WO2013166165A1

WO2013166165A1 - Derivation of endothelial cells from human pluripotent stem cells

Info

Publication number: WO2013166165A1
Application number: PCT/US2013/039088
Authority: WO
Inventors: Rodolfo Gonzalez; Ibon Garitaonandia; Ruslan SEMECHKIN
Original assignee: International Stem Cell Corporation
Priority date: 2012-05-02
Filing date: 2013-05-01
Publication date: 2013-11-07
Also published as: CN104302764A

Abstract

The present invention is based in part on a chemically defined method of generating endomesodermal precursor cells (EMPCs) from human pluripotent stem cells (hPSCs). The invention further provides methods for generating endothelial cells (ECs) from EMPCs or hPSCs. The present invention also provides reagents and kits useful for the derivation of EMPCs and ECs from human pluripotent stem cells.

Description

DERIVATION OF ENDOTHELIAL CELLS FROM HUMAN PLURIPOTENT STEM

CELLS

FIELD OF THE INVENTION

[0001] The present invention relates generally to stem cells and more specifically to methods for deriving endomesodermal precursor cells and endothelial cells from human pluripotent stem cells.

BACKGROUND INFORMATION

[0002] Human embryonic stem cells (ES) cells are pluripotent cells that can differentiate into a large array of cell types. Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ- specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.

[0003] During embryonic development, stem cells form the tissues of the body from three major cell populations: ectoderm, mesoderm and definitive endoderm. Mesoderm gives rise to blood cells, endothelial cells, cardiac and skeletal muscle, and adipocytes. Definitive endoderm generates liver, pancreas and lung. Ectoderm gives rise to the nervous system, skin and adrenal tissues.

[0004] A potential application of stem cells, is making cells and tissues for medical therapies. Today, donated organs and tissues are often used to replace those that are diseased or destroyed. Unfortunately, the number of people needing a transplant far exceeds the number of organs available for transplantation. Stem cells offer the possibility of a renewable source of replacement cells and tissues to treat a myriad of diseases, conditions, and disabilities including Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury, burns, heart disease, diabetes, and arthritis.

[0005] The endothelium is the thin layer of cells that line the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. Vascular disease is a pathological state of large and medium sized muscular arteries and is triggered by endothelial cell dysfunction. Because of factors like pathogens, oxidized LDL particles and other inflammatory stimuli endothelial cells become activated and start to excrete cytokines and chemokines and express adhesion molecules on their surface. This process eventually leads to the thickening of vessel walls, forming a plaque consisting of proliferating smooth muscle cells, macrophages and various types of lymphocytes. This plaque results in obstructed blood flow leading to diminished amounts of oxygen and nutrients that reach the target organ. In the final stages, the plaque may also rupture causing the formation of clots, and as a result strokes. There is a need for new treatments for vascular diseases. Provided herein is a method for generating a high purity population f endomesodermal precursor cells and endothelial cells which may be useful in the treatment of vascular diseases.

SUMMARY OF THE INVENTION

[0006] The present invention is based in part on a chemically defined method of generating endomesodermal precursor cells (EMPCs) and endothelial cells (ECs) from human pluripotent stem cells (hPSCs). The present invention also provides reagents and kits useful for the derivation of endomesodermal precursor cells and endothelial cells from human pluripotent stem cells.

[0007] The present invention provides for the generation of endomesodermal precursor cells (EMPCs) by treating human pluripotent stem cells (hPSCs) with at least one

endomesodermal induction compound and assaying the cells for endomesodermal cell markers. The present invention also provides for the derivation of a high-purity population, homogenous population of endothelial cells (ECs) from hPSCs in a robust and reproducible manner via an endomesodermal stage, where the cells can be expanded and cryopreserved, using newly developed chemically directed differentiation methods.

[0008] In one embodiment, the present invention provides a method to generate endomesodermal cells (EMPCs). The method includes treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor and analyzing the cells for endomesodermal cell markers. In one aspect, the hPSCs are treated with the GSK-3 inhibitor for about 24 hours. In another aspect, the GSK-3 inhibitor is Chir99021 or salts or analogs thereof. Other antagonists include 3F8, A1070722, AR-A014418, BIO, FRATide, 10Z- Hymenialdsine, Indirubin-3'-oxime, Kenpaullone, L803, NSC693868, SB216763, SB415286, TC-G24, TCS202, TCS 21311, and TWS119, all available through Glaxo Smith Kline.

[0009] In an aspect, the hPSCs are human embryonic stem cells (hESCs), human parthenogenetic stem cells (hpSCs) or induced pluripotent stem cells (iPSCs), or cell lines derived therefrom.

[0010] In one aspect, the endomesodermal precursor cell markers include MIXL1, NODAL, and BRACHYURY, for example, or any combination thereof.

[0011] In one embodiment, the present invention provides for endomesodermal precursor cells (EMPCs). The subject EMPCs are generated by treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor and analyzing the cells for endomesodermal cell markers. In one aspect, the hPSCs are treated with the GSK-3 inhibitor for about 24 hours, or from 12-36 hours. In another aspect, the GSK-3 inhibitor is Chir99021 and the endomesodermal marker is Brachyury.

[0012] In another embodiment, the present invention provides a method to generate endothelial cells (ECs). The method includes treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor; identifying endomesodermal precursor cells (EMPCs) by assaying the treated hPSCs for endomesodermal cell markers; treating the EMPCs with at least one growth factor and analyzing the cells for endothelial cell markers. In one aspect, the hPSCs are treated with the GSK-3 inhibitor for about 24 hours. In certain aspects, the GSK-3 inhibitor is Chir99021 or salts or analogs thereof. In a further aspect, the EMPCs are treated with the growth factor for about 72 hours, or from about 48-96 hours. In an additional aspect the growth factors maybe bFGF, VEGF or BMP4 or any combination thereof. In a preferred aspect, the growth factors are bFGF and VEGF.

[0013] In an aspect, the hPSCs are human embryonic stem cells (hESCs), human parthenogenetic stem cells (hpSCs) or induced pluripotent stem cells (iPSCs), or cell lines derived therefrom.

[0014] In one aspect of the method, the endomesodermal cell markers maybe MIXL1, NODAL, BRACHYURY, for example, or any combination thereof.

[0015] In an additional aspect of the method, the endothelial cell markers maybe VE Cadhedrin, PECAM1, ACE/CD 143 ,MCAM/CD146, Clq R1/CD93, Nectin-2/CDl 12, VE- CflHhfirin PD-FCGF/Thymidine Phosphorylase, CC Chemokine Receptor D6, Podocalyxin, CD31/PECAM-1, Podoplanin, CD34, SlPl/EDG-1, CD36/SR-B3, S1P2/EDG-5, CD151, S1P3/EDG-3, CD160, S1P4/EDG-6, CD300LG/Nepmucin, S1P5/EDG-8, CL-Kl/COLECl 1, E-Selectin/CD62E, CL-P1/COLEC12, E-Selectin (CD62E)/P-Selectin (CD62P), Coagulation Factor Ill/Tissue Factor, P-Selectin/CD62P, DC-SIGNR/CD299, SLAM/CD 150,

DCBLD2/ESDN, Stabilin-1, EMMPRIN/CD147, Stabilin-2, Endoglin/CD105,

TEM7/PLXDC 1 , Endomucin, TEM8/ANTXR1, Endosialin/CD248,

Thrombomodulin/BDCA-3, EPCR, THSD1, Erythropoietin R, Tie-2, ESAM, TNF

RI/TNFRSF1A, FABP5/E-FABP, TNF RII/TNFRSF1B, FABP6, TRA-1-85/CD147, ICAM- 1/CD54, TRAIL R1/TNFRSF10A, ICAM-2/CD102, TRAIL R2/TNFRSF10B, IL-1 RI, VCAM-1/CD106, IL-13 R alpha 1, VE-Statin, Integrin alpha 4/CD49d, VEGF Rl/Flt-1, Integrin alpha 4 beta 1, VEGF R2/KDR/Flk-1, Integrin alpha 4 beta 7/LPAM-l, VEGF R3/Flt-4, Integrin beta 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 or any combination thereof. In a specific aspect, the endothelial cell markers are VE cadherin and PECAM1.

[0016] In a further embodiment, the present invention provides for endothelial cells (ECs). The subject ECs are generated by treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor; identifying endomesodermal precursor cells (EMPCs) by assaying the treated hPSCs for endomesodermal cell markers; treating the EMPCs with at least one growth factor and analyzing the cells for endothelial cell markers. In certain aspects, the hPSCs are treated with a GSK-3 inhibitor for 24 hours and the EMPCs are treated with growth factors for 72 hours. In specific aspects, the GSK-3 inhibitor is Chir99021 and the growth factors are bFGF and VEGF. In an additional aspect, the endomesodermal marker is Brachyury and the endothelial cell markers are VE cadhedrin and PECAM1.

[0017] In another embodiment, the present invention provides a kit for the derivation of EMPCs. The kit may include a glycogen synthase kinase 3 (GSK-3) inhibitor, reagents to identify endomesodermal precursor cell markers and directions for the generation of EMPCs from hPSCs. In one aspect, the GSK-3 inhibitor is Chir99021 or salts or analogs thereof. The kit may include reagents to identify MIXL1, NODAL, BRACHYURY cell markers or any combination thereof.

[0018] In another embodiment, the present invention provides a kit for the production of ^pnHnthfilifll r.^plls fFCs). The kit includes a glycogen synthase kinase 3 (GSK-3) inhibitor, reagents to identify endomesodermal precursor cell markers, growth factors, reagents to identify endothelial cell markers and directions for the production of endothelial cells from hPSCs. In one aspect, the GSK-3 inhibitor is Chir99021 or salts or analogs thereof. In another aspect the growth factors are bFGF and VEGF. The kit may include reagents to END016, FoxA, GATAE, SMIP, Hex, P19 and Brachyury endomesodermal precursor cell markers or any combination thereof and VE Cadhedrin, PECAM1, ACE/CD 143

,MCAM/CD146, Clq R1/CD93, Nectin-2/CDl 12, VE-Cadherin, PD-ECGF/Thymidine Phosphorylase, CC Chemokine Receptor D6, Podocalyxin, CD31/PECAM-1, Podoplanin, CD34, SlPl/EDG-1, CD36/SR-B3, S1P2/EDG-5, CD151, S1P3/EDG-3, CD160,

S1P4/EDG-6, CD300LG/Nepmucin, S1P5/EDG-8, CL-K1/COLEC11, E-Selectin/CD62E, CL-P1/COLEC12, E-Selectin (CD62E)/P-Selectin (CD62P), Coagulation Factor Ill/Tissue Factor, P-Selectin/CD62P, DC-SIGNR/CD299, SLAM/CD 150, DCBLD2/ESDN, Stabilin-1, EMMPRIN/CD147, Stabilin-2, Endoglin/CD105, TEM7/PLXDC1, Endomucin,

TEM8/ANTXR1, Endosialin/CD248, Thrombomodulin/BDCA-3, EPCR, THSD1,

Erythropoietin R, Tie-2, ESAM, TNF RI/TNFRSF1A, FABP5/E-FABP, TNF

RII/TNFRSF1B, FABP6, TRA-1-85/CD147, ICAM-1/CD54, TRAIL R1/TNFRSF10A, ICAM-2/CD102, TRAIL R2/TNFRSF10B, IL-1 RI, VCAM-1/CD106, IL-13 R alpha 1, VE- Statin, Integrin alpha 4/CD49d, VEGF Rl/Flt-1, Integrin alpha 4 beta 1, VEGF R2/KDR/Flk- 1, Integrin alpha 4 beta 7/LPAM-l, VEGF R3/Flt-4, Integrin beta 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 endothelial markers or any combination thereof. In preferred aspects, the endomesodermal marker is Brachyury and the endothelial markers are VE cadhedrin and PECAM1.

[0019] In an additional embodiment, the present invention provides a method of treatment for vascular diseases and disorders. The method includes the administration of EMPCs to a subject with a vascular disease or disorder.

[0020] In a further embodiment, the present invention provides a method of treating vascular diseases and disorders. The method includes the administration of ECs to a subject with a vascular disease or disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 depicts scheme for the derivation of endothelial cells from hPSCs. Diirina stat^p 1 lm differentiated feeder free hPSCs are treated with 1) bFGF+BMP4, 2) bFGF+BMP4+VEGF for two days, 3) Chir99021+bFGF+BMP4+VEGF for two days, 4) Chir99021 for one day, 5) Chir99021 for two days, or 6) StemPro hESC media for two days. During stage 2, all of the cells were treated with bFGF+VEGF for three days. After stage 1 the cells are assayed for Brachyury expression. After stage 2, the cells are assayed for VE- CAM and PEC AMI expression.

[0022] Figure 2 is a graph showing the screening of cells after stage 2 by RT-PCR for CFH5, KDPv, MIXL, Oct4, PECAM1, SOX2, T, and VWF after treatment for 3 days with bFGF and VEGF.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention is based in part on a chemically defined method of generating endomesodermal precursor cells (EMPCs) and endothelial cells (ECs) from human pluripotent stem cells (hPSCs). The present invention also provides reagents and kits useful for the derivation of endomesodermal precursor cells and endothelial cells from human pluripotent stem cells.

[0024] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0025] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0026] 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 invention, the preferred methods and materials are now described. [0027] The present invention provides for the derivation of endomesodermal precursor cells (EMPCs) by treating hPSCs with at least one endomesodermal precursor cell inducing compound and assaying the cells for endomesodermal precursor cell markers. The present invention also provides for the derivation of a high-purity, homogenous population of endothelial cells (EC)s from hPSCs in a robust and reproducible manner via a stable EMPC stage, where the cells can be expanded and cryopreserved, using newly developed chemically directed differentiation methods.

[0028] Generation of endomesodermal precursor cells (EMPCs) from human pluripotent stem cells (hPSCs), including but not limited to human embryonic stem cells (hESCs), human parthenogenetic stem cells (hpSCs), induced pluripotent stem cells (iPSCs), is a vital component of cell-based strategies for treatment of vascular diseases. Before hPSC-derived EMPCs can be administered in therapeutic modalities, however, chemically defined culture conditions must be developed that reproducibly and robustly induce the generation of EMPCs. Here, the small molecule Chir99021, a reported inhibitor of glycogen synthase kinase 3 (GSK-3), was identified as an endomesodermal precursor cell inducing compound and used to develop a chemically defined differentiation method to differentiate hPSCs into EMPCs. Two growth factors were identified, bFGF and VEGF, which induced the differentiation of EMPCs into ECs. The chemical method reported herein provides instructions for the generation of endothelial cells from hPSCs that can be used for vascular disease cellular therapy or drug discovery.

[0029] The chemical method reported herein provides for the generation of a

homogenous population of EMPCs from hPSCs that can be further differentiated into mature endothelial cells (ECs) for cell therapies or drug discovery.

[0030] Generation of EMPCs and ECs from human pluripotent stem cells (hPSCs), including but not limited to human embryonic stem cells (hESC), human parthenogenetic stem cells (hpSCs), induced pluripotent stem cells (iPSCs), is a vital component of cell-based strategies for treatment of vascular disease. Before hPSC-derived EMPCs and ECs can be administered in therapeutic modalities, however, chemically defined culture conditions must be developed that reproducibly and robustly induce endothelial cell differentiation.

[0031] The methods of deriving endomesodermal precursor cells (EMPCs), endothelial ^pplls fF.C.«fi and ih^p resulting cells that are described herein are generated from human pluripotent stem cells (hPSCs), such as embryonic stem cells. As used herein, "embryonic" refers to a range of developmental stages of an organism beginning with a single zygote and ending with a multicellular structure that no longer comprises pluripotent or totipotent cells other than developed gametic cells. In addition to embryos derived by gamete fusion, the term "embryonic" refers to embryos derived by somatic cell nuclear transfer. Human stem cells can be maintained in culture in a pluripotent state without substantial differentiation using methods that are known in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,453,357, 5,670,372, 5,690,926 5,843,780, 6,200,806 and 6,251,671 the disclosures of which are incorporated herein by reference in their entireties.

[0032] As used herein, "multipotent" or "multipotent cell" refers to a cell type that can give rise to a limited number of other particular cell types. Examples of multipotent cells include ectodermal cells, endodermal cells, mesodermal cells and neural stem cells which can give rise to limited number of other cells.

[0033] As used herein, a "pluripotent cell" refers to a cell that can be maintained in vitro for prolonged, theoretically indefinite period of time in an undifferentiated state, which can give rise to different differentiated tissue types, i.e., ectoderm, mesoderm, and endoderm. Human pluripotent stem cells (hPSCs) include, but are not limited to, human embryonic stem cells (hESCs), human parthenogenetic stem cells (hpSCs) and induced pluripotent stem cells (iPSCs). Methods of obtaining such hPSCs are well known in the art.

[0034] One method of obtaining hPSCs is by parthenogenesis. "Parthenogenesis" ("parthenogenically activated" and "parthenogenetically activated" are used herein

interchangeably) refers to the process by which activation of the oocyte occurs in the absence of sperm penetration, and refers to the development of an early stage embryo comprising trophectoderm and inner cell mass that is obtained by activation of an oocyte or embryonic cell, e.g., blastomere, comprising DNA of all female origin. In a related aspect, a "parthenote" refers to the resulting cell obtained by such activation. In another related aspect, "blastocyst: refers to a cleavage stage of a fertilized of activated oocyte comprising a hollow ball of cells made of outer trophoblast cells and an inner cell mass (ICM). In a further related aspect, "blastocyst formation" refers to the process, after oocyte fertilization or activation, where the oocyte is subsequently cultured in media for a time to enable it to develop into a hollow ball nf PPI I S mads nf miter trophoblast cells and ICM (e.g., 5 to 6 days). [0035] Another method of obtaining hPSCs is through nuclear transfer. As used herein, "nuclear transfer " refers to the fusion or transplantation of a donor cell or DNA from a donor cell into a suitable recipient cell, typically an oocyte of the same or different species that is treated before, concomitant or after transplant or fusion to remove or inactivate its endogenous nuclear DNA. The donor cell used for nuclear transfer include embryonic and differentiated cells, e.g., somatic and germ cells. The donor cell may be in a proliferative cell cycle (Gl, G2, S or M) or non-proliferating (GO or quiescent). Preferably, the donor cell or DNA from the donor cell is derived from a proliferating mammalian cell culture, e.g., a fibroblast cell culture. The donor cell optionally may be transgenic, i.e., it may comprise one or more genetic addition, substitution or deletion modifications.

[0036] A further method for obtaining hPSCs is through the reprogramming of cells to obtain induced pluripotent stem cells. Takahashi et al. (Cell 131, 861-872 (2007)) have disclosed methods for reprogramming differentiated cells, without the use of any embryo or ES (embryonic stem) cell, and establishing an inducible pluripotent stem cell having similar pluripotency and growing abilities to those of an ES cell. Takahashi et al. describe various different nuclear reprogramming factors for differentiated fibroblasts, which include products of the following four genes: an Oct family gene; a Sox family gene; a Klf family gene; and a Myc family gene.

[0037] The pluripotent state of the cells is preferably maintained by culturing cells under appropriate conditions, for example, by culturing on a fibroblast feeder layer or another feeder layer or basic fibroblast growth factor (bFGF)The pluripotent state of such cultured cells can be confirmed by various methods, e.g., (i) confirming the expression of markers characteristic of pluripotent cells; ; (ii) injection of cells into animals, e.g., SCID mice, with the production of different differentiated cell types in vivo; and (iii) observation of the differentiation of the cells (e.g., when cultured in the absence of feeder layer or LIF) into embryoid bodies and other differentiated cell types in vitro.

[0038] The pluripotent state of the cells used in the present invention can be confirmed by various methods. For example, the cells can be tested for the presence or absence of characteristic ES cell markers. In the case of human ES cells, examples of such markers are identified supra, and include SSEA-4, SSEA-3, TRA-1-60, TRA-1-81 and OCT 4, and are known in†hs art [0039] Also, pluripotency can be confirmed by injecting the cells into a suitable animal, e.g., a SCID mouse, and observing the production of differentiated cells and tissues. Still another method of confirming pluripotency is using the subject pluripotent cells to generate chimeric animals and observing the contribution of the introduced cells to different cell types.

[0040] Yet another method of confirming pluripotency is to observe ES cell

differentiation into embryoid bodies and other differentiated cell types when cultured under conditions that favor differentiation (e.g., removal of fibroblast feeder layers). This method has been utilized and it has been confirmed that the subject pluripotent cells give rise to embryoid bodies and different differentiated cell types in tissue culture.

[0041] The resultant pluripotent cells and cell lines, preferably human pluripotent cells and cell lines have numerous therapeutic and diagnostic applications. Such pluripotent cells may be used for cell transplantation therapies or gene therapy (if genetically modified) in the treatment of numerous disease conditions.

[0042] Human pluripotent stem cells (hPSCs) include, but are not limited to, human embryonic stem cells (hESCs), human parthenogenetic stem cells (hpSCs), induced pluripotent stem cells (iPSCs) and cell lines produced by such cells. hPSCs are maintained in culture in a pluripotent state by routine passage until it is desired that endomesodermal precursor cells be derived.

[0043] An "EMPC" (also referred to as a "multipotent endomesodermal cell") exhibits one or more of the following properties: 1) expression of MIXL1, NODAL, and/or

BRACHYURY 2) ability to differentiate into mesodermal and endodermal cell lineages; and 3) morphological characteristics typical for EMPCs.

[0044] EMPCs are multipotent cells that generate cells comprising the main cellular phenotypes of mesoderm and endoderm.

[0045] The endomesoderm is an embryonic blastomere or cell layer not yet differentiated into mesoderm and endoderm but destined to give rise to both. The innermost of the cell layers that develop from the embryonic disk of the inner cell mass of the blastocyst. From the endoderm arises the epithelium of the trachea, bronchi, lungs, GI tract, liver, pancreas, urinary bladder, anal canal, pharynx, thyroid, tympanic cavity, tonsils, and parathyroid glands. The endoderm thus comprises the lining of the cavities and passages of the body and ih^p mvfflflo nf most of the internal organs. The mesoderm is the middle of the three primary germ layers of the embryo, lying between the ectoderm and endoderm; from it are derived the connective tissue, bone, cartilage, muscle, blood and blood vessels, lymphatics, lymphoid organs, notochord, pleura, pericardium, peritoneum, kidneys, and gonads. Mesoderm gives rise to blood cells, endothelial cells, cardiac and skeletal muscle, and adipocytes.

[0046] Endomesodermal precursor cells can be identified by detecting increased expression of endomesodermal precursor cell markers, which include, but are not limited to: MIXL1, NODAL, and Brachyury or any combination thereof

[0047] The T-box gene family consists of members that share a unique DNA binding domain. The best characterized T-box (Tbx) gene, Brachyury or T, encodes a transcription factor that plays an important role in early vertebrate development. Tbx genes are a family of developmental regulators with more than 20 members recently identified among invertebrates and vertebrates. Mutations in Tbx genes have been found to cause several human diseases. The understanding of functional mechanisms of Tbx products has come mainly from the prototypical T/Brachyury protein, which is a transcription activator. The T-domain is a highly conserved DNA-binding motif originally defined in Brachyury and characteristic of the Tbx family of transcription factors. Brachyury is required in the early determination and differentiation of mesoderm. Brachyury is essential for the formation of the posterior body in all vertebrates. Mutations in brachyury have been shown to result in vertebral malformations. In addition, dysregulation of brachyury may be involved in the formation of chordomas, malignant tumors in the spine.

[0048] The present invention provides methods for the derivation of endomesodermal precursor cells (EMPCs) by treating hPSCs with at least one endomesodermal precursor cell induction compound and assaying the cells for endomesodermal precursor cell markers.

[0049] As used herein, "endomesodermal cell inducing compound" is a compound that induces hPSCs to become an EMPC. Such compounds include, but are not limited to, glycogen synthase kinase 3 inhibitors.

[0050] Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. First discovered in 1980 as a regulatory kinase for its namesake, Glycogen synthase, GSK-3 has since been identified as a kinase for over forty different proteins in a variety of different nathwfl s Tn mammals GSK-3 is encoded by two known genes, GSK-3 alpha (GSK3A) and GSK-3 beta (GSK3B). GSK-3 has recently been the subject of much research because it has been implicated in a number of diseases, including Type II diabetes (Diabetes mellitus type 2), Alzheimer's Disease, inflammation, cancer, and bipolar disorder. GSK-3 is active in a number of central intracellular signaling pathways, including cellular proliferation, migration, inflammation and immune responses, glucose regulation, and apoptosis.

[0051] Classes of chemical compounds which may inhibit GSK-3 include, but are not limited to, maleimide derivatives, staurosporine and organometallic inhibitors, indole derivatives, paillone derivatives, pyrazolamide derivatives, pyrimidine and furopyrimidine derivatives, thiazole derivatives, pyrroloazepine, flavone, benzazepinone, bis-indole, pyrrolopyrazine, thiadiazolidinone, pyridyloxadiazole, aminopyrimidine,

pyrazoloquinoxaline, oxindole (indolinone), thiazole, bisindolylmaleimide,

azaindolylmaleimide, arylindolemaleimide, anilinomaleimide, phenylaminopyrimidine, triazole, pyrrolopyrimidine, pyrazolopyrimidine and chloromethyl thienyl ketone.

[0052] Specific GSK-3 inhibitors include, but are limited to, Lithium chloride,

Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone, Azakenpaullone, Indirubin-30- oxime, 6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound 12, Pyrazolopyridine 18, Pyrazolopyridine 9,

Pyrazolopyridine 34, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516, ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX, SB216763, SB415286, 15, CGP60474, Compound 8b, TWS119, Compound 1A, Compound 17, Lithium Atom

(competition with Mg2C), Beryllium Atom (competition with Mg2C and ATP) and Zinc Atom (uncompetitive).

[0053] Chir99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-lH-imidazol-2-yl)-2- pyrimidinyl] amino] ethy-1] amino] -3 -pyridinecarbonitrile ) (Tocris, Bristol, United Kingdom) is a potent and highly selective inhibitor of glycogen synthase kinase 3 (GSK-3) (IC50 values are 6.7 and 10 nM for GSK-3P and GSK-3a respectively). This compound exhibits >500- fold selectivity for GSK-3 over closely related kinases; and also displays >800-fold selectivity against 45 additional enzymes and receptors. In combination with

tranylcypromine,Chir99021 enables reprogramming of mouse embryonic fibroblasts, transduced by Oct4 and Klf4 only, into iPSCs. The compound also enhances mouse and human ESC self-renewal when used in combination with PD 0325901.

[0054] The present invention includes Chir99021 in the form of salts, including acid addition salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts will normally be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable salts may be of utility in the preparation and purification of the compound in question. Basic addition salts may also be formed and be pharmaceutically acceptable. For a more complete discussion of the preparation and selection of salts, refer to Pharmaceutical Salts: Properties, Selection, and Use (Stahl, P. Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

[0055] The present invention also includes analogs of Chir99021. As used herein the term "analog" is a chemical compound that is structurally similar to another compound but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group).

[0056] In one embodiment, the present invention provides a method to generate endomesodermal cells (EMPCs). The method includes treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor and analyzing the cells for endomesodermal cell markers. In one aspect, the hPSCs are treated with the GSK-3 inhibitor for about 24 hours or for 12-36 hours. In another aspect, the GSK-3 inhibitor is Chir99021 or salts or analogs thereof.

[0057] In an aspect, the hPSCs are human embryonic stem cells (hESCs), human parthenogenetic stem cells (hpSCs) or induced pluripotent stem cells (iPSCs), or cell lines derived therefrom.

[0058] In one aspect, the endomesodermal precursor cell markers include MIXL1, NODAL and Brachyury or any combination thereof.

[0059] EMPCs derived from hPSCs can be readily identified using methods well known to one of skill in the art. These methods include identifying endomesodermal precursor cell markers using immunohistochemistry, FACS analysis and measurement of RNA expression levels.

[0060] In one embodiment, the present invention provides for endomesodermal

nr^ppursnr r.^plls fFMPCs). The subject EMPCs are generated by treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor and analyzing the cells for endomesodermal cell markers. In one aspect, the hPSCs are treated with the GSK-3 inhibitor for 24 hours. In another aspect, the GSK-3 inhibitor is Chir99021 and the endomesodermal marker is Brachyury.

[0061] Once EMPCs are derived, the cells should be used or cryopreserved, retaining the ability to differentiate into other mesodermal and endodermal cell types. As shown in the Examples, a GSK-3 inhibitor, Chir99021, was identified as inducing hPSCs to differentiated into EMPCs. The hPSC derived EMPC population was positive for Brachyury, MIXLl and NODAL cell markers. These EMPCs are suitable for further expansion, cryopreservation and differentiation, making them a practical source for ECs. As such, the present invention demonstrates the disclosed methods produced EMPCs from hPSCs.

[0062] The present invention also provides for the generation of endothelial cells (ECs) from hPSCs with under defined chemical conditions.

[0063] As used herein, "differentiation" refers to a change that occurs in cells to cause those cells to assume certain specialized functions and to lose the ability to change into certain other specialized functional units. Cells capable of differentiation may be any of totipotent, pluripotent or multipotent cells. Differentiation may be partial or complete with respect to mature adult cells.

[0064] "Differentiated cell" refers to a non-embryonic cell that possesses a particular differentiated, i.e., non-embryonic, state. The three earliest differentiated cell types are endoderm, mesoderm, and ectoderm.

[0065] EMPCs derived from hPSCs are multipotent and can be differentiated into several cell types including endothelial cells.

[0066] Endothelial cells comprise the endothelium. The endothelium is the thin layer of cells that lines the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. The cells that form the endothelium are called endothelial cells. Endothelial cells in direct contact with blood are called vascular endothelial cells, whereas those in direct contact with lymph are known as lymphatic endothelial cells.

[0067] Endothelial cells line the interior of all blood vessels, composing the endothelium. A ll ^pnHnthfilifll PPI I S are derived from a common angioblast precursor and subsequently develop organ specific properties. Embryonic endothelial cells exhibit much heterogeneity within, and between, organs. Differentiation of endothelial cells is governed by several factors, including the immediate microenvironment, interactions with surrounding cells, and the local release of cytokines and growth factors. Adult endothelial cells retain remarkable plasticity and are known to reprogram in response to IL-1, TNF, VEGF, and FGF. Because endothelial cells provide many essential functions, endothelial dysfunction leads to vascular disease and can promote the development of chronic inflammatory conditions, such as atherosclerosis.

[0068] Vascular endothelial cells line the entire circulatory system, from the heart to the smallest capillaries. These cells have very distinct and unique functions that are paramount to vascular biology. These functions include fluid filtration, such as in the glomeruli of the kidney, blood vessel tone, hemostasis, neutrophil recruitment, and hormone trafficking. Endothelium of the interior surfaces of the heart chambers are called endocardium.

[0069] The present invention provides method for EC differentiation from EMPCs.

[0070] In another embodiment, the present invention provides a method to generate endothelial cells (ECs). The method includes treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor; identifying endomesodermal precursor cells (EMPCs) by assaying the treated hPSCs for endomesodermal cell markers; treating the EMPCs with at least one growth factor and analyzing the cells for endothelial cell markers. In one aspect, the hPSCs are treated with the GSK-3 inhibitor for 24 hours. In certain aspects, the GSK-3 inhibitor is Chir99021 or salts or analogs thereof. In a further aspect, the EMPCs are treated with the growth factor for about 48-96 hours, preferably about 72 hours. In an additional aspect, the growth factors maybe bFGF, VEGF or BMP4 or any combination thereof. In a preferred aspect, the growth factors are bFGF and VEGF.

[0071] In an aspect, the hPSCs are human embryonic stem cells (hESCs), human parthenogenetic stem cells (hpSCs) or induced pluripotent stem cells (iPSCs), or cell lines derived therefrom.

[0072] In one aspect of the method, the endomesodermal cell markers maybe END016, FoxA, GATAE, SMIP, Hex, P19 and Brachyury or any combination thereof.

[0073] In an additional aspect of the method, the endothelial cell markers maybe VE C^ Arin P CAM1 , ACE/CD 143 ,MCAM/CD146, Clq R1/CD93, Nectin-2/CDl 12, VE- Cadherin, PD-ECGF/Thymidine Phosphorylase, CC Chemokine Receptor D6, Podocalyxin, CD31/PECAM-1, Podoplanin, CD34, SlPl/EDG-1, CD36/SR-B3, S1P2/EDG-5, CD151, S1P3/EDG-3, CD160, S1P4/EDG-6, CD300LG/Nepmucin, S1P5/EDG-8, CL-Kl/COLECl 1, E-Selectin/CD62E, CL-P1/COLEC12, E-Selectin (CD62E)/P-Selectin (CD62P), Coagulation Factor Ill/Tissue Factor, P-Selectin/CD62P, DC-SIGNR/CD299, SLAM/CD 150,

DCBLD2/ESDN, Stabilin-1, EMMPRIN/CD147, Stabilin-2, Endoglin/CD105,

TEM7/PLXDC 1 , Endomucin, TEM8/ANTXR1, Endosialin/CD248,

Thrombomodulin/BDCA-3, EPCR, THSD1, Erythropoietin R, Tie-2, ESAM, TNF

RI/TNFRSF1A, FABP5/E-FABP, TNF RII/TNFRSF1B, FABP6, TRA-1-85/CD147, ICAM- 1/CD54, TRAIL R1/TNFRSF10A, ICAM-2/CD102, TRAIL R2/TNFRSF10B, IL-1 RI, VCAM-1/CD106, IL-13 R alpha 1, VE-Statin, Integrin alpha 4/CD49d, VEGF Rl/Flt-1, Integrin alpha 4 beta 1, VEGF R2/KDR/Flk-1, Integrin alpha 4 beta 7/LPAM-l, VEGF R3/Flt-4, Integrin beta 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 or any combination thereof. In a specific aspect, the endothelial cell markers are VE cadherin and PEC AMI or any combination thereof.

[0074] A growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation. Usually, it is a protein or a steroid hormone. Growth factors are important for regulating a variety of cellular processes. Growth factors typically act as signaling molecules between cells. Examples are cytokines and hormones that bind to specific receptors on the surface of their target cells. They often promote cell differentiation and maturation, which varies between growth factors. For example, bone morphogenic proteins stimulate bone cell differentiation, while fibroblast growth factors and vascular endothelial growth factors stimulate blood vessel differentiation (angiogenesis). Examples of growth factors and families of growth factors include, but are not limited to: Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line- derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor- 9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF),

TnsiiHn-Hkfi ornwth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-a), Transforming growth factor beta(TGF-P), Tumor necrosis factor-alpha(TNF-a), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway, placental growth factor (P1GF), Fetal Bovine

Somatotrophin (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, bFGF and VEGF.

[0075] In a further embodiment, the present invention provides for endothelial cells (ECs). The subject ECs are generated by treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor; identifying endomesodermal precursor cells (EMPCs) by assaying the treated hPSCs for endomesodermal cell markers; treating the EMPCs with at least one growth factor and analyzing the cells for endothelial cell markers. In certain aspects, the hPSCs are treated with a GSK-3 inhibitor for 24 hours and the EMPCs are treated with growth factors for 72 hours. In specific aspects, the GSK-3 inhibitor is Chir99021 and the growth factors are bFGF and VEGF. In an additional aspect, the endomesodermal marker is Brachyury and the endothelial markers are VE cadhedrin and PECAM1.

[0076] As shown in the Examples, the disclosed methods resulted in the production of fully functional endothelial cells. The ECs were generated by treating hPSCs with

Chir99021, identifying EMPCs by assaying for Brachyury expression, treating the EMPCs with bFGF and VEGF and assaying for VE cadherin and PEC AMI expression. The resulting ECs cells expressed VE cadhedrin and PE CAM1 as measured by RT-PCR and

immunofluorescence. Further the hPSC derived ECs grown on matrigel coated plates for > 6 passages continued to express VE-Cadhedrin and PECAM1. This demonstrates the production endothelial cells from hPSCs using the disclosed methods.

[0077] EMPCs and ECs may play a vital role in the treatment of vascular diseases and disorders. Endothelial dysfunction, or the loss of proper endothelial function, is a hallmark for vascular diseases, and is often regarded as a key early event in the development of atherosclerosis. Impaired endothelial function, causing hypertension and thrombosis, is often seen in patients with coronary artery disease, diabetes mellitus, hypertension,

hypercholesterolemia, as well as in smokers. Endothelial dysfunction has also been shown to be predictive of future adverse cardiovascular events, and is also present in inflammatory His^pfls^p sur.h as rheumatoid arthritis and systemic lupus erythematosus. One of the main mechanisms of endothelial dysfunction is the diminishing of nitric oxide, often due to high levels of asymmetric dimethylarginine, which interfere with the normal L-arginine-stimulated nitric oxide synthesis and so leads to hypertension. The most prevailing mechanism of endothelial dysfunction is an increase in reactive oxygen species, which can impair nitric oxide production and activity via several mechanisms. The signalling protein ER 5 is essential for maintaining normal endothelial cell function. A further consequence of damage to the endothelium is the release of pathological quantities of von Willebrand factor, which promote platelet aggregation and adhesion to the subendothelium, and thus the formation of potentially fatal thrombi.

[0078] Vascular disease is a pathological state of large and medium sized muscular arteries and is triggered by endothelial cell dysfunction. Because of factors like pathogens, oxidized LDL particles and other inflammatory stimuli endothelial cells become activated. This leads to change in their characteristics: endothelial cells start to excrete cytokines and chemokines and express adhesion molecules on their surface. This in turn results in recruitment of white blood cells (monocytes and lymphocytes), which can infiltrate the blood vessel wall. Stimulation of smooth muscle cell layer with cytokines produced by endothelial cells and recruited white blood cells causes smooth muscle cells to proliferate and migrate towards the blood vessel lumen. The process causes thickening of the vessel wall, forming a plaque consisting of proliferating smooth muscle cells, macrophages and various types of lymphocytes. This plaque result in obstructed blood flow leading to diminished amounts of oxygen and nutrients that reaches the target organ. In the final stages, the plaque may also rupture causing the formation of clots, and as a result strokes.

[0079] In an additional embodiment, the present invention provides a method of treatment for vascular diseases and disorders. The method includes the administration of EMPCs to a subject with a vascular disease or disorder.

[0080] In a further embodiment, the present invention provides a method of treating vascular diseases and disorders. The method includes the administration of ECs to a subject with a vascular disease or disorder.

[0081] In another embodiment, the present invention provides a kit for the derivation of EMPCs. The kit may include a glycogen synthase kinase 3 (GSK-3) inhibitor, reagents to iHfinti ^pnHnm^psndermal precursor cell markers and directions for the generation of EMPCs from hPSCs. In one aspect, the GSK-3 inhibitor is Chir99021 or salts or analogs thereof. The kit may include reagents to identify END016, FoxA, GATAE, SMIP, Hex, P19 and Brachyury endomesodermal precursor markers or any combination thereof.

[0082] In another embodiment, the present invention provides a kit for the production of endothelial cells (ECs). The kit includes a glycogen synthase kinase 3 (GSK-3) inhibitor, reagents to identify endomesothelial precursor cell markers, growth factors, reagents to identify endothelial cell markers and directions for the production of endothelial cells from hPSCs. In one aspect, the GSK-3 inhibitor is Chir99021 or salts or analogs thereof. In another aspect, the growth factors are bFGF and VEGF. The kit may include reagents to END016, FoxA, GATAE, SMIP, Hex, P19 and Brachyury endomesodermal precursor cell markers or any combination thereof and VE Cadhedrin, PECAM1, ACE/CD 143

TEM8/ANTXR1, Endosialin/CD248, Thrombomodulin/BDCA-3, EPCR, THSD1,

Erythropoietin R, Tie-2, ESAM, TNF RI/TNFRSF1A, FABP5/E-FABP, TNF

RII/TNFRSF1B, FABP6, TRA-1-85/CD147, ICAM-1/CD54, TRAIL R1/TNFRSF10A, ICAM-2/CD102, TRAIL R2/TNFRSF10B, IL-1 RI, VCAM-1/CD106, IL-13 R alpha 1, VE- Statin, Integrin alpha 4/CD49d, VEGF Rl/Flt-1, Integrin alpha 4 beta 1, VEGF R2/KDR/Flk- 1, Integrin alpha 4 beta 7/LPAM-l, VEGF R3/Flt-4, Integrin beta 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 endothelial cell markers or any combination thereof.

[0083] The following examples are intended to illustrate, but not limit the invention.

EXAMPLE 1

FEEDER GROWTH OF hPSCs

[0084] The hPSCs maintained on mitomycin-C inactivated mouse embryonic fibroblast (Millipore) feeder layer in embryonic stem medium: Knock Out DMEM/F12 (Life

7m M L-glutamine (GlutaMax-I, Invitrogen), 0.1 mM MEM nonessential amino acids (Life Technology), 0.1 mM β-mercaptoethanol (Life Technologies), penicillin/streptomycin/amphotericin B (100 U/100 μg/250 ng) (MP Biomedicals) and 5 ng/ml bFGF (Peprotech). Cells were passaged with dispase or collagenase IV (both Life Technologies) every 5-7 days with split ratio of 1 :4 or 1 :6.

EXAMPLE 2

FEEDER FREE GROWTH OF hPSCs

[0085] The hPSCs were then transferred to Matrigel (BD Biosciences) coated plates and grown with Stem Pro hESC SFM medium (Invitrogen): DMEM/F12 with GlutaMAX, IX STEMPRO hESC SFM Growth Supplement, 1.8% Bovine Serum Albumin, 8 ng/mL bFGF and O.lmM 2-Mercaptoethanol.

EXAMPLE 3

DERIVATION OF ENDOTHELIAL CELLS FROM hPSCs

[0086] hPSCs cells growing for 4 passages under feeder-free culture conditions were dissociated with Accutase (Sigma) and plated into Matrigel coated plates and grown with N2/B27 medium [DMEM/F12 with GlutaMAX, 1X N2/B27 Supplement (Invitrogen)] under 6 different (stage 1) treatments were performed (1) bFGF plus BMP4, (2)

bFGF+BMP4+VEGF for two days, (3) Chir99021+bFGF+BMP4+VEGF for two days, (4) Chir99021 for one day, (5) Chir99021 for two days and (6) Stempro hESC Medium for two days follow by (stage 2) treatment with bFGF (50ng/mL)+VEGF (50ng/mL) for 3 days

[Figure 1]. hPSCS cells treated with Οώ·99021(10μΜ) for 1 day, induced them to differentiate them into endomesoderm precursors that express Brachyury. Further treatment of hPSCs that have been treated with Οώ·99021(10μΜ) for 1 day (hPSC derived

endomesoderm precursors) with N2B27 + bFGF (50ng/ml) +VEGF (50ng/ml) for 3 days differentiates them into endothelial cells that express VE-CAM and PEC AMI as measured by RT-PCR [Figure 2] and by immunofluorescence.

EXAMPLE 4

EXPANSION OF ENDOTHLIAL CELLS DERIVED FROM hPSCs

[0087] Endothelial Cells derived from hPSCs can be dissociated with Accutase (Sigma) and grown on Matrigel coated plates in N2B27 medium 50ng/ml bFGF and 50ng/ml VEGF for > 6 passages. The hPSC-ECs at passaged 6 expressed VE-Cadherin and PECAM as m^pflsnr^pH bv immunofluorescence. [0088] RT-PCR analysis. Total RNA from at least triplicate samples with around 1 million cells each was isolated using either QIAsymphony automatic purification system or RNeasy Plus Mini kit, according to manufacturer's instructions (Qiagen). Total RNA was used for reverse transcription with the iScript cDNA synthesis kit (Biorad) and Px2 Thermal Cycler (Thermo Scientific). To analyze gene expression, PCR reactions were performed in duplicate using 1 /25-th of the cDNA per reaction and the QuantiTect Primer Assay and Quantitest SYBR Green master mix (Qiagen). qPCR was performed using the Rotor-Gene Q (Qiagen) 5 min at 95°C, 5 s at 92°C, and 20 s at 60°C for 37 cycles followed by melt to check the specificity of the amplicons from 50 to 99°C raising by 1°C each step. Relative quantification was performed against a standard curve and quantified values were normalized against the input determined by PPIG. The primers used for the analysis are listed in

Supplementary Table SI . The results are presented as presented as mean ± s.e.m. and statistical analysis was performed using a confidence level of 95% (a = 0.05) with the two- tailed Student's t-test for comparing two groups or one-factor AN OVA with Dunnett test for comparing multiple groups against control and P < 0.05 was considered significant.

[0089] Immunocvtochemistry. Around 100,000 cells per sample were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS, and permeabilized and blocked for 1 hour at room temperature in 0.3% Triton X-100, 5% normal donkey serum, and 1% BSA in PBS. The cells were incubated overnight at 4°C with primary antibodies in 0.3% Triton X-100, 2% BSA in PBS. Cells are washed three times with PBS and incubated for 1 hour at room temperature with secondary antibodies in 0.3% Triton X-100, 5% normal donkey serum, and 1% BSA in PBS. The nuclei were stained with DAPI.

[0090] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A method of producing endomesodermal precursor cells comprising:

(a) treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor; and

(b) assaying the cells for endomesodermal cell markers.

2. The method of claim 1, wherein the hPSCs are treated with the GSK-3 inhibitor for 24 hours.

3. The method of claim 2, wherein the GSK-3 inhibitor is Chir99021 or salts or analogs thereof

4. The method of claim 1, wherein the endomesodermal precursor cell markers are selected from the group consisting of: MIXLl, NODAL, and Brachyury or any combination thereof.

5. The method of claim 4, wherein the endomesodermal precursor cell marker is Brachyury.

6. The method of claim 1, wherein the hPSCs are selected from the group consisting of: are human embryonic stem cells (hESCs), human parthenogenetic stem cells (hpSCs) or induced pluripotent stem cells (iPSCs), or cell lines derived therefrom.

7. An endomesodermal precursor cell derived by the method of claim 1.

8. The endomesodermal precursor cell of claim 7, wherein the GSK-3 inhibitor is Chir99021 or salts or analogs thereof.

9. The endomesodermal precursor cell of claim 7, wherein the endomesodermal precursor cell marker is Brachyury.

10. A method of producing endothelial cells comprising:

(a) treating human pluripotent stem cells (hPSCs) with a glycogen synthase kinase 3 (GSK-3) inhibitor;

(b) identifying endomesodermal precursor cells (EMPCs) by identifying

endomesodermal cell markers;

(c) treating the EMPCs with at least one growth factor; and

(d) analyzing the cells for endothelial cell markers.

11. The method of claim 10, wherein the hPSCs are treated with the GSK-3 inhibitor for 24 hours.

12. The method of claim 11, wherein the GSK-3 inhibitor is Chir99021 or salts or analogs thereof.

13. The method of claim 10, wherein the endomesodermal precursor cell markers are selected from the group consisting of: MIXL1, NODAL, and Brachyury or any combination thereof.

14. The method of claim 13, wherein the endomesodermal precursor cell marker is Brachyury.

15. The method of claim 10, wherein the EMPCs are treated with the growth factor for 72 hours.

16. The method of claim 10, wherein the growth factor is selected from the group consisting of: bFGF, VEGF and BMP4 or any combination thereof.

17. The method of claim 16, wherein the growth factors are bFGF and VEGF.

18. The method of claim 10, wherein the endodermal cell markers are selected from the group consisting of: VE Cadhedrin, PECAMl, ACE/CD143 ,MCAM/CD146, Clq R1/CD93, Nectin-2/CD112, VE-Cadherin, PD-ECGF/Thymidine Phosphorylase, CC Chemokine Receptor D6, Podocalyxin, CD31/PECAM-1, Podoplanin, CD34, SlPl/EDG-1, CD36/SR- B3, S1P2/EDG-5, CD151, S1P3/EDG-3, CD160, S1P4/EDG-6, CD300LG/Nepmucin, S1P5/EDG-8, CL-K1/COLEC11, E-Selectin/CD62E, CL-P1/COLEC12, E-Selectin

(CD62E)/P-Selectin (CD62P), Coagulation Factor Ill/Tissue Factor, P-Selectin/CD62P, DC- SIGNR/CD299, SLAM/CD 150, DCBLD2/ESDN, Stabilin-1, EMMPRIN/CD147, Stabilin-2, Endoglin/CD105, TEM7/PLXDC1, Endomucin, TEM8/ANTXR1, Endosialin/CD248, Thrombomodulin/BDCA-3, EPCR, THSD1, Erythropoietin R, Tie-2, ESAM, TNF

Rl/TNFRSFIA, FABP5/E-FABP, TNF RII/TNFRSF1B, FABP6, TRA-1-85/CD147, ICAM- 1/CD54, TRAIL R1/TNFRSF10A, ICAM-2/CD102, TRAIL R2/TNFRSF10B, IL-1 RI, VCAM-1/CD106, IL-13 R alpha 1, VE-Statin, Integrin alpha 4/CD49d, VEGF Rl/Flt-1, Integrin alpha 4 beta 1, VEGF R2/KDR/Flk-1, Integrin alpha 4 beta 7/LPAM-l, VEGF R3/Flt-4, Integrin beta 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 or any combination thereof.

19. The method of claim 18, wherein the endothelial cell markers are VE Cadhedrin and PECAMl .

20. An endothelial cell produced by the method of claim 10.

21. The endothelial cell of claim 20, wherein the GSK-3 inhibitor is Chir99021 and the growth factors are bFGF and VEGF.

22. The endothelial cell of claim 20, wherein the endomesodermal precursor cell marker is Brachyury and the endothelial cell markers are VE cadhedrin and PECAM1.

23. A kit for the production of endomesodermal precursor comprising:

(a) glycogen synthase kinase 3 (GSK-3) inhibitor;

(b) reagents to assay for endomesdormal precursor cell markers; and

(c) directions for the derivation of endomesodermal precursor cells from hPSCs.

24. A kit for the production of endothelial cells comprising:

(a) glycogen synthase kinase 3 (GSK-3) inhibitor;

(b) reagents to assay for endomesdormal precursor cell markers;

(c) at least one growth factor;

(d) reagents to assay for endothelial cell markers; and

(e) directions for the production of endothelial cells from hPSCs.