtID P19022), sos1 (UniProtID Q07889), sprouty2 (UniProtID 043597), sre (UniProtID P12931 ), statl (UniProtID P42224), stat3 (UniProtID P40763), stat5b (UniProtID P51692), syndecan-2 (UniProtID P34741 ), syndecan-4 (UniProtID P31431 ), upa (UniProtID P00749), upar (UniProtID Q03405), and the like.

[0124] Activators of the FGF pathway and / or the MAPK / ERK pathway include but are not limited to, e.g., FGF family ligands (e.g., FGF1 , FGF2, FGF-3, FGF-4, FGF-5, FGF-6, KGF / FGF-7, FGF-8, FGF-9, FGF-10, FGF-1 1 , FGF-12, FGF-13, FGF-15, FGF-16, FGF-17, FGF-19, FGF-20, FGF-21 , FGF-22, FGF-23, etc.), SUN 1 1602 (4-[[4-[[2-[(4-Amino-2, 3,5,6- tetramethylphenyl)amino]acetyl]methylamino]-1 -piperidinyl]methypenzamide), t-Butylhydroquinone, U-46619, 02 Ceramide, Lactosyl Ceramide, Angiotensin II, Baicalin, and the like. In some instances, activation of the FGF pathway and / or the MAPK / ERK pathway may be achieved through repression of the a FGF pathway and / or the MAPK / ERK pathway inhibitor, e.g., including but not limited to the use of an inhibitory nucleic acid targeting an inhibitor of the FGF pathway and / or the MAPK / ERK pathway or an antibody or small molecule directed to a FGF pathway inhibitor and / or MAPK / ERK pathway inhibitor. In some embodiments an FGF activator is an FGF protein, e.g. human basic FGF (FGF2).

[0125] In some instances, an inducing agent useful in a particular induction composition may include an activator of the BMP pathway. Activators of the BMP pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate at least one component of the BMP pathway resulting in a corresponding activation in cellular BMP signaling. Components and downstream effectors of the BMP pathway include but are not limited to, e.g., bambi (UniProtID Q13145), bmp2 (UniProtID P12643), bmp4 (UniProtID P12644), bmp6 (UniProtID P22004), bmp7 (UniProtID P18075), bmprl a (UniProtID P36894), bmprl b (UniProtID 000238), bmpr2 (UniProtID Q13873), cer1 (UniProtID 095813), chrd (UniProtID Q9H2X0), chrdU (UniProtID Q9BU40), endofin (UniProtID Q7Z3T8), erk2 (UniProtID P28482), fetua (UniProtID P02765), fs (UniProtID P19883), gadd34 (UniProtID 075807), greml (UniProtID 060565), gsk3beta (UniProtID P49841 ), nog (UniProtID Q13253), nup214 (UniProtID P35658), ppml a (UniProtID P35813), pppl ca (UniProtID P62136), rgma (UniProtID Q96B86), rgmb (UniProtID Q6NW40), rgmc (UniProtID Q6ZVN8), scp1 (UniProtID Q9GZU7), scp2 (UniProtID 014595), scp3 (UniProtID 015194), ski (UniProtID P12755), smadl (UniProtID Q15797), smad4 (UniProtID Q13485), smad5 (UniProtID Q99717), smad6 (UniProtID 043541 ), smad7 (UniProtID 015105), smad8a (UniProtID 015198), smurfl (UniProtID Q9HCE7), smurf2 (UniProtID Q9HAU4), tab1 (UniProtID Q15750), tab2 (UniProtID Q9NYJ8), tak1 (UniProtID 043318), usagl (UniProtID Q6X4U4), xiap (UniProtID P98170), and the like.

[0126] Activators of the BMP pathway include but are not limited to, e.g., BMP family ligands e.g., BMP2, BMP4, BMP7, etc., Alantolactone, FK506, isoliquiritigenin, 4’-hydroxychalcone, and the like. In some instances, activation of the BMP pathway may be achieved through repression of a BMP pathway inhibitor, e.g., including but not limited to the use of an inhibitory nucleic acid targeting an inhibitor of the BMP pathway or an antibody or small molecule directed to a BMP pathway inhibitor. In some embodiments BMP activator is an BMP protein, e.g. human BMP4.

[0127] In some instances, an inducing agent useful in a particular induction composition may include an inhibitor of Rho-associated protein kinase (ROCK), which inhibitors are commonly used in cell culture to modulate cellular processes influenced by ROCK activity, such as cell motility, proliferation, and morphology. Inhibitors of ROCK pathway include small molecule inhibitors, peptide inhibitors, antibodies, nucleic acid inhibitors, and the like that inhibit at least one component of the ROCK pathway resulting in a corresponding activation or inhibition in cellular ROCK signaling. Inhibitors include, without limitation, the pyridine Y-27632, which inhibits ROCK by competing with ATP for binding to the kinase domain; and the isoquinolone Fasudil. In some embodiments the ROCK inhibitor is Y-27632.

[0128] In some instances, an inducing agent useful in a particular induction composition may include an activator of the VEGF pathway. Activators of the VEGF pathway include small molecule activators, peptide activators, antibodies, nucleic acid activators, nucleic acid activators, and the like that activate at least one component of the VEGF pathway resulting in a corresponding activation in cellular VEGF signaling. VEGF is a dimeric, disulfide-linked 46- kDa glycoprotein related to Platelet-Derived Growth Factor ("PDGF"). It is produced by normal cell lines and tumor cell lines; is an endothelial cell-selective mitogen; shows angiogenic activity in in vivo test systems (e.g., rabbit cornea); is chemotactic for endothelial cells and monocytes; and induces plasminogen activators in endothelial cells, which are involved in the proteolytic degradation of the extracellular matrix during the formation of capillaries. A number of isoforms of VEGF are known, which while they show comparable biological activity, differ in the type of cells that secrete them and in their heparin-binding capacity. In addition, there are other members of the VEGF family, such as Placenta Growth Factor ("PGF") and VEGF- C.

[0129] The cellular receptors of VEGFs (VEGFRs) are transmembranous receptor tyrosine kinases. They are characterized by an extracellular domain with seven immunoglobulin-like domains and an intracellular tyrosine kinase domain. Various types of VEGF receptor have been characterized, including VEGFR-1 (also known as flt-1 ), VEGFR-2 (also known as KDR), and VEGFR-3. In some embodiments a VEGF activator is a VEGF protein, e.g. human VEGF165.

[0130] In some instances, an inducing agent useful in a particular induction composition may include an inhibitor of the CDK pathway. Inhibitors include small molecule inhibitors, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that inhibit at least one component of the CDK pathway. Representative CDK inhibitors include BI1347, Flavopiridol (Alvocidib, L868275, HMR-1275), R-roscovitine (CYC202, Seliciclib, Roscovitine), Dinaciclib (SCH 727965, SCH-727965), P276-00 (Riviciclib hydrochloride, P276), TG02 (SB1317, TG-02; SB-1317), AT7519 (AT 7519, AT-75190), Roniciclib (BAY1000394), RGB- 286638, PHA-793887, ZK304709, Xylocydine, SNS032 (BMS-387032), R547 (Ro 4584820), RGB286147, Purvalanol A (NG60), Purvalanol B (NG95, NG-95), Olomoucine, Olomoucine II, NVP-LCQ195, Meriolin 3, Kenpaullone (NSC664704, 9-Bromopaullone), JNJ-7706621 , lndirubin-3’-Monoxime, Indirubin (Isoindigotin; Indigopurpurin), AZD-5438, AZD 5597, Bohemine, Butyrolactone, CYC065, 10Z-Hymenialdisine, 5-lodo-indirubin-3’- monoxime, AG024322, Aloisine A, Aloisine B, Alsterpaullone (9-Nitropaullone, Aminopurvalanol, R-CR8, Voruciclib.

[0131] In some instances, an inducing agent useful in a particular induction composition may include an inhibitor of myosin light chain kinase (MLCK). Inhibitors include small molecule inhibitors, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that inhibit at least one component of the MLCK pathway. In mammals, myosin light chain kinase (MLCK) is encoded by the mylkl and mylk2 genes. mylk2 encodes an MLCK isoform that is exclusively expressed in skeletal muscle cells. Mylkl gene coding products are expressed in diverse cell types and tissues including muscle, platelets, and secretory and brain cells. Most MLCK inhibitors act by competitive binding at or near the ATP-binding site on the MLCK molecule. Examples include ML-9, ML-7, K-252a, KT592, Wortmannin, Quercetin, Genistin, Wogonin, Capsaicin, Salvianolic acid B, and lithium. In some embodiments the inhibitor is ML-9 [1 -(5-chloronaphthalene-1 -sulfonyl)-1 H-hexahydro-1 ,4- diazepine]. In some embodiments the inhibitor is ML-7 [1 -(5-iodonaphthalene-1 -sulphonyl) 1 H-hexahydro 1 , 4-diazepine hydrochloride].

[0132] In some embodiments human platelet lysate (hPL) is included in culture medium, and may be provided as a substitute for serum, e.g. fetal calf serum. Platelet lysate (PL) is one of the sources of bioactive molecules found in platelet releasate. It is used in cell growth and proliferation and is a good alternative to fetal bovine serum. PL is considered to be a novel and multifactorial material containing comparably more growth factors than other blood- derived products and can be stored for longer periods of time in lower temperatures in a freezer, whereas platelet-rich plasma (PRP) is a temperature sensitive mixture and cannot be preserved below 4°C. The acellular nature of PL can potentially surpass the traditional PRP in terms of non-autologous application and patient variability.

[0133] In some instances, induction, or pathway modulating agents, as described above and including pathway activators and pathway inhibitors include, e.g., those that are commercially available, e.g., from such suppliers such as Tocris Bioscience (Bristol, UK), Sigma-Aldrich (St. Louis, Mo.), PeproTech (Thermo Fisher Scientific, MA), Santa Cruz Biotechnology (Santa Cruz, Calif.), and the like.

[0134] Pluripotent progenitors and derivatives thereof may be contacted with induction agents by any convenient means. Generally, an induction agent is added to culture media, as described herein, within which cells of the instant disclosure are grown or maintained, such that the induction agent is present, in contact with the cells, at an effective concentration to produce the desired effect, e.g., induce a desired lineage restriction event. In other instances, e.g., where the existing culture media is not compatible with a particular induction agent, the culture media in which the cells are being grown is replaced with fresh culture media containing the particular induction agent present in the fresh media at an effectiveconcentration to produce the desired effect. In instances where fresh or specific culture media is provided with a particular induction agent the culture agent may, in some instances, be specifically formulated for the particular induction agent, e.g., containing one or more specific additional reagents to, e.g., aid in the delivery of the induction agent, aid in the solubility of the induction agent, aid in the stability of the induction agent, etc.

[0135] The effective concentration of a particular induction agent will vary and will depend on the agent. In addition, in some instances, the effective concentration may also depend on the cells being induced, the culture condition of the cells, other induction agents co-present in the culture media, etc. As such, the effective concentration of induction agents will vary and may range from 1 ng / mL to 10 pg / mL or more, including but not limited to, e.g., 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 1 1 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 1 -5 ng / mL, 1 -10 ng / mL, 1 -20 ng / mL, 1 -30 ng / mL, 1 -40 ng / mL, 1 -50 ng / mL, 5-10 ng / mL, 5-20 ng / mL, 10-20 ng / mL, 10-30 ng / mL, 10-40 ng / mL, 10-50 ng / mL, 20-30 ng / mL, 20-40 ng / mL, 20-50 ng / mL, 30-40 ng / mL, 30-50 ng / mL, 40-50 ng / mL, 1 -100 ng / mL, 50-100 ng / mL, 60-100 ng / mL, 70-100 ng / mL, 80-100 ng / mL, 90- 100 ng / mL, 10-100 ng / mL, 50-200 ng / mL, 100-200 ng / mL, 50-300 ng / mL, 100-300 ng / mL, 200-300 ng / mL, 50-400 ng / mL, 100-400 ng / mL, 200-400 ng / mL, 300-400 ng / mL, 50-500 ng / mL, 100-500 ng / mL, 200-500 ng / mL, 300-500 ng / mL, 400 to 500 ng / mL, 0.001 -1 pg / mL, 0.001 -2 pg / mL, 0.001 -3 pg / mL, 0.001 -4 pg / mL, 0.001 -5 pg / mL, 0.001 -6 pg / mL, 0.001 -7 pg / mL, 0.001 -8 pg / mL, 0.001 -9 pg / mL, 0.001 -10 pg / mL, 0.01 -1 pg / mL, 0.01 -2 pg / mL, 0.01 -3 pg / mL, 0.01 -4 pg / mL, 0.01 -5 pg / mL, 0.01-6 pg / mL, 0.01 -7 pg / mL, 0.01 -8 pg / mL, 0.01 -9 pg / mL, 0.01 -10 pg / mL, 0.1 -1 pg / mL, 0.1 -2 pg / mL, 0.1 -3 pg / mL, 0.1 -4 pg / mL, 0.1 -5 pg / mL, 0.1 - 6 pg / mL, 0.1-7 pg / mL, 0.1 -8 pg / mL, 0.1 -9 pg / mL, 0.1 -10 pg / mL, 0.5-1 pg / mL, 0.5-2 pg / mL, 0.5-3 pg / mL, 0.5-4 pg / mL, 0.5-5 pg / mL, 0.5-6 pg / mL, 0.5-7 pg / mL, 0.5-8 pg / mL, 0.5-9 pg / mL, 0.5-10 pg / mL, and the like.

[0136] In some instances, the effective concentration of an induction agent in solution, e.g., cell culture media, may range from 1 nM to 100 pM or more, including but not limited to, e.g., 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 1 -2 nM, 1 -3 nM, 1 -4 nM, 1 -5 nM, 1 -6 nM, 1-7 nM, 1 -8 nM, 1 -9 nM, 1 -10 nM, 1 .5 nM, 1.5-2 nM, 1.5- 3 nM, 1 .5-4 nM, 1 .5-5 nM, 1.5-6 nM, 1 .5-7 nM, 1.5-8 nM, 1 .5-9 nM, 1.5-10 nM, 2-3 nM, 2-4nM, 2-5 nM, 2-6 nM, 2-7 nM, 2-8 nM, 2-9 nM, 2-10 nM, 3-4 nM, 3-5 nM, 3-6 nM, 3-7 nM, 3-8 nM, 3-9 nM, 3-10 nM, 4-5 nM, 4-6 nM, 4-7 nM, 4-8 nM, 4-9 nM, 4-10 nM, 5-6 nM, 5-7 nM, 5-8 nM, 5-9 nM, 5-10 nM, 6-7 nM, 6-8 nM, 6-9 nM, 6-10 nM, 7-8 nM, 7-9 nM, 7-10 nM, 8-9 nM, 8- 10 nM, 9-10 nM, 5-15 nM, 5-20 nM, 5-25 nM, 5-30 nM, 5-35 nM, 5-40 nM, 5-45 nM, 5-50 nM, 10-15 nM, 10-20 nM, 10-25 nM, 10-30 nM, 10-35 nM, 10-40 nM, 10-50 nM, 15-20 nM, 15-25 nM, 15-30 nM, 15-35 nM, 15-40 nM, 15-45 nM, 15-50 nM, 20-25 nM, 20-30 nM, 20-35 nM, 20-40 nM, 20-45 nM, 20-50 nM, 25-30 nM, 25-35 nM, 25-40 nM, 25-45 nM, 25-50 nM, 30-35 nM, 30-40 nM, 30-45 nM, 30-50 nM, 35-40 nM, 35-45 nM, 35-50 nM, 40-45 nM, 40-50 nM, 45-50 nM, 10-100 nM, 20-100 nM, 30-100 nM, 40-100 nM, 50-100 nM, 60-100 nM, 70-100 nM, 80-100 nM, 90-100 nM, 50-150 nM, 50-200 nM, 50-250 nM, 50-300 nM, 50-350 nM, 50- 400 nM, 50-450 nM, 50-500 nM, 10-150 nM, 10-200 nM, 10-250 nM, 10-300 nM, 10-350 nM, 10-400 nM, 10-450 nM, 10-500 nM, 100-150 nM, 100-200 nM, 100-250 nM, 100-300 nM, 100- 350 nM, 100-400 nM, 100-450 nM, 100-500 nM, 200-500 nM, 300-500 nM, 400-500 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM,650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 200-400 nM, 300-500 nM, 400-600 nM, 500-700 nM, 600-800 nM, 700-900 nM, 800 nM to 1 pM, 0.5-1 pM, 0.5-1.5 pM, 0.5-2 pM, 0.5-2.5 pM, 0.5-3 pM, 0.5-3.5 pM, 0.5-4 pM, 0.5-4.5 pM, 0.5-5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 1 1 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 21 pM, 22 pM, 23 pM, 24 pM, 25 pM, 26 pM, 27 pM, 28 pM, 29 pM, 30 pM, 31 pM, 32 pM, 33 pM, 34 pM, 35 pM, 36 pM, 37 pM, 38 pM, 39 pM, 40 pM, 41 pM, 42 pM, 43 pM, 44 pM, 45 pM, 46 pM, 47 pM, 48 pM, 49 pM, 50 pM, 1 -2 pM, 1 -3 pM, 1 -4 pM, 1-5 pM, 1 -6 pM, 1 -7 pM, 1-8 pM, 1 -9 pM, 1 -10 pM, 1 .5 pM, 1 .5-2 pM, 1.5-3 pM, 1 .5-4 pM, 1.5-5 pM, 1.5-6 pM, 1.5-7 pM, 1 .5-8 pM, 1.5-9 pM, 1.5-10 pM, 2-3 pM, 2-4 pM, 2-5 pM, 2-6 pM, 2-7 pM, 2-8 pM, 2-9 pM, 2-10 pM, 3-4 pM, 3-5 pM, 3-6 pM, 3-7 pM, 3-8 pM, 3-9 pM, 3- 10 pM, 4-5 pM, 4-6 pM, 4-7 pM, 4-8 pM, 4-9 pM, 4-10 pM, 5-6 pM, 5-7 pM, 5-8 pM, 5-9 pM, 5-10 pM, 6-7 pM, 6-8 pM, 6-9 pM, 6-10 pM, 7-8 pM, 7-9 pM, 7-10 pM, 8-9 pM, 8-10 pM, 9-10 pM, 5-15 pM, 5-20 pM, 5-25 pM, 5-30 pM, 5-35 pM, 5-40 pM, 5-45 pM, 5-50 pM, 10-15 pM, 10-20 pM, 10-25 pM, 10-30 pM, 10-35 pM, 10-40 pM, 10-50 pM, 15-20 pM, 15-25 pM, 15-30 pM, 15-35 pM, 15-40 pM, 15-45 pM, 15-50 pM, 20-25 pM, 20-30 pM, 20-35 pM, 20-40 pM, 20-45 pM, 20-50 pM, 25-30 pM, 25-35 pM, 25-40 pM, 25-45 pM, 25-50 pM, 30-35 pM, 30-40 pM, 30-45 pM, 30-50 pM, 35-40 pM, 35-45 pM, 35-50 pM, 40-45 pM, 40-50 pM, 45-50 pM, 10-100 pM, 20-100 pM, 30-100 pM, 40-100 pM, 50-100 pM, 60-100 pM, 70-100 pM, 80-100 pM, 90-100 pM, and the like.

[0137] In some instances, the effective concentration of an induction agent will be below a critical concentration such that the induction produces the desired effect without undesirable effects. As used herein, the term “critical concentration” refers to a concentration of induction agent above which undesirable effects are produced. Undesirable effects that may be theresult of a concentration exceeding the critical concentration include but are not limited to, e.g., off-target effects (off-target activation of signaling, off-target inhibition of signaling), reduction or loss of function (e.g., loss of desired activator function, loss of desired inhibitor function) reduction of cell viability, increase in cell mortality, lineage restriction towards an undesired cell type, differentiation into an undesired cell type, loss of expression of a particular desired marker, etc. Whether a particular induction agent will have a critical concentration and what the critical concentrations of those agents having a critical concentration are will depend on the agent and the specific conditions in which the agent is used.

[0138] In some instances, cells of the instant disclosure may be contacted with multiple induction agents and / or multiple induction compositions in order achieve a desired endothelial cell type and terminally differentiated derivative thereof. In some instances, a particular induction composition will contain two or more induction agents such that a particular cell culture is simultaneously contacted with multiple induction agents. In some instances, a particular series of induction compositions may be used, one at a time, in generating a desired endothelial cell such that a particular cell culture is successively contacted with multiple induction agents.

[0139] The duration of contact of a particular induction composition with a particular cell type will vary and will depend, e.g., the cell type being induced, and the components of the induction composition. In some instances, a particular induction composition may be introduced for different exposure times depending on the context of use, e.g., cell type X may be contacted with induction composition Y for time Z whereas cell type A may be contacted with induction composition Y for time B, wherein cell type X is different than cell type A and time Z is different than time B. As such, the time cells are contacted with a particular induction composition may vary, e.g., when being used on different cells, when being used to generate different cells, or when being used at different steps of a differentiation process.

[0140] The duration of contact of a particular induction composition with a particular cell type, in some instances, may be referred to as the “exposure time” and exposure times may range from a day to weeks or more, including but not limited to e.g., 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, 10 days, 1 1 days, 12, days, 13, days, 14 days, 15, days, etc. As used herein, exposure times are, in some instances, referred as consisting essentially of, e.g., 24 hours, indicating that the exposure time may be longer or shorter than that specified including those exposure times that are longer or shorter but do not materially affect the basic outcome of the particular exposure. As such, in some instances where a particular exposure is more time sensitive such that under or over exposure, e.g., of more or less than 1 hour, materially affects the outcome of the exposure, a time period consisting essentially of, e.g., 24 hours, will be interpreted to refer to a time period ranging from about 23 hours to about 25hours. In some other instances where a particular exposure is less time sensitive such that under or over exposure, e.g., of more than 12 hours, does not materially affect the outcome of the exposure, a time period consisting essentially of, e.g., 24 hours will mean a time period ranging from about 12 hours or less to about 36 hours or more. In some instances, depending on the context, an exposure period consisting essentially of 24 hours may refer to an exposure time of 22-26 hours, 21 -27 hours, 20-28 hours, 19-29 hours, 18-30 hours, etc.

[0141] In some instances, time periods of exposure may be pre-determined such that cells are contacted with an induction composition according to a schedule set forth prior to the contacting. In some instances, the time period of exposure, whether pre-determined or otherwise, may be modulated according to some feature or characteristic of the cells and / or cell culture, including but not limited to, e.g., cell morphology, cell viability, cell appearance, cellular behaviors, cell number, culture confluence, marker expression, etc.

[0142] Methods of modification of cells, including modification of pluripotent cells and modification of endothelial cell are well-known in the art and include but are not limited to e.g., genetic modification (e.g., through deletion mutagenesis, through substitution mutagenesis), through insertional mutagenesis (e.g., through the introduction of heterologous nucleic acid into the pluripotent cell, etc.), non-mutagenic genetic modification (e.g., the non-mutagenic insertion of heterologous nucleic acid, etc.), epigenetic modification (e.g., through the treatment with one or more specific or general epigenetic modifying agents (e.g., methylation inhibitors, methylation activators, demethylases, etc.), other modifications (e.g., non-genetic labeling, etc.).

[0143] Modifications of cells may be transient or stable. In some instances, a modification of a particular pluripotent cell or mesodermal progenitor cell may be stable such that the modification persists through derivation of a desired endothelial cell from the pluripotent cell or progenitor cell as described herein. In some instances, stable modifications may persist through introduction of a derived cell type into a host. In some instances, stable modifications may persist through proliferation of the cell such that all progenitors of a particular modified cell also contain the subject modification. In some instances, a modification of a particular pluripotent cell or progenitor cell may be transient such that the modification is lost after derivation of a cell type of interest from the transiently modified pluripotent cell. In certain instances, transient modifications may persist through one or more rounds of proliferation of the modified cell such that some but not all of the progeny of the modified cell contain the subject modification. In some instances, a transient modification will not persist during proliferation such that none of the progeny of a modified cell will contain the subject modification. In some instances, a transiently modified cell may be configured such that the modification persists through certain aspects of derivation of the cell type of interest, e.g.,through derivation of a particular cell type of interest, but is lost prior to introduction of the derived cell into a host.Generation of Endothelial Cells

[0144] Methods are provided for the efficient in vitro differentiation of hPSCs into ECs in a feeder layer- and animal component-free culture. This method exclusively relies on extracellular signaling agents, which take the form of either small molecules or recombinant proteins, as disclosed herein, to guide the differentiation process. This method employs an efficient stepwise differentiation of hPSCs into mesodermal cells, endothelial progenitor cells, and ultimately mature and pure endothelial cells within a span of within about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days.

[0145] In an initial step, human pluripotent stem cells, including without limitation human iPSC or human ES cells, are induced to a mesodermal fate. The term “population of mesodermlineage cells” as used herein means a cell population containing mesodermal cells themselves and / or cells generated by differentiation from mesodermal cells. The cells generated by differentiation from mesodermal cells include hemangioblasts, mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells, cardiac progenitor cells, and the like. Preferably, these mesodermal-lineage cells exhibit a uniform spindle-shaped morphology and express higher levels of MESP1 , ISL1 , HAND1 , KDR, and SOX17. The methods outlined for generating mesodermal-lineage cells in step are not specifically limited, and an appropriate method may be chosen from known methods. A preferred method for the present invention is as follows.

[0146] In a first step, human pluripotent stem cells, including without limitation human iPSC or human ES cells are induced to a mesodermal fate. The hPSCs are seeded at a density of, for example, from 0.5x104to 2.5x104cells / cm2, and preferably from 1 x104to 1 .6x104cells / cm2to a culture vessel pre-coated with a matrix, such as vitronectin, laminin 521 , or iMatrix-51 1 . After allowing time to adhere to the vessel, the cells are cultured with media comprising an effective amount of a GSK inhibitor, for example and without limitation, CHIR99021 . The medium may further comprise one or more of a Wnt inhibitor. In some embodiments the Wnt inhibitor is Wnt-C59. The medium may further comprise a CDK8 inhibitor. In some embodiments the CDK8 inhibitor is BI1347. The cells are cultured from about 24 to about 72 hours, from about 36 to about 60 hours and may be for about 48 hours. In some embodiments the medium is serum free. In some embodiments the medium is based on,, CDM-HSA (1 / 2 IMDM and1 / z Ham’s F12 nutrient mix-based medium), as described in Table 3 of the examples, etc.

[0147] In some embodiments a strategy is employed for mesoderm induction that effectively modulates Wnt activity consistently across various hPSC lines or through iterativedifferentiations of the same hPSC line within the same effective range. This is achieved by providing in the culture medium an effective dose of a Wnt agonist, a Wnt inhibitor, and a CDK8 inhibitor at carefully titrated concentrations. Culture may be in the absence of BMP4 and activin A.

[0148] The effective dose of a Wnt agonist may be a concentration equivalent to from about 1 uM to about 25 uM CHIR99021 , from about 4 uM to about 15 M, and may be about 8 pM CHIR99021 . The effective dose of a Wnt inhibitor may be a concentration equivalent to from about 0.1 uM to about 10 LIM Wnt-C59, from about 0.5 pM to about 5 pM, and may be about 1 pM Wnt-C59. The effective dose of a CDK8 inhibitor may be a concentration equivalent to from about 0.05 pM to about 5 pM BI1347, from about 0.1 pM to about 1 pM, and may be about 0.25 pM BI1347. In addition, other growth factors and the like may be added to the medium within the range so as not to impair the effects of the present invention.

[0149] In a second step, endothelial progenitor cells are induced in culture from mesodermal- lineage cells of the first step. The term "endothelial progenitor cells", as used herein, refers to cells whose differentiation is directed towards endothelial cells. The identification of endothelial progenitor cells can be confirmed by analyzing the expression patterns of transcriptional factors or cell-surface antigens. For instance, the expression levels of transcriptional factors or cell-surface antigens, either individually or in combination, are measured. These levels are typically low or undetected before the induction of differentiation but significantly increase after the induction of differentiation. Effective markers for confirming endothelial progenitor cells include kinase insert domain receptor (KDR), CD34, SOX17, and similar markers. Preferably, endothelial progenitor cells are characterized by the expression of KDR, also known as vascular endothelial growth factor receptor-2 (VEGFR-2) or Flk-1 , which plays a crucial role in the vascularization process.

[0150] The mesodermal-lineage cells are dissociated into single cells and replated in the medium comprising an effective dose of an FGF agonist, a BMP agonist, a VEGF agonist, and a TGFp inhibitor. The medium may further comprise an effective dose of a Wnt inhibitor, a ROCK inhibitor, a myosin light chain kinase inhibitor, and human platelet lysate. The cells are cultured from about 24 to about 72 hours, from about 36 to about 60 hours and may be cultured for about 48 hours to produce a substantially pure population of endothelial progenitor cells. Other growth factors and the like may be added to the medium within a range so as not to impair the effects exhibited by the present invention. Culture is carried out preferably in the absence of activin A. In some embodiments the medium is serum free. In some embodiments the medium is based on CDM-HSA (1 / z IMDM and Vz Ham’s F12 nutrient mix-based medium), as described in Table 3 of the examples, etc.

[0151] As one preferred embodiment of the present invention, mesodermal-lineage cells are dissociated, and seeded at a density of, for example, from 0.5x104to 3x104cells / cm2, and preferably from 1 x 104to 2x 104cells / cm2to a culture vessel pre-coated with a suitable matrix.

[0152] The effective dose of a Wnt inhibitor may be a concentration equivalent to from about 0.1 pM to about 10 pM Wnt-C59, from about 0.5 pM to about 5 pM, and may be about 1 pM Wnt-C59. The concentration of a BMP agonist may be a concentration equivalent to from about 0.5 to about 100 ng / ml BMP4 protein, from about 1 to about 50 ng / ml, from about 5 to about 10 ng / ml BMP4. The concentration of a VEGF agonist may be a concentration equivalent to from about 10 to about 500 ng / ml VEGF, from about 25 to about 100 ng / ml. The concentration of an FGF agonist may be a concentration equivalent to from about 1 to about 200 ng / ml FGF2 protein, from about 2 to about 100 ng / ml, from about 10 to about 50 ng / ml FGF2. The effective dose of a TGFp inhibitor may be a concentration equivalent to from about 1 to about 20 pM SB431542, from about 5 to about 10 pM SB431542. The effective dose of a ROCK inhibitor may be a concentration equivalent to from about 1 to about 20 pM Y27632, from about 5 to about 10 pM Y27632. The effective dose of a myosin light chain kinase inhibitor may be a concentration equivalent to from about 0.5 to 20 pM ML-7, from about 2 to about 10 pM ML-7. The effective dose of human platelet lysate may be from about 0.05% (vol / vol) to about 2%, from about 0.1 % to about 0.5%.

[0153] The endothelial cells may be skewed to arterial endothelial cells, or to vein endothelial cells. In some embodiments, where vein endothelial cells are desired, the medium at this stage further comprises an effective dose of a notch inhibitor, e.g. and without limitation, R04929097. Where arterial endothelial cells are desired the medium may lack a notch inhibitor.

[0154] In a third step, the endothelial progenitor cells of step 2 are induced to differentiate into mature endothelial cells. The term "endothelial cells", as used herein, refers to cells expressing at least one of the endothelial cell markers, e.g. PECAM1 (CD31 ), VE-cadherin (CD144), Endoglin (CD105), von Willebrand factor (vWF), etc. The endothelial cells may express one or both of CD31 and CD144.

[0155] The endothelial cells may be skewed to arterial endothelial cells, or to vein endothelial cells. In some embodiments, where vein endothelial cells are desired, the medium at this stage further comprises an effective dose of a notch inhibitor, e.g. and without limitation, R04929097. Where arterial endothelial cells are desired the medium may lack a notch inhibitor.

[0156] In some embodiments the medium is serum free. In some embodiments the medium is based on CDM-HSA (1 / 2 IMDM and 1 / 2 Ham’s F12 nutrient mix-based medium), as described in Table 3 of the examples, etc, or commercial xeno-free endothelial cell expansion medium, EndoGO. Endothelial cell expansion and maturation medium may comprise, for example, oneor more of VEGF, EGF, IGF- 1 , FGF, ascorbic acid, and hydrocortisone. A TGFp inhibitor may also be included. Medium include, for example EGM-2 (Lonza), ECM (ScienCell), or EndoGO XF medium (Sartorius) supplemented with, for example, 0.5% to 5% human platelet lysate for 24 to 72 hours. The medium may alternatively be prepared by appropriately combining basal CDM-HSA medium using in the first and second steps and factors. The cells are induced to a substantially pure population of endothelial progenitor cells in a culture period of from about 24 to about 72 hours, from about 24 to about 60 hours and may be cultured for about 24-48 hours. The cells may be cultured for longer periods of time without losing the endothelial cell phenotype, e.g. greater than 1 week, greater than 2 weeks, etc.

[0157] Expansion of endothelial cells may proceed in the medium utilized for induction of mature endothelial cells. Optionally, an effective dose of an EndoMT repressor is present in the medium. In some embodiments the repressor is one or more of simvastatin, GGTi298, chlorpromazine, and cyclosporin A. In some embodiments the repressor is present in the medium at a concentration of from about 0.5 to about 5 pM, e.g. from around 1 LIM to around 2 pM.

[0158] The endothelial cells may be cultured to generate an organoid, by culture in a medium such as EndoGo (Sartorius), EGM-2 (Lonza), ECM (ScienCell), or CDM-HSA. The medium may be supplemented with an effective dose of a VEGF agonist, and an FGF agonist. In some embodiments the VEGF agonist is VEGF. In some embodiments the FGF agonist is FGF2. The period of time for generating a vessel organoid may be from about 1 , 2, 3, 4, 5, 6, 7, 8 or more days following endothelial cell induction.

[0159] The methods of the disclosure provide an EC population, designated as passage 0 (P0), that forms a compact monolayer with high purity, for example exceeding 98% endothelial cells. The ECs can be further expanded in the same medium at a split ratio of about 1 :4 for at least 6 passages. The appropriate culturing time may be determined based on the state of cells or specific culturing conditions. Additionally, medium exchange or subculturing may be appropriately performed during the culturing period.

[0160] The ratio of ECs (CD31 positive ratio) is substantially maintained by the maintenance culturing condition mentioned above within the first 3 to 4 passages. For example, after the maintenance subculturing is carried out for 3 times, the ratio of the ECs (CD31 positive ratio) is 90% or more. EndoMT markers, such as SOX9, SLUG, and TWIST1 , are 1 % or less.

[0161] Endothelial cells may be classified according to differentiation stages. Cells that are at the early stage (for example, within the first week) of differentiation may be referred to as “immature ECs,” and cells with progressed differentiation may be referred to as “mature ECs.” The differentiation stage of ECs can be confirmed by analyzing expression patterns of transcriptional factors or cell-surface antigens. In an embodiment, the expression patterns of transcriptional factors or cell-surface antigens, alone or in a combination, are measured. Markers effective for confirming the differentiation stage of ECs can be retrieved from the single-cell dataset specifically examining the gene expression profiles of fetal and adult ECs. ECs generated using the methods of the disclosure have an adult EC phenotype.

[0162] According to vascular bed-specific marker expression patterns, endothelial cells produced by the methods of the disclosure in the absence of a notch inhibitor are found to be predominantly arterial ECs, with a very small portion of cells (less than 5%) expressing venous EC markers. A negligible amount of lymphatic or corneal ECs may be produced. Addition of a notch inhibitor results in predominantly vein endothelial cells.

[0163] Optionally endothelial cells are purified from the cell population generated by the methods of the disclosure. In general, this is not required because the cell populations produced by the methods disclosed herein are substantially pure. However, if desired, isolation of the ECs may be carried out by selectively capturing cells expressing endothelial cell surface markers with a fluorescence-activated cell sorting (FACS), a magnetic-activated cell sorting (MACS), or the like, and then collecting the captured cells. In these separating means, it is advantageous that an antibody that specifically binds to ECs is utilized. Therefore, for example, an antibody or a fragment thereof that binds to KDR, CD34, PECAM1 , CD144 or other markers expressed on a surface of ECs can be utilized.

[0164] For example, a cell population comprising ECs can be treated with a suitably labeled antibody that specifically binds to an EC surface cell marker, such as an anti-CD31 antibody. Subsequently, the isolation of ECs is achieved by collecting the labeled cells. For example, antibody-bound cells can be isolated by subjecting a cell population treated with a fluorescence-labeled antibody to sorting through Fluorescence-Activated Cell Sorting (FACS). Alternatively, if an antibody labeled with a magnetic material is used, Magnetic-Activated Cell Sorting (MACS) or the like can be employed. Furthermore, the aforementioned antibody may be combined with another antibody (secondary antibody) specifically binding to the label attached to the initial antibody or the antibody itself. In this case, the separation of ECs is carried out by using a label attached to the secondary antibody. A commercially available antibody can be used as the above anti-CD31 antibody or the secondary antibody. For example, “anti-CD31 antibody bonded with a PE fluorescent label” and “magnetic beadsembedding anti-PE antibodies” can be used, without being particularly limited thereto. The ratio of the isolated ECs (CD31 positive ratio) is, preferably 98% or more.Screening

[0165] Aspects of the instant disclosure include methods of screening pharmacological agents using endothelial cells, organoids, or panels of cells derived according to the methods described herein. In some instances, a plurality of cell populations derived according to the methods as described herein are contacted with a plurality of pharmacological agents in order to screen for agents producing a cellular response of interest. A cellular response of interest may be any cellular response including but not limited to, e.g., cell death, cell survival, cell self-renewal, proliferation, differentiation, expression of one or more markers, loss of expression of one or more markers, change in morphology, change in cellular physiology, cellular engraftment, change in cell motility, change in cell migration, production of a particular cellular component, cease of production of a particular cellular component, change in metabolic output, response to stress, and the like.

[0166] Screening pharmacological agents using cells described herein may be performed in vitro, e.g., in a tissue culture chamber, on a slide, etc., or may be performed in vivo, e.g., in an animal host, etc. Cells used in such screening assays may be genetically altered or may an unaltered cell. In some instances, cells generated according to the methods as described herein are used in multiplexed in vitro pharmacological screening. Methods for evaluating cellular responses during in vitro screening are well-known in the art and include but are not limited to, e.g., microscopic methods (e.g., light microscopy, electron microscopy, etc.), expression assays, enzymatic assays, cytological assays (e.g., cellular staining), genomics, transcriptomics, metabolomics, and the like.

[0167] In some instances, cells generated according to the methods as described herein are introduced into a host animal and the host animal may be administered a pharmacological agent in order to screen for a response from the introduced cells. In some instances, the cells of the in vivo assay may be directly evaluated, e.g., for an intrinsic response to a pharmacological agent. In some instances, the host animal of the in vivo assay may be evaluated as an indirect measurement of the response of the cells to the pharmacological agent.

[0168] In certain embodiments, the subject disclosure includes screening cells derived according to the methods described herein as a method of therapy of an animal model of disease and / or a human disease. Methods of screening cells derived according to the methods described herein as a method of therapy may be, in some instances, performed according to those methods described below regarding using such cells in therapeutic protocols.

[0169] In certain embodiments, the subject disclosure includes screening cells derived according to the methods described herein introduced to a host animal as a method of directly evaluating the cells or particular cellular behaviors, e.g., due to an introduced genetic modification or a naturally derived mutation. In one embodiment, genetically modified cells, e.g., having at least one modified genomic locus, derived according to the methods described herein may be introduced into a host animal and the ability of the cells to differentiate into a particular tissue or cell type may be evaluated. In another embodiment, genetically modified cells derived according to the methods described herein may be introduced into a host animal and the behavior of the cells within the host animal and / or within a tissue of the host animal may be evaluated. In another embodiment, cells derived from a donor organism having a particular mutation or phenotype and lineage restricted according to the methods described herein may be introduced into a host animal and the behavior of the cells within the host animal and / or within a tissue of the host animal may be evaluated, including, e.g., the ability of the cells to differentiate into one or more tissue or cell types. The cells may be introduced into the host animal in an autologous graft, an allograft, or a xenograft such that the introduced cells may be derived from the host animal, a separate donor of the same species as the host animal, or a separate donor of a different species as compared to the host animal, respectively.

[0170] In some embodiments a panel of genetically distinct human ECs are provided, which may be referred to as an “endothelial cell village”. A panel may comprise 5, 10, 15, 20, 25, 30 or more genetically distinct cells. In addition to endothelial cells, a panel may comprise earlier stage cells, for example, iPSCs, mesodermal lineage cells and endothelial progenitor cells, which can be generated according to the methods of the disclosure. In some embodiments a genetic difference comprises a targeted change in a gene of interest, e.g. a naturally occurring mutation, an introduced mutation, and the like. In some embodiments a genetic difference comprises a different ancestry, a different sex, a different region of origin, and the like.

[0171] In some embodiments, screening is performed on a panel as described above. As demonstrated in the Examples, genetic background can influence drug-induced cellular phenotypes (e.g., cytotoxicity), mimicking real-world population variability observed in clinical drug trials. Unlike traditional human clinical trials, the cell models of the disclosure offer precise environmental control, eliminating confounding factors such as diet and lifestyle. The system is also scalable, enabling high-throughput screening of multiple compounds across a genetically diverse “population”. Moreover, the significant ancestry-driven differences in response validate the utility of iPSC models for pharmacogenetics research. This approach allows prediction of drug efficacy and toxicity in underrepresented populations, addressing an urgent need in precision medicine.Therapy

[0172] Aspects of the disclosure include methods for lessening the symptoms of and / or ameliorating a dysfunction in an endothelial cell or a disease of endothelial origin, herein referred to as endothelial dysfunction or disorder. Non-limiting examples of endothelial cell types and / or tissues of endothelial origin that may be subject to disease or dysfunction that may be treated according to the method described herein include but are not limited to cardiac, arterial and venous endothelial cells, and the like.

[0173] Treatment methods described herein include therapeutic treatments, in which the subject is inflicted prior to administration and prophylactic treatments. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of having an increased likelihood of becoming inflicted (e.g., relative to a standard, e.g., relative to the average individual, e.g., a subject may have a genetic predisposition to endothelial dysfunction or disorder and / or a family history indicating increased risk of endothelial dysfunction or disorder), in which case the treatment can be a prophylactic treatment. In some embodiments, the individual to be treated is an individual with endothelial dysfunction or disorder. As used herein endothelial dysfunction or disorder” includes any form of dysfunction of an endothelial derived tissue or cell type. Any and all forms of endothelial dysfunction, whether treated or untreated, or resulting from any primary condition, whether treated or untreated, are suitable endothelial dysfunctions or disorders to be treated by the subject methods described herein. For example, conditions affecting cardiac tissue, such as ischemic conditions, may be treated.

[0174] In some instances, the treatment methods described herein include the alleviation or reduction or prevention of one or more symptoms of endothelial dysfunction or disorder. Symptoms of endothelial dysfunction or disorder will vary, may be infrequent, occasional, frequent, or constant.

[0175] The methods of treatment described herein include administering a therapeutically effective amount of a population, e.g., an essentially homogenous population, of endothelial cells derived by the methods of the disclosure, to a subject in need thereof.

[0176] The effective amount administered varies depending upon the goal of the administration, the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g., human, non-human primate, primate, etc.), the degree of resolution desired (e.g., the amount of alleviation or reduction of symptoms), the formulation of the cell composition, the treating clinician’s assessment of the medical situation, and other relevant factors.

[0177] A “therapeutically effective dose” or “therapeutic dose” is an amount sufficient to affect desired clinical results (i.e., achieve therapeutic efficacy) or reduce, alleviate, or prevent symptoms to a desired extent as determined by the patient or the clinician. A therapeutically effective dose can be administered in one or more administrations. For purposes of this disclosure, a therapeutically effective dose of cells and / or an organoid composition is anamount that is sufficient, when administered to (e.g., transplanted into) the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state.

[0178] In some embodiments, a therapeutically effective dose of cells is one cell or more (e.g., 1 x102or more, 5x102or more, 1 x103or more, 5x103or more, 1x104cells, 5x104or more, 1 x105or more, 5x105or more, 1 x 106or more, 2x106or more, 5x106or more, 1 x107cells, 5x107or more, 1 x108or more, 5x108or more, 1 x 109or more, 5x109or more, or 1 x1010or more).

[0179] In some embodiments, a therapeutically effective dose of cells is in a range of from 1 x103cells to 1 x1010cells (e.g., from 5x103cells to 1 x1010cells, from 1 x104cells to 1 x1010cells, from 5x104cells to 1 x1010cells, from 1x105cells to 1 x1010cells, from 5x105cells to 1 x1010cells, from 1 x10® cells to 1 x1010cells, from 5x106cells to 1 x1010cells, from 1 x107cells to 1 x1010cells, from 5x107cells to 1x1010cells, from 1x10scells to 1 x101° cells, from 5x108cells to 1 x1010, from 5x103cells to 5x109cells, from 1 x104cells to 5x109cells, from 5x104cells to 5x109cells, from 1 x105cells to 5x109cells, from 5x105cells to 5x109cells, from 1 x106cells to 5x109cells, from 5x106cells to 5x109cells, from 1 x107cells to 5x109cells, from 5x107cells to 5x109cells, from 1 x108cells to 5x109cells, from 5x108cells to 5x109, from 5x103cells to 1 x109cells, from 1 x104cells to 1 x109cells, from 5x104cells to 1 x109cells, from 1 x105cells to1 x109cells, from 5x105cells to 1 x109cells, from 1 x106cells to 1 x109cells, from 5x106cells to1 x109cells, from 1 x107cells to 1 x109cells, from 5x107cells to 1 x109cells, from 1 x108cells to1 x109cells, from 5x108cells to 1 x109, from 5x103cells to 5x108cells, from 1 x104cells to 5x108cells, from 5x104cells to 5x108cells, from 1 x105cells to 5x108cells, from 5x105cells to 5x108cells, from 1 x106cells to 5x108cells, from 5x106cells to 5x108cells, from 1x107cells to 5x108cells, from 5x107cells to 5x108cells, or from 1 x108cells to 5x108cells).

[0180] In some embodiments, the concentration of cells to be administered is in a range of from 1 x 105cells / ml to 1 x 109cells / ml (e.g., from 1 x 105cells / ml to 1 x 108cells / ml, from 5 x 105cells / ml to 1 x 108cells / ml, from 5 x 105cells / ml to 5 x 107cells / ml, from 1 x 106cells / ml to 1 x 108cells / ml, from 1 x 106cells / ml to 5 x 107cells / ml, from 1 x 106cells / ml to 1 x 107cells / ml, from 1 x 106cells / ml to 6 x 106cells / ml, or from 2 x 106cells / ml to 8 x 106cells / ml).

[0181] In some embodiments, the concentration of cells to be administered is 1 x 105cells / ml or more (e.g., 1 x 105cells / ml or more, 2 x 105cells / ml or more, 3 x 105cells / ml or more, 4 x 105cells / ml or more, 5 x 105cells / ml or more, 6 x 105cells / ml or more, 7 x 105cells / ml or more, 8 x 105cells / ml or more, 9 x 105cells / ml or more, 1 x 106cells / ml or more, 2 x 106cells / ml or more, 3 x 10® cells / ml or more, 4 x 10® cells / ml or more, 5 x 10® cells / ml or more, 6 x 10® cells / ml or more, 7 x 10® cells / ml or more, or 8 x 10® cells / ml or more).

[0182] A therapeutically effective dose of cells may be delivered or prepared and any suitable medium, including but not limited to, e.g., those described herein. Suitable medium for the delivery of a therapeutically effective dose of cells will vary and may depend on, e.g., the typeof endothelial cells from which the effective dose of cells is derived or the type of derived cells of the effective dose. In some instances, a suitable medium may be a basal medium. “Cell medium” as used herein are not limited to liquid media may, in some instances, include nonliquid components or combinations of liquid media and non-liquid components. Non-liquid components that may find use a delivery or preparation medium include those described herein and those known in the art. In some instances, non-liquid components include natural or synthetic extra cellular matric components including but not limited to, e.g., basement membrane matrix components and the like.

[0183] In some instances, an effective dose of the cells described herein may be coadministered with one or more additional agents (e.g., prepared in a suitable medium). Additional agents useful in such co-administration include agents that improve the overall effectiveness of the effective dose of cells or decrease the dose of cells necessary to achieve an effect essentially equal to administration of an effective dose of the cells without the additional agent. Non-limiting examples of additional agents that may be co-administered include: conventional agents for treating diseases, additional cell types, pro-survival factors, pro-engraftment factors, functional mobilization agents, and the like.

[0184] By pro-survival factors is meant a factor or agent that may be added to culture media, delivery excipient, or storage solution that promotes the survival of a desired cell type. Such pro-survival factors may be general pro-survival factors that generally promote the survival of most cell types or may be specific pro-survival factors that only promote the survival of certain specific cell types. In some instances, pro-survival factors of the subject disclosure include but are not limited to, e.g., Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g., low does, i.e., sub-immunosuppressive dose, cyclosporine), Z-VAD-FMK, pro-survival cytokines (e.g., insulin-like growth factor-1 (IGF-1 )), extra cellular matrix (ECM) components, hydrogels, Matrigel, collagen, gelatin, agarose, alginate, polyethylene glycol), hyaluronic acid, etc.

[0185] By pro-engraftment factors is meant a factor or agent that may be added to the administered dose or the delivery excipient or the cell storage solution that, upon delivery of the cells into a subject for treatment, increase the engraftment of the administered cells into the tissue targeted for engraftment and therapy. In some instances, pro-engraftment factors include factors that physically retain the administered cells at the delivery site, e.g., the injection site in the case of direct injection to the affected area, including but not limited to, e.g., gels, polymers, and highly viscous liquids that have physical properties that prevent the administered cells from freely diffusing. Such gels, polymers, and highly viscous liquids include but are not limited to e.g., extracellular matrix components, hydrogels, Matrigel, collagen, gelatin, agarose, alginate, polyethylene glycol), and the like.

[0186] The terms “co-administration” and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject’s body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.

[0187] The cells may be introduced by injection, catheter, intravenous perfusion, or the like. The cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being capable of use upon thawing. Once thawed, the cells may be expanded by use of growth factors and / or feeder cells or in feeder-free conditions associated with progenitor cell proliferation and differentiation. In some instances, the cells may be administered fresh such that the cells are expanded and differentiated and administer without being frozen.

[0188] The cells and / or compositions of this disclosure can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient or buffer or media prepared under sufficiently sterile conditions for human administration. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. Choice of the cellular excipient and any accompanying elements of the composition will be adapted in accordance with the route and device used for administration. The composition may also comprise or be accompanied with one or more other ingredients that facilitate the engraftment or functional mobilization of the cells. Suitable ingredients include matrix proteins that support or promote adhesion of the cells, or complementary cell types.

[0189] Cells of the subject methods may be autologously derived. By autologously derived it is meant that the cells are derived from the subject that is to be treated with the cells. The cells may be derived from a tissue sample obtained from the subject including but not limited to, e.g., a blood sample (e.g., a peripheral blood sample), a skin sample, a bone marrow sample, and the like. In some instances, the sample from which cells are derived may be a biopsy or swab, e.g., a biopsy or swab collected to diagnose, monitor, or otherwise evaluate the subject,e.g., diagnose the subject for a endothelial dysfunction or deficiency, e.g., bone disease or a muscle disease or a cartilage disease or a related condition, or for cell collection. In some instances, the autologous sample from which the cells are derived may be a previously collected and stored sample, e.g., a banked tissue sample, from the subject to be treated, including but not limited to e.g., banked cardiac tissue or cells, banked musculoskeletal tissue or cells, banked reproductive tissue or cells, banked skin tissue or cells, banked bone tissue or cells, banked bone marrow tissue or cells, banked vascular tissue or cells, banked umbilical cord blood tissue or cells, and the like.

[0190] In some instances, cells of the subject methods may be non-autologously derived. By non-autologously derived, it is meant that the cells are not derived from the subject that is to be treated with the cells. In some instances, non-autologously derived cells may be xeno- derived (i.e., derived from a non-human animal) or allo-derived (i.e., derived from a human donor other than the subject to be treated). Non-autologously derived cells or tissue may be derived from any convenient source of cells or tissue collected by any convenient means.

[0191] Whether to use autologously derived or non-autologously derived cells may be determined according to the discretion of the subject’s clinician and may depend on, e.g., the health, age, genetic predisposition or other physical state of the subject. In some instances, autologous cells may be preferred, including, e.g., to decrease the risk or immune rejection of the transplanted cells. In some instances, non-autologous cells may be preferred, including, e.g., when the subject has a genetic defect that affects endothelial tissues.

[0192] In some instances, methods as described herein may be performed using newly derived non-autologous pluripotent progenitor cells or newly derived autologous pluripotent progenitor cells including but not limited to, e.g., newly derived embryonic stem cells (ESC) (including, e.g., those derived under xeno-free conditions as described in, e.g., Lei et al. (2007) Cell Research, 17:682-688) and newly derived induced pluripotent stem cells (iPSC), followed by culture with the methods of the disclosure. General methods of inducing pluripotency to derive pluripotent progenitor cells are described in, e.g., Rodolfa KT, (2008) Inducing pluripotency, StemBook, ed. The Stem Cell Research Community, doi / 10.3824 / stembook.1.22.1 and Selvaraj et al. (2010) Trends Biotechnol, 28(4)214-23, the disclosures of which are incorporated herein by reference. In some instances, pluripotent progenitor cells, e.g., iPS cells, useful in the methods described herein are derived by reprogramming and are genetically unmodified, including e.g., those derived by integration- free reprogramming methods, including but not limited to those described in Goh et al. (2013) PLoS ONE 8(1 1): e81622; Awe et al (2013) Stem Cell Research & Therapy, 4:87; Varga (2014) Exp Cell Res, 322(2)335-44; Jia et al. (2010) Nat Methods, 7(3):197-9; Fusaki et al. (2009) Proc Jpn Acad Ser B Phys Biol Sci. 85(8) :348-62; Shao & Wu, (2010) Expert Opin Biol Then 10(2):231 -42; the disclosures of which are incorporated herein by reference. In someinstances, the derived or obtained pluripotent progenitor cells are prepared, dissociated, maintained and / or expanded in culture prior to being differentiated and / or lineage restricted as described herein.

[0193] In some instances, before differentiation or lineage restriction of the pluripotent progenitor cells are dissociated, e.g., to generate a single-cell suspension. In some instances, the dissociation of the pluripotent progenitors is chemical, molecular (e.g., enzyme mediated), or mechanical dissociation. Methods of chemical, molecular, and / or enzyme mediated dissociation will vary and, in some instances, may include but are not limited to the use of, e.g., trypsin, TrypLE Express™, TrypLE Select™, Accutase®, StemPro® (Life Technologies, Inc., Grand Island, NY), calcium- and magnesium-free media, low calcium and magnesium medium, and the like. In some instances, the dissociation media may further include prosurvival factors including but not limited to, e.g., Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g., low does, i.e., sub-immunosuppressive dose, cyclosporine), Z-VAD-FMK, pro-survival cytokines (e.g., insulin-like growth factor-1 (IGF-1 )), Thiazovivin, etc.

[0194] In some instances, methods of culturing endothelial cells include xeno-free culture conditions wherein, e.g., human cells are not cultured with any reagents derived from nonhuman animals. In some instances, methods culturing of endothelial stem cells include feeder- free culture conditions, wherein the endothelial cells are cultured under conditions that do not require feeder cells and / or in feeder cell free medium, including e.g., commercially available feeder-free mediums, such as, e.g., those available from STEMCELL Technologies, Inc. (Vancouver, BC). In some instances, methods of culturing endothelial cells include culture conditions that include supplemental serum, including supplement of autologously derived serum, as described in Stute et al. (2004) Exp Hematol, 32(12):1212-25. In some instances, methods of culturing of endothelial cells or derivatives thereof include culture conditions that are serum-free, meaning the culture media does not contain animal, mammal, or human derived serum. Serum-free culture conditions may be performed for only a portion of the life of the culture or may performed for the entire life of the culture. In some instances, serum-free culture conditions are used for a particular method step or procedure, e.g., during differentiation, during lineage restriction, prior to or during harvesting, etc. As is known in the art, in some instances, cells may be cultured in two-dimensional or three-dimensional formats (e.g., on non-coated or coated surfaces or within a solid or semi-solid matrix). Instances where two dimensional or three-dimensional culture is appropriate for use in the methods as described herein, e.g., to promote survival or differentiation of a desired cell type, will be readily apparent to the ordinary skilled artisan. In some instance, the endothelial cell media include one or more pro-survival factors, e.g., including those described herein.

[0195] In some instances, the endothelial cells used according to the methods described herein may be genetically unmodified. By “genetically unmodified” is meant that essentially no modification of the genome of the cells transplanted into the subject has been performed. Encompassed within the term genetically unmodified are instances wherein transient genetic modification is performed at some point during the derivation of the cells but essentially no genetic modification persists in the cells that are eventually transplanted into the subject (i.e., the cells are essentially indistinguishable before the transient genetic modification and after the course of the transient modification). Also encompassed within the term genetically unmodified are instances wherein the genome of the cells is not transiently or stably modified, e.g., where the cells are manipulated, e.g., pluripotent progenitors are derived or cells are transformed, without genetic modification (e.g., modification of the nucleotide sequence of the genome) of the cells.

[0196] For further elaboration of general techniques useful in the practice of this disclosure, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, and embryology. With respect to tissue culture and stem cells, the reader may wish to refer to Teratocarcinomas and embryonic stem cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al. eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31 , 1998).Systems

[0197] Also provided are systems for use in practicing the subject methods. Systems of the subject disclosure may include a cell production system, e.g., for the production of a homogenous or highly pure population of derived endothelial cells from pluripotent progenitor cells.

[0198] In some instances, the cell production system includes a cell culture chamber or cell culture vessel for the culture of desired cell types. Such cell culture chambers may be configured for the expansion of pluripotent progenitor cells and for the differentiation and / or lineage restriction of such pluripotent progenitor cells into desired cell types, e.g., derived endothelial cell progenitors and / or differentiated endothelial cells. In some instances, the cell culture chamber is also configured for the expansion of endothelial cells. In certain embodiments, the cell culture chamber or cell culture vessel may be an open culture system, including but not limited to e.g., tissue culture dishes, tissue culture plates, tissue culture multiwell plates, tissue culture flasks, etc. In certain embodiments, the cell culture chamber or cellculture vessel may be a closed culture system, including e.g., a bioreactor, a stacked tissue culture vessel (e.g., CellSTACK Culture Chambers, Corning, NY). In some instances, culture media and or other factors or agents may be exchanged in and out of the cell culture chamber through the use of one or more pumps (e.g., syringe pumps, peristaltic pumps, etc.) or gravity flow devices. In instances where the cells are cultured under sterile conditions, the culture system may allow for the sterile exchange of culture media, e.g., through the use of sterile tubing connected, sealed, and reconnected through the use of a sterile devices, including but not limited to, e.g., a sterile tube welder and / or a sterile tube sealer. The cell culture system may be configured to control certain environmental conditions, including but not limited to e.g., temperature, humidity, light exposure, air composition (e.g., oxygen levels, carbon dioxide levels, etc.) to achieve the conditions necessary for expansion and / or differentiation of desired cell types. In some instances, the cell culture chamber may include a cell culture vessel that includes one or more patterned cell culture substrates or one or more arrays of patterned cell culture substrates as described herein.

[0199] The cell culture chamber may be configured for the production of cells for clinical use, e.g., according to current good manufacturing practice (cGMP) compliant cell culture practices, including the methods and configurations described in e.g., Fekete et al. PLoS ONE (2012) 7(8): e43255; Pham et al. (2014) J Trans Med 12:56; Gastens et al. (2007) Cell Transplant 16(7):685-96; Fernandes et al. (2013) Stem Cell Bioprocessing: For Cellular Therapy, Diagnostics and Drug Development, Burlington, Oxford: Elsevier Science: Woodhead Publishing, the disclosures of which are incorporated herein by reference.

[0200] The cell production system may, in some instances, by computer-controlled and / or automated. Automated and / or computer-controlled cell production systems may include a “memory” that is capable of storing information such that it is accessible and retrievable at a later time or date by a computer. Any convenient data storage structure may be chosen, based on the means used to access the stored information. In certain aspects, the information may be stored in a “permanent memory” (i.e., memory that is not erased by termination of the electrical supply to a computer or processor) or “non-permanent memory”. Computer harddrive, CD-ROM, floppy disk, portable flash drive and DVD are all examples of permanent memory. Random Access Memory (RAM) is an example of non-permanent memory. A file in permanent memory may be editable and re-writable.

[0201] In certain instances, a computer-controlled and / or automated cell culture system may include a module or program stored in memory for production of cells according to the methods described herein. Such a module may include instructions for the administration of induction agent and / or induction compositions, e.g., at particular timing intervals or according to a particular schedule, in order to generate a desired cell type. In some instances, such a computer module may further include additional modules for routine cell culture tasks includingbut not limited to, e.g., monitoring and record keeping, media changes, environmental monitoring, etc.

[0202] Systems of the present disclosure include components and / or devices for delivering cells produced according to the methods described herein to a subject in need thereof. For example, in some instances a system for treating a subject with a tissue dysfunction or deficiency includes a cell injection system for delivering cells in a carrier, with or without optional adjuvants, to a desired injection site, including diseased tissue, adjacent to diseased tissue, and / or within, on or near a dysfunctioning organ. Such systems utilize known injection devices (e.g., including but not limited to needles, bent needles, cannulas, syringes, pumps, infusion devices, diffusion devices, etc.) and techniques (e.g., including but not limited to intramuscular injection, subcutaneous injection, device-guided injection, etc.). In some instances, a device or technique used for the delivery of a cell scaffold or other bioengineered device may be configured or adapted for use in a cell delivery system for use in delivering cells derived according to the methods described herein.

[0203] In addition to the above-described components systems of the subject disclosure may include a number of additional components, such as data output devices, e.g., monitors and / or speakers, data input devices, e.g., interface ports, keyboards, etc., fluid handling components, power sources, controllers, etc.Compositions and Kits

[0204] Also provided are compositions and kits for use in the subject methods. The subject compositions and kits include any combination of components for performing the subject methods. In some embodiments, a composition can include, but is not limited to and does not require, the following: cell dissociation agents and / or media, cell reprogramming agents and / or media, pluripotent progenitor cells, cell culture agents and / or media, cell differentiation agents and / or media; lineage restriction agents (e.g., induction agents) and / or media; conventional agents for treating diseases and / or dysfunctions, pro-survival factors, pro-engraftment factors, functional mobilization agents and any combination thereof.

[0205] In some embodiments, a kit can include, but is not limited to and does not require, the following: any of the above described composition components, a sample collection container, a sample collection device (e.g., a sample collection container that includes a sample enrichment mechanism including, e.g., a filter), a tissue collection device (e.g., a biopsy device), a tissue dissociation device, a cell culture vessel, a cell production system; and any combination thereof.

[0206] In some embodiments, a kit can include, but is not limited to and does not require, a cell delivery system and / or a cell injection system configured for delivery of cells derived according to the methods described herein. For example, a kit may include a cell injectionsystem configured for injection or delivery of cells into a desired area of the subject in order to effectively treat the subject for a tissue dysfunction or deficiency, e.g., through delivery of cells to the tissue. Such kits may include a cell delivery or injection system, as described herein, including individual components of such systems in assembled or unassembled form. In some instances, cells derived according to the methods described herein may be “preloaded” into a cell injection or delivery system such that the system is provided in a “ready-to-use" configuration. In other instances, a cell injection or delivery system may be provided in an “unloaded” configuration such that cells derived according to the methods described herein must be loaded into the system, with any desired carrier or vehicle, prior to use.

[0207] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is electronic, e.g., a website address which may be used via the internet to access the information at a removed site.EXAMPLES

[0208] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., room temperature (RT); base pairs (bp); kilobases (kb); picoliters (pl); seconds (s or sec); minutes (m or min); hours (h or hr); days (d); weeks (wk or wks); nanoliters (nl); microliters (ul); milliliters (ml); liters (L); nanograms (ng); micrograms (ug); milligrams (mg); grams (g), in the context of mass); kilograms (kg); equivalents of the force of gravity (g), in the context of centrifugation); nanomolar (nM); micromolar (pM), millimolar (mM); molar (M); amino acids (aa); kilobases (kb); base pairs (bp); nucleotides (nt); intramuscular (i.m.); intraperitoneal (i.p.); subcutaneous (s.c.); and the like.

[0209] Methods are provided for producing mesodermal-lineage cells, endothelial progenitor cells, and terminally differentiated endothelial cells. Also provided are methods of screening for cellular responses and treating a subject for a condition using the produced endothelial cells.Efficient and Rapid Generation of Endothelial Cells from Human Pluripotent Stem Cells

[0210] Here, we report a stepwise approach to generate pure endothelial cells from human induced pluripotent stem cells (iPSCs), which were shown to resemble primary cells at molecular, cellular, and functional levels.

[0211] Multiple protocols were tested in development, as shown in the figures, and as summarized below. The nomenclature “brew” refers to the commercially available StemMAC iPS-Brew XF, human medium for induced pluripotent stem cell maintenance.

[0212] Version 1.0, shown in FIG. 2, used a chemically defined medium (CDM) containing bovine serum albumin (BSA) for iPSC differentiation. Mesodermal cells were induced by activating Wnt signaling through the glycogen synthase kinase (GSK) 3 inhibitor, CHIR99021 (Gi). However, treatment with CHIR99021 alone resulted in a heterogeneous population of mesodermal cells, compromising the efficiency of endothelial progenitor cell (EPC) differentiation. Once mesodermal cells were generated, we further treated them with three commonly used morphogens to induce an endothelial fate: FGF2 (fibroblast growth factor 2 / basic fibroblast growth factor), BMP4 (bone morphogenetic protein 4), and VEGF (vascular endothelial growth factor). Additionally, we included a TGF (transforming growth factor) inhibitor, SB431542, to prevent endothelial cell-to-mesenchymal transition (EndoMT). Collectively, we have four components (C4) in the endothelial progenitor cell induction medium in the version 1 .0 protocol. This method achieves >85% efficiency in generating endothelial cells using most iPSC or ESC lines.

[0213] Version 2.0, shown in FIG. 5: Mesodermal cells were generated in the same way in version 1.0. However, we additionally included a ROCK inhibitor, Y27632, and a myosin light chain kinase inhibitor, ML7, in the endothelial progenitor cell induction medium (C6) to further improve the yield of EPCs. This method achieves >90% efficiency in generating endothelial cells using most iPSC or ESC lines.

[0214] Version 3.0, shown in FIG. 8: By leveraging single-cell RNA sequencing, we observed that Wnt5a activation is commonly found in non-endothelial clusters. Therefore, we additionally incorporated a Wnt inhibitor, Wnt-C59, into the endothelial progenitor cell induction medium (C7) to enhance the yield of EPCs. This method achieves >92% efficiency in generating endothelial cells using most iPSC or ESC lines.

[0215] Version 4.0, shown in FIG. 22: In comparison to version 3.0, we substituted bovine serum albumin (BSA) with human serum albumin (HSA) in the chemically defined medium(CDM) for iPSC differentiation. This alteration was demonstrated to significantly enhance the differentiation efficiency of all intermediate cell types. To enhance the homogeneity of mesodermal cells, we employed a combination of concentration-titrated small molecules, comprising a Wnt signaling activator, CHIR99021 , a Wnt inhibitor, Wnt-C59, and a CDK8 inhibitor, BI1347. We termed this approach “baseline Wnt activation (GiWiCi)”. The homogeneity of mesoderm cells was confirmed by uniform expression of nuclear p-catenin and similar global gene expression profiles across various iPSC lines. The seven components used to induce EPCs were the same in version 3.0, expect the HSA-containing CDM was used. This method achieves >98% efficiency in generating endothelial cells using most iPSC or ESC lines.

[0216] Version 5.0 is shown in FIG. 27: In this version, we found that human platelet lysate (hPL) significantly enhances the survival of MES after replating and the proliferation rate of emerged EPCs. Consequently, we supplemented the endothelial progenitor cell induction medium (C8) with concentration-titrated hPL. Additionally, we replaced Lonza endothelial growth medium (EGM2) with a xeno-free endothelial cell growth medium (EndoGO) to further expedite EC proliferation and improve purity. This change allowed us to shorten the differentiation process from the original 14 days to 5-6 days. By further using recombinant vitronectin as the coating matrix, the differentiation system employed in version 5.0 is now 100% xeno-free. This method achieves >98.5% efficiency in generating endothelial cells using any iPSC or ESC lines.

[0217] The multiple protocols are summarized below.Table 1

[0218] Activities of the added factors are as follows: Endothelial Cell Growth Medium 2 is a low-serum (2% V / V) medium that lacks Endothelial Cell Growth Supplement (ECGS, bovine hypothalamic extract) but contains Insulin-like Growth Factor (Long R3 IGF) and Vascular Endothelial Growth Factor (VEGF).

[0219] CDM-BSA and CDM-HSA are described below in Table 3.

[0220] EndoGo™ XF Medium is a xeno-free culture medium designed for the long-term expansion of endothelial cells. The complete endothelial cell culture media consists of two components: EndoGo™ XF Medium and EndoGo™ XF Supplement Mix. In addition, the medium required the use of human AB serum (off the clot) or human platelet lysate.

[0221] Activities of the factors utilized in these cultures is shown in Table 2.Table 2

[0222] To derive a homogenous population of mesodermal cells with a specified hematopoietic cell fate across different iPSC lines or iterative differentiations, we subjected 1 .6X104 / cm2iPSCs to a cocktail comprising 8 pM CHIR99021 , 1 pM Wnt-C59, and 0.25 |uM BI1347 in a human serum albumin containing chemically defined medium (Table 1 ) for 48 hours (FIG. 20-21 ) to generate mesodermal lineage cells. Matrigel or recombinant vitronectin is used for culture vessel coating.

[0223] After 48 hours, mesodermal-lineage cells were dissociated into single cells using 1X TrypLE Select for 3-5 min. Cells at the density of 1 .6X104 / cm2were then resuspend in endothelial progenitor cell induction medium containing 50 ng / ml VEGF165, 20 ng / ml FGF2, 10 ng / ml BMP4, 1 pM Wnt-C59, 10 pM SB431542, 10 pM Y27632, 5 pM ML7, and 0.2% human platelet lysate (hPL) in a human serum albumin containing chemically defined medium (Table 1 ) for 48 to 72 hours (FIG. 23).

[0224] Subsequently, endothelial progenitor cell induction medium was replaced by EndoGO complete medium plus 2% hPL and 5 pM SB431542 for another 24 to 48 hours, or until cells reach >90% confluency (FIG. 27).

[0225] Confluent endothelial cells then can be dissociated using 1 X TrypLE Select for 5 to 7 min to get single cells. The cells can be either cryopreserved for later use or replated for furtherexpansion (FIG. 29). The culture vessels used for cell expansion can be coated with either vitronectin, 0.1 % gelatin, or fibronectin.

[0226] To repress the potential activation of the EndoMT program, 5 pM SB431542 can be added to the EndoGO medium plus 2% hPL. Simvastatin, GGTi298, chlorpromazine, or cyclosporin A at the concentrations of 1 pM, 1 pM, 2 pM, or 2 pM can be added to further repress EndoMT.

[0227] The derived endothelial cells are positive for CD31 and VE-cadherin and can uptake acetylated low-density lipoprotein (FIG. 32), negative for mural cell and fibroblast markers (FIG. 33), sensitive to pro-inflammatory cytokine-mediated secretion of adhesion molecules (FIG. 34-35), and capable of forming tubular structures when being seeded on Matrigel (FIG. 36).

[0228] Our single-cell data show that these endothelial cells predominantly acquire an arterial cell fate by default (FIG. 37) and closely resemble the transcriptomic profiles of adult primary cardiac endothelial cells (Fig. 38-39).Materials and Methods

[0229] Culture medium: Essential 8 (ThermoFisher, cat#A1517001 ) or Stemmacs™ iPS-Brew XF (Miltenyi Biotec cat#130-104-368); Ham’s F12 Nutrient Mix (Gibco, cat#1 1765-054); IMDM (Gibco, cat#12440-053); EGM™-2 Endothelial Cell Growth Medium-2 BulletKit (Lonza, cat#CC-3162) or EndoGO™ XF medium and supplement mix (Sartorius, cat#05-400-1 A and 05-410-1-25).

[0230] Chemicals, growth factors, and small molecules. Chemically defined lipid concentrate (Gibco, cat#1 1905-031 ); Bovine serum albumin (BSA, MilliporeSigma, cat#A9418); 20% Human serum albumin (HSA, Sartorius, cat#05-730-1 E); Glutamax™-! (Gibco, cat#35050- 061 ); Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (100X) (ThermoFisher, cat#51500056); Monothioglycerol (MilliporeSigma, cat#M6145); Gelatin solution (0.1% wt / vol, MilliporeSigma, cat#G1393); L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (25 mg / ml, MilliporeSigma, cat#A8960); Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, phenol red-free; LDEV-free (0.4% wt / vol, Corning, cat#356231 );vitronectin (Thermo Fisher Scientific, cat#A14700);TrypLE™ Express Enzyme (1 X), phenol red (Thermo Fisher Scientific, cat#12605036).

[0231] CHIR99021 (10 mM, Selleck Chemicals, cat#S2924); SB431542 (10 mM, SelleckChemicals, cat#S1067); Y27632 2HCI (10 mM, Selleck Chemicals, cat#S1049); BI-1347 (2.5 mM, Selleck Chemicals, cat#S1058); Wnt-C59 (10 mM, Selleck Chemicals, cat#S7037); ML- 7 HCI (20 mM, Selleck Chemicals, cat#S8388); RO4929097 (2 mM, Selleck Chemicals, cat# S1575), Simvastatin (1 mM, Sigma, cat#PHR1438), GGTi298 (1 mM, Selleck Chemicals, cat#E7318), chlorpromazine (2 mM, Selleck Chemicals, cat#S5749), cyclosporin A (2 mM,Selleck Chemicals, cat#S2286), Hydrocortisone (2 mM, Sigma, cat#H0888) VEGF165 (50 pg / ml, PeproTech, cat#100-20); FGF2 (100 pg / ml, PeproTech, cat#100-18B); BMP4 (100 pg / ml, PeproTech, cat#120-05 ET); EGF (100 pg / ml, PeproTech, cat# AF-100-15), IGF (200 pg / ml, PeproTech, cat#100-ll), CD144 (VE-Cadherin) MicroBeads, human (Miltenyi, cat#130- 097-857); LS COLUMNS 25 / PK (Miltenyi Biotec, Cat# 130-042-401 ).Table 3Procedure

[0232] Mesoderm induction: Passage iPSCs using standard protocols to maintain the pluripotency of the cells. When cells become -80% confluency, dissociate iPSCs using 1 X TrypLE Select (5 min) to get single-cell suspension.

[0233] Count and seed cells at 1 .0-1 .2 million per,T75 flask. Gently shake the flask in three quick back-and-forth and left-and-right motions to distribute iPSCs evenly across the flask in the incubator. Add ROCK inhibitor Y27632 at the final concentration of 5-10 pM to improve cell viability. [Note: Make sure iPSC viability is >90% to achieve successful differentiation]

[0234] Early mesoderm (MES) induction: Prepare MES induction medium in CDM [To make 50 ml MES induction medium, add 40 pl CHIR (8 pM) + 5 pl Wnt-C59 (1 pM) + 5 pl BI-1347 (0.25 pM) to 50 ml of CDM], Wash cells with DPBS and add 12 ml MES induction medium to each flask. Change medium after 48 hours (can be extended up to 60 hours). The cells, after 48-60 hours of CHIR treatment, should be in an elongated form and grow out from dense and round “cell aggregates” as observed on day 1 of induction.

[0235] Endothelial progenitor cell (EPC) specification: Prepare EPC induction medium in CDM [8 components / C8]: To make 50 ml of EPC medium, add 50 pl VEGF165 (50 ng / ml) + 50 pl SB431542 (10 pM)+ 50 pl Y27632 (10 pM) + 12.5 pl ML-7 (5 pM)+ 5 pl Wnt-C59 (1 pM) + 10 pl FGF2 (20 ng / ml)+ 5 pl BMP4 (10 ng / ml) + 100 pl human platelet lysate (hPL, 0.2% vol / vol). Dissociate Day 2 mesodermal cells into singlets using 1 X TrypLE Select for 5 min and seed 50K (CDM-BSA) or 100K (CDM-HSA) cells / well of 6-well plates; 250K (CDM-BSA) or 500K (CDM-HSA) cells / 10-cm dish; 500K-680K (CDM-BSA) or 1 M-1 .2M (CDM-HSA) cells / T75 flask (Matrigel or vitronectin-coated). Gently shake the dishes in three quick back- and-forth and left-and-right motions to distribute iPSCs evenly across the wells in the incubator. Add 2 ml per well, 8 ml per 10-cm dish, or 12 ml per flask. Maintain cells in the medium for 2-3 days without medium change.

[0236] EC maturation and expansion: At day 5, switch to EndoGo medium+ 2% hPL-i- 5 pM of SB431542 to promote EC proliferation and maturation. Alternatively, ECs can be expanded in CDM-HSA-based EC expansion medium, as shown in Table 3. It normally takes 1 -2 days before cells reach > 90% confluency. If cell purification is required, proceed to steps [00207- 00212]; otherwise, dissociate ECs using 1 X TrypLE Select for 5-7 min, and replate cells at 1 .5X105 / well in gelatin- or fibronectin-coated 6-well plates (or 1 :4 ratio in 10-cm dishes or T75 flasks).

[0237] EC purification using the MACS sorting system: MACS buffer: Prepare a solution containing Ca2+, Mg2+-free phosphate-buffered saline (PBS), pH 7.2, 0.5% BSA or HSA, and 2 mM EDTA. Keep buffer sterile and cold (4 °C).

[0238] Save the spent EndoGO medium from the original plates / dishes / flasks before cell dissociation. Wash cells with PBS. Dissociate cells using 1 X TrypLE for 5-7 min until almostall cells become singlets. Pool cells from up to 6 of 10-cm dishes or 4 of T75 flasks after dissociation at this point. Neutralizing the enzyme by transferring cell-containing IX TrypLE to saved spent EndoGO medium. You may count the total cell numbers, and for every 107cells, add 80 pl MACS buffer and 20 pl anti-VE-cadherin (CD144+) antibody magnetic beads.

[0239] Incubate the cells for 15-20 minutes at 4°C.

[0240] Add 10 ml of MACS buffer to wash the anti-VE-cadherin antibody labeled cells. Centrifuge cells at 300X g for 5 min. Aspirate supernatant completely.

[0241] Resuspend the cell pellet in 0.5 ml of MACS buffer. Follow the sorting protocol as per the manufacturer's instructions.

[0242] The cell pellet is resuspended in 1 -2 ml of EndoGO medium plus 2% hPL. After cell counting, seed cells at 1.5X105 / well in 0.1% gelatin- or fibronectin-coated 6-well plates (or scale up the total cell number if you want to seed cells in 10-cm dishes or T75 flasks). To improve cell growth and suppress EMT, add 5 pM SB431542 to the EndoGo medium. Simvastatin, GGTi298, chlorpromazine, or cyclosporin A at the concentrations of 1 pM, 1 pM, 2 pM, or 2 pM can be added to further repress EndoMT.EC culture maintenance: Refresh EndoGO +2% hPL + 5 pM SB431542 medium every other day. When cells become >85% confluent, split cells at a 1 :3-1 :4 ratio. Alternatively, ECs can be expanded in CDM-HSA-based EC expansion medium, as shown in Table 3.Protocol for generating vessel organoids (adapted from iPSC-EC protocol version 5.0)

[0243] [Day 0 to Day 2] Mesoderm (MES) induction: same steps / procedures as described above.

[0244] [Day 3-4] Embodied body (EB) formation using MES: Pre-treat round bottom ultra-low attachment 96-well plates (Coring, cat#7007) with 20 pl of anti-adherence rinsing solution (STEMCELL Technologies, cat#07010) or 0.1% Pluronic F-127 (Sigma, cat#P2443) for at least 10 min at 37 °C.

[0245] Prepare endothelial progenitor cell (EPC) induction medium: To make 50 ml of EPC medium, add 50 pl VEGF165 (50 ng / ml) + 50 pl SB431542 (10 pM)+ 50 pl Y27632 (10 pM) + 12.5 pl ML-7 (5 pM)+ 5 pl Wnt-C59 (1 pM) + 10 pl FGF2 (20 ng / ml)+ 5 pl BMP4 (10 ng / ml) + 100 pl human platelet lysate (hPL, 0.2% vol / vol) to 50 ml of CDM-HSA medium (as described above).

[0246] Dissociate MES into single cells using 1X TrypLE Select (3 min). Resuspend MES into EPC induction medium and adjust the cell density to (3-5) X104 / ml. Aspirate anti-adherence resining solution or 0.1% Pluronic F-127 from 96-well plate and dispense 100 pl of MES suspension into each well. Spin down the 96-well plate at 400 xg for 5 min. Leave the plate undisturbed in an incubator for 48 hours.

[0247] After 48 hours, freshly prepare collagen l-Matrigel solution for MES-EB embedding: Mix 300 pl of 0.1 M NaOH (Sigma, cat#S8045), 125.2 pl of 10X DMEM (ThermoFisher, cat#11430030), 25.2 pl of HEPES (1 M, ThermoFisher, cat#15630080), 20 pl of 7.5% sodium bicarbonate (ThermoFisher, cat#25080094), 12.4 pl of GlutaMax (ThermoFisher, cat#35050061 ), 184 pl of Ham’s F-12 Nutrient Mix (ThermoFisher, cat#11765062), and 1 .332 ml of PureCol Type I Collagen Solution (3 mg / ml, Advanced Biomatrix, cat#5005) on ice. Mix the collagen I solution well before adding 0.67 ml of growth factor-reduced Matrigel (Corning, cat#354230) on ice and mix gently.

[0248] Gently transfer all EBs using a wide-bore and low-retention 1000 pl tip from the 96- well plate to a 35-mm dish.

[0249] Keep the original 96-well plate on ice and use a wide-bore and low-retention 200 pl tip to transfer each EB back to individual well. Minimize the volume of medium to be transferred to each well. Add 20 pl of collagen-Matrigel solution to each well of 96-well plate. Spin down the plate at 400 xg for 5 min.

[0250] Put the 96-well plate back to an incubator for 2 hours.

[0251] Prepare vessel organoid sprouting medium: add 100 ng / ml VEGF165 and 100 ng / mlFGF2 to complete EGM2 medium (Lonza), EndoGO (Sartorius) supplemented with 2% hPL, or CDM-HSA endothelial cell expansion medium.

[0252] [Day 5-12] Vessel organoid maturation. After 2 hours, add 200 pl of vessel organoid sprouting medium to each well. Change the medium every 2-3 days.

[0253] Vessel organoids can be harvested for functional assays at day 12 of differentiation or may be maintained in the same medium described above for up to one month. Immunofluorescence staining of endothelial cell markers (e.g. CD31 and CD144) and pericyte markers (e.g., NG2 and PDGFRp) can be performed either on OCT-sectioned or tissue- cleared whole mount vessel organoids to validate the vascular network formation.

[0254] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed thatperform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

THAT WHICH is CLAIMED IS:1 . An in vitro method for method of producing a population of endothelial cells, the method comprising(a) contacting a population of pluripotent stem cells in culture with mesodermal cell (MES) induction medium to induce a population of MES cells;(b) contacting the population of MES cells with endothelial progenitor cell (EPC) induction medium to induce a population of endothelial progenitor cells; and(c) contacting the population of endothelial progenitor cells with endothelial maturation (EC) medium to induce a population of endothelial cells.

2. The method of claim 1 , wherein the population of endothelial cells that is produced is greater than about 90% pure.

3. The method of claim 1 , wherein the population of endothelial cells that is produced is greater than about 98% pure.

4. The method of any of claims 1 -3, wherein the endothelial cells are CDH5 (CD144, VE-cadherin)+and PECAM1 (CD31 )+.

5. The method of any of claims 1 -4, wherein each of steps (a), (b) and (c) are of a time period from about 12 to 60 hours.

6. The method of any of claims 1 -5, wherein each of steps (a), (b) and (c) are of a time period from about 24 to 48 hours.

7. The method of any of the previous claims, wherein the MES induction medium comprises an effective dose of a Wnt pathway activator.

8. The method of claim 7, wherein the pathway activator is present at a concentration equivalent to from about 1 uM to about 25 pM CHIR99021 .

9. The method of claim 7 or claim 8, wherein the MES induction medium further comprises an effective dose of a Wnt pathway inhibitor, and a CDK8 inhibitor.

10. The method of claim 9, wherein the Wnt pathway inhibitor is present at a concentration equivalent to from about 0.1 pM to about 10 pM Wnt-C59.

11. The method of claim 9 or claim 10, wherein the CDK8 inhibitor is present at a concentration equivalent to from about 0.05 pM to about 5 pM BI1347.

12. The method of any of the previous claims, wherein the EPC induction medium comprises an effective dose of an effective dose of an FGF agonist, a BMP agonist, a VEGF agonist, and a TGFp inhibitor.

13. The method of claim 12, wherein the EPC induction medium further comprises an effective dose of a Wnt inhibitor, a ROCK inhibitor, a myosin light chain kinase inhibitor, and human platelet lysate.

14. The method of claim 13, wherein the Wnt inhibitor is present at a concentration equivalent to from about 0.1 pM to about 10 pM Wnt-C59.

15. The method of any of claims 12-14, wherein the BMP agonist is present at a concentration equivalent to from about 0.5 to about 100 ng / ml BMP4 protein.

16. The method of any of claims 12-15, wherein the VEGF agonist is present at a concentration equivalent to from about 10 to about 500 ng / ml VEGF165.

17. The method of any of claims 12-16, wherein the FGF agonist is present at a concentration equivalent to from about 1 to about 200 ng / ml FGF2 protein.

18. The method of any of claims 12-17, wherein the TGFp inhibitor is present at a concentration equivalent to from about 2 to about 100 ng / ml SB431542.

19. The method of any of claims 13-18, wherein the ROCK inhibitor is present at a concentration equivalent to from about 1 to about 20 pM Y27632.

20. The method of any of claims 13-19, wherein the myosin light chain kinase inhibitor is present at a concentration of from about 0.5 to 20 pM ML-7.21 . The method of any of claims 13-20, wherein the human platelet lysate is present at a concentration of from about 0.05% (vol / vol) to about 2%.

22. The method of any of the previous claims, wherein the EC maturation medium comprises an effective dose of human platelet lysate, a VEGF pathway activator, and a TGF- beta pathway inhibitor.

23. The method of any of the previous claims, further comprising:(d) expanding the population of endothelial cells in endothelial cell medium.

24. The method of any of the previous claims wherein the medium in one or both of step (c) and (d) comprises an effective dose of a repressor of Endothelial-to-Mesenchymal Transition (EndoMT).

25. The method of claim 24, wherein the repressor is one or more of simvastatin, GGTi298, chlorpromazine, and cyclosporin A.

26. The method of any of the previous claims, wherein the medium in one or both of step (c) and (d) comprises an effective dose of a notch inhibitor, and wherein the endothelial cells are induced to vein endothelial cells.

27. The method of any of claims 23-26, wherein the medium in (d) comprises an effective dose of a VEGF agonist and an FGF agonist.

28. The method of claim 27, wherein a vascular organoid is produced in (d).

29. A kit for use in the methods of any of claims 1-28.

30. A substantially pure population of endothelial cells produced by the methods of any of claims 1 -28.31 . A panel of genetically distinct endothelial cells produced by the methods of any of claims 1 -28.

32. Use of the substantially pure population of endothelial cells for transplantation to a patient in need thereof.

33. A method of screening a candidate agent for activity on endothelial cells, the method comprising:contacting one or a panel of endothelial cells of claim 30 or 31 with the candidate agent, and determining response of the cells to the agent.

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