CN104302764A

CN104302764A - Derivation of endothelial cells from human pluripotent stem cells

Info

Publication number: CN104302764A
Application number: CN201380024629.3A
Authority: CN
Inventors: R·冈萨雷斯; I·加里陶南迪亚; R·斯莫切肯
Original assignee: International Stem Cell Corp
Current assignee: International Stem Cell Corp
Priority date: 2012-05-02
Filing date: 2013-05-01
Publication date: 2015-01-21
Also published as: WO2013166165A1

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

Endotheliocyte is derived by human pluripotent stem cells

Invention field

Present invention relates in general to stem cell, and more specifically to being derived the method for endomesoderm precursor cell and endotheliocyte by human pluripotent stem cells.

Background information

Human embryo stem cell (ES) cell is the pluripotent cell that can be divided into a large series of cell type.Stem cell is different from other cell type because of two key properties.First, they be can by cell fission, be the non-specialized cell of renewal after long-term inactivation itself sometimes.The second, under some physiology or experiment condition, they can be induced to become the tissue-or organ-specific cell with specific function.In some organs of such as intestines and marrow, stem cell divides to repair and the tissue changed wearing and tearing or damage regularly.But in other organ of such as pancreas and heart, stem cell is only divided under special conditions.

During fetal development, stem cell is by three kinds of main cell colonies: ectoderm, mesoderm and definitive entoderm form bodily tissue.Mesoderm produces hemocyte, endotheliocyte, cardiac muscle and skeletal muscle and adipocyte.Definitive entoderm produces liver, pancreas and lung.Ectoderm produces neural system, skin and adrenal tissue.

The potential application of stem cell manufactures the biological cells and tissues for medical therapy.Now, the Organ and tissue of donations is through being usually used in changing those Organ and tissues that are ill or that destroy.Regrettably, need the number of graft considerably beyond the organ number that can be used for transplanting.Stem cell provides the possibility of renewable source of replacing biological cells and tissues comprising Parkinson's disease (Parkinson's disease), amyotrophic lateral sclerosis, Spinal injury, burn, heart trouble, diabetes and arthritic various diseases, symptom and obstacle in order to treatment.

Endothelium is liner blood vessel and vasculolymphatic internal surface thus blood circulation in the lumen or form the cell thin at interface between lymph liquid and the remainder of tube wall.Vascular disease are the pathological state of large-scale and medium-sized muscular artery and trigger because of endothelial dysfunction.Due to the factor of such as pathogenic agent, the LDL particle of oxidation and other inflammatory stimulus endotheliocyte are activated and start secrete cytokines and chemokine and expression of adhesion molecules over their surface.Described process finally causes tube wall thickening, thus forms the patch be made up of propagation smooth muscle cell, scavenger cell and various types of lymphocyte.Described patch causes blood flow to be obstructed, thus causes the amount of the oxygen and nutrient substance arriving target organ to reduce.In terminal stage, patch also may break, thus causes forming grumeleuse, and result causes apoplexy.Need the novel therapeutic for vascular disease.A kind of method for generation of being applicable to the treatment endomesoderm precursor cell of vascular disease and the high purity colony of endotheliocyte is provided herein.

Summary of the invention

The present invention is based in part on a kind of method being produced the chemical formula of endomesoderm precursor cell (EMPC) and endotheliocyte (EC) by human pluripotent stem cells (hPSC).The present invention also provides the reagent and the test kit that are applicable to be derived endomesoderm precursor cell and endotheliocyte by human pluripotent stem cells.

The invention provides by measuring described cell for endomesoderm cell marker produce endomesoderm precursor cell (EMPC) with at least one endomesoderm inducing compounds handler's multipotential stem cell (hPSC).The present invention also provide use the chemically-directed differentiation method of newly-developed via can by cell amplification and the endomesoderm stage of freezen protective to be derived high purity colony, the homogeneous population of endotheliocyte (EC) by hPSC in sane and reproducible mode.

In one embodiment, the invention provides a kind of method for generation of endomesoderm cell (EMPC).Described method comprises with glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC) and analyzes described cell for endomesoderm cell marker.On the one hand, by hPSC GSK-3 inhibitor process about 24 hours.On the other hand, GSK-3 inhibitor is Chir99021 or its salt or analogue.Other antagonist comprises 3F8, A1070722, AR-A014418, BIO, FRATide, 10Z-Hymenialdsine, isatin-3 '-oxime, Kan Paluo ketone (Kenpaullone), L803, NSC693868, SB216763, SB415286, TC-G24, TCS202, TCS 21311 and TWS119, all buy through Glaxo Smith Kline.

On the one hand, described hPSC be human embryo stem cell (hESC), people's parthenogenesis stem cell (hpSC) or induction multipotential stem cell (iPSC) or from its derivative clone.

On the one hand, described endomesoderm precursor cell marker comprises such as MIXL1, NODAL and BRACHYURY or its arbitrary combination.

In one embodiment, the invention provides endomesoderm precursor cell (EMPC).Described tested EMPC is by analyzing described cell to produce for endomesoderm cell marker with glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC).On the one hand, by hPSC GSK-3 inhibitor process about 24 hours or 12-36 hour.On the other hand, GSK-3 inhibitor is Chir99021 and endomesoderm marker is Brachyury.

In another embodiment, the invention provides a kind of method for generation of endotheliocyte (EC).Described method comprises with glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC); Endomesoderm precursor cell (EMPC) is differentiated by the hPSC handled by measuring for endomesoderm cell marker; Described cell is analyzed with EMPC described in the process of at least one somatomedin for endothelial cell marker thing.On the one hand, by hPSC GSK-3 inhibitor process about 24 hours.In some aspects, GSK-3 inhibitor is Chir99021 or its salt or analogue.On the other hand, by EMPC somatomedin process about 72 hours or about 48-96 hour.On the other hand, somatomedin can be bFGF, VEGF or BMP4 or its arbitrary combination.In preferred at one, somatomedin is bFGF and VEGF.

On the one hand, hPSC be human embryo stem cell (hESC), people's parthenogenesis stem cell (hpSC) or induction multipotential stem cell (iPSC) or from its derivative clone.

In the one side of described method, endomesoderm cell marker can be such as MIXL1, NODAL, BRACHYURY or its arbitrary combination.

In the another aspect of described method, endothelial cell marker thing can be VE Cadhedrin, PECAM1, ACE/CD143, MCAM/CD146, C1q R1/CD93, Fibronectin-2/CD112, VE-cadherin (Cadherin), PD-ECGF/ thymidine phosphorylase, CC-chemokine receptor D6, foot glycocalicin (Podocalyxin), CD31/PECAM-1, flatfoot albumen (Podoplanin), CD34, S1P1/EDG-1, CD36/SR-B3, S1P2/EDG-5, CD 151, S1P3/EDG-3, CD160, S1P4/EDG-6, CD300LG/Nepmucin, S1P5/EDG-8, CL-K1/COLEC11, E-Selectin/CD62E, CL-P1/COLEC12, E-Selectin (CD62E)/palatelet-selectin (CD62P), thromboplastin/tissue factor, palatelet-selectin/CD62P, DC-SIGNR/CD299, SLAM/CD150, DCBLD2/ESDN, immobilon (Stabilin)-1, EMMPRIN/CD147, immobilon-2, Endoglin (Endoglin)/CD105, TEM7/PLXDC1, endothelium Saliva Orthana (Endomucin), TEM8/ANTX R1, endosialin (Endosialin)/CD248, thrombomodulin (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 α 1, VE-statin (Statin), integrin alpha-4/CD49d, VEGF R1/Flt-1, integrin alpha-4 β 1, VEGF R2/KDR/Flk-1, integrin alpha-4 β 7/LPAM-1, VEGF R3/Flt-4, integrin β 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 or its arbitrary combination.In concrete at one, endothelial cell marker thing is VE cadherin and PECAM1.

In another embodiment, the invention provides endotheliocyte (EC).Tested EC is by using glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC); Endomesoderm precursor cell (EMPC) is differentiated by the hPSC handled by measuring for endomesoderm cell marker; Analyze described cell to produce for endothelial cell marker thing with EMPC described in the process of at least one somatomedin.In some aspects, by hPSC with GSK-3 inhibitor process 24 hours and by EMPC somatomedin process 72 hours.In concrete, GSK-3 inhibitor is Chir99021 and somatomedin is bFGF and VEGF.On the other hand, endomesoderm marker is Brachyury and endothelial cell marker thing is VE cadherin and PECAM1.

In another embodiment, the invention provides a kind of test kit for derivative EMPC.Described test kit can comprise glycogen synthase kinase 3 (GSK-3) inhibitor, for differentiating the reagent of endomesoderm precursor cell marker and the guidance for being produced EMPC by hPSC.On the one hand, GSK-3 inhibitor is Chir99021 or its salt or analogue.Described test kit can comprise the reagent for differentiating MIXL1, NODAL, BRACHYURY cell marker or its arbitrary combination.

In another embodiment, the invention provides a kind of test kit for generation of endotheliocyte (EC).Described test kit comprises glycogen synthase kinase 3 (GSK-3) inhibitor, for differentiating the reagent of endomesoderm precursor cell marker, somatomedin, for differentiating the reagent of endothelial cell marker thing and being produced the guidance of endotheliocyte by hPSC.On the one hand, GSK-3 inhibitor is Chir99021 or its salt or analogue.On the other hand, somatomedin is bFGF and VEGF.Described test kit can comprise the reagent for differentiating the following: ENDO16, FoxA, GATAE, SMIP, Hex, P19 and Brachyury endomesoderm precursor cell marker or its arbitrary combination and VE Cadhedrin, PECAM1, ACE/CD143, MCAM/CD146, C1q R1/CD93, Fibronectin-2/CD112, VE-cadherin, PD-ECGF/ thymidine phosphorylase, CC-chemokine receptor D6, foot glycocalicin, CD31/PECAM-1, flatfoot albumen, CD34, S1P1/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)/palatelet-selectin (CD62P), thromboplastin/tissue factor, palatelet-selectin/CD62P, DC-SIGNR/CD299, SLAM/CD150, DCBLD2/ESDN, immobilon-1, EMMPRIN/CD147, immobilon-2, Endoglin/CD105, TEM7/PLXDC1, endothelium Saliva Orthana, 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 α 1, VE-statin, integrin alpha-4/CD49d, VEGF R1/Flt-1, integrin alpha-4 β 1, VEGF R2/KDR/Flk-1, integrin alpha-4 β 7/LPAM-1, VEGF R3/Flt-4, integrin β 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 endothelial marker thing or its arbitrary combination.In preferred, endomesoderm marker is Brachyury and endothelial marker thing is VE cadherin and PECAM1.

In another embodiment, the invention provides a kind of method for the treatment of vascular disease and illness.Described method comprises the experimenter being applied to by EMPC and suffering from vascular disease or illness.

In another embodiment, the invention provides a kind of method for the treatment of vascular disease and illness.Described method comprises the experimenter being applied to by EC and suffering from vascular disease or illness.

Accompanying drawing is sketched

Fig. 1 describes the scheme being derived endotheliocyte by hPSC.During the stage 1, by undifferentiated without raising the hPSC of thing (feeder) with 1) bFGF+BMP4 process, 2) bFGF+BMP4+VEGF process two days, 3) Chir99021+bFGF+BMP4+VEGF process two days, 4) Chir99021 process one day, 5) Chir99021 process two days, or 6) StemProhESC medium treatment two days.During the stage 2, by all cells bFGF+VEGF process three days.After stage 1, for Brachyury expression, cell is measured.After the stage 2, express for VE-CAM and PECAM1 and cell is measured.

Fig. 2 be illustrate with after bFGF and VEGF process 3 days by the figure of RT-PCR for CFH5, KDR, MIXL, Oct4, PECAM1, SOX2, T and VWF cell screening after the stage 2.

Detailed Description Of The Invention

The present invention is based in part on the method for the chemical formula being produced endomesoderm precursor cell (EMPC) and endotheliocyte (EC) by human pluripotent stem cells (hPSC).The present invention also provides the reagent and the test kit that are applicable to be derived endomesoderm precursor cell and endotheliocyte by human pluripotent stem cells.

Before the description present composition and method, should be understood that and the invention is not restricted to described concrete composition, method and experiment condition, because described composition, method and condition can change.Should also be understood that term used herein only for describing the object of particular, and be not intended to have restricted, because scope of the present invention only limits by appended claims.

As in this specification and in the appended claims use, unless the context clearly indicates otherwise, otherwise " one (a) " of singulative, " a kind of (an) " and " described (the) " comprise plural reference.Therefore, such as " described method " quote the step comprising one or more methods and/or type described herein, after reading disclosure etc., this will become apparent to those skilled in the art.

Unless otherwise defined, otherwise all technical terms used herein and scientific terminology have the identical implication usually understood with the those of ordinary skill in field belonging to the present invention.Although can use similar in practice of the present invention or test or be equal to any method and the material of those methods described herein and material, now preferred method and material are described.

The invention provides by measuring described cell for endomesoderm precursor cell marker carry out derivative endomesoderm precursor cell (EMPC) with at least one endomesoderm precursor cell inducing compounds process hPSC.The present invention also provide use the chemically-directed differentiation method of newly-developed by can by cell amplification and the stable EMPC stage of freezen protective to be derived the high purity homogeneous population of endotheliocyte (EC) by hPSC in sane and reproducible mode.

By include but not limited to hESC (hESC), people's parthenogenesis stem cell (hPSC), induction the human pluripotent stem cells (hPSC) of multipotential stem cell (iPSC) produce the important component part that endomesoderm precursor cell (EMPC) is the strategy based on cell being used for the treatment of vascular disease.But, can treat before form uses at the EMPC that hPSC is derivative, must develop reproducibly and the culture condition of the chemical formula of inducing EMPC to produce steadily.At this, the differentiation method of small molecules Chir99021 (i.e. the report inhibitor of glycogen synthase kinase 3 (GSK-3)) discriminating for endomesoderm precursor cell inducing compounds and for developing chemical formula is divided into EMPC to make hPSC.Authenticated two kinds of somatomedin: bFGF and VEGF, its induction EMPC is divided into EC.The chemical process reported in this article is provided for producing by hPSC the specification sheets that can be used for the endotheliocyte of vascular disease cell therapy or drug discovery.

The chemical process reported in this article provides the homogeneous population being produced EMPC by hPSC, and described EMPC can be divided into ripe endotheliocyte (EC) further for cell therapy or drug discovery.

By include but not limited to human embryo stem cell (hESC), people's parthenogenesis stem cell (hpSC), induction the human pluripotent stem cells (hPSC) of multipotential stem cell (iPSC) produce the important component part that EMPC and EC is the strategy based on cell being used for the treatment of vascular disease.But, can treat before form uses at EMPC and EC that hPSC is derivative, must develop reproducibly and the culture condition of the chemical formula of inducing endothelial cell differentiation steadily.

Derivative endomesoderm precursor cell (EMPC) described herein, endotheliocyte (EC) and the method for cell that produces are produced by the human pluripotent stem cells (hPSC) of such as embryonic stem cell.As used herein, " embryo " refers to that organism starts with single zygote and no longer to comprise the etap scope that multipotency except the gametid [cell of growing or the multi-cellular structure of totipotent cell terminate.Except the embryo derived by gametogamy, term " embryo " also refers to by the derivative embryo of body-cell neucleus transplanting.Methods known in the art can be used to be maintained in culture by human stem cell when breaking up without substance and to be in multipotency state.Described method is described in such as U.S. Patent number 5,453,357,5,670,372,5,690,926,5,843,780,6,200,806 and 6,251, and in 671, its disclosure by reference entirety is incorporated to herein.

As used herein, " pluripotency (multipotent) " or " multipotential cell " refer to the cell type of other particular cell types that can produce limited quantity.The example of pluripotent cell comprises ectoderm cell, endoderm cell, mesoblastema and neural stem cell, and they can produce other cell of limited quantity.

As used herein, " pluripotent cell " refers to and can maintain cell that extend, the theory unlimited time with undifferentiated state in vitro, and it can produce different differentiated tissue's types, i.e. ectoderm, mesoderm and entoderm.The multipotential stem cell (iPSC) that human pluripotent stem cells (hPSC) includes but not limited to human embryo stem cell (hESC), people's parthenogenesis stem cell (hpSC) and induces.The method obtaining described hPSC is well known in the art.

A kind of method of hPSC of acquisition passes through parthenogenesis." parthenogenesis " (" activating (parthenogenically activated) in parthenogenesis mode " and " activating (parthenogenetically activated) in parthenogenesis mode " is used interchangeably herein) refers to there is not sperm and penetrate the process of lower generation activation of oocytes by it, and refers to and comprise trophectoderm and the growth by the body early embryo that activates the ovocyte or embryonic cell (such as blastomere) that comprise the DNA in all female sources and the inner cell mass obtained.At a related aspect, " parthenote " refers to the cell produced obtained by described activation.In another related fields, " blastaea " refer to fertilization or the cleavage stage of ovocyte that activates, described ovocyte comprises the cell hollow ball be made up of outer trophocyte and inner cell mass (ICM).In another related fields, " Blastocyst formation " refers to the process after oocyte fertilization or activation, ovocyte is cultivated for some time (such as subsequently in the medium in this process, 5-6 days), the cell hollow ball be made up of outer trophocyte and ICM can be budded into make it.

The another kind of method obtaining hPSC passes through nuclear transplantation.As used herein, " nuclear transplantation " refers to by donorcells or from the DNA of donorcells and merges or migrate in the suitable acceptor cell of ovocyte of normally identical or different species, by described recipient cell in transplanting or before merging, simultaneously or carry out afterwards processing to remove its endogenous core DNA or make its endogenous core DNA inactivation.Donorcells for nuclear transplantation comprises the cell of embryonic cell and differentiation, such as somatocyte and sexual cell.Described donorcells can be in proliferative cell cycle (G1, G2, S or M) or for non-proliferative (G0 or static).Preferably, donorcells or the DNA from donorcells derive from proliferating mammalian cells culture, such as fibroblast cell cultures.Described donorcells optionally can be genetically modified, that is, it can comprise a place or the interpolation of many places gene, replace or disappearance modification.

The another kind of method obtaining hPSC is to obtain the multipotential stem cell of induction by cell reprogrammed.The people such as Takahashi (Cell 131,861-872 (2007)) have disclosed for the reprogrammed noble cells when not using any embryo or ES (embryo does) cell and have formed the method with the derivable multipotential stem cell of the versatility similar with ES cell and energy for growth.The people such as Takahashi describe the fibroblastic various different nuclear reprogramming factor for breaking up, and it comprises the product of following four kinds of genes: Oct family gene; Sox family gene; Klf family gene; With Myc family gene.

The multipotency state of cell preferably by culturing cell under proper condition, such as, by maintaining at fibroblast feeder layer or another feeder layer or the upper cultivation of Prostatropin (bFGF).The multipotency state of described culturing cell confirms by following various method, and such as, (i) confirms the expression of the marker feature of pluripotent cell; (ii) cell is injected animal (such as SCID mouse), wherein produce different differentiated cell types in vivo; And (iii) observation of cell (when such as, cultivating when there is not feeder layer or LIF) vitro differentiation becomes the cell type of embryoid and other differentiation.

The multipotency state of the cell used in the present invention confirms by different methods.Such as, described cell presence or absence characteristic ES cell marker can be tested.When people ES cell, the example of described marker is determined above, and comprises SSEA-4, SSEA-3, TRA-1-60, TRA-1-81 and OCT 4, and is well known in the art.

Versatility is also by injecting proper animal (such as SCID mouse) by described cell and the generation observing the biological cells and tissues of differentiation confirms.Confirm that another method of versatility uses tested pluripotent cell to produce chimeric animal and to observe the cell introduced to the effect of different cell type.

Confirm that another method of versatility observes ES cell is divided into embryoid and other differentiation cell type when cultivating under the condition being conducive to breaking up (such as, removing fibroblast feeder layer).This method has been utilized and has confirmed that tested pluripotent cell produces the cell type of embryoid and different differentiation in tissue culture.

Gained pluripotent cell and clone, preferably people's pluripotent cell and clone have numerous treatment and diagnostic use.Described pluripotent cell can for cell transplantation therapy or gene therapy (if genetic modification) in the treatment of numerous disease event.

Human pluripotent stem cells (hPSC) includes but not limited to human embryo stem cell (hESC), people's parthenogenesis stem cell (hpSC), the multipotential stem cell (iPSC) of inducing and the clone produced by described cell.HPSC maintains multipotency state by routine passage in culture, until need derivative endomesoderm precursor cell.

" EMPC " (being also called as " multipotency endomesoderm cell ") shows one or more following character: 1) express MIXL1, NODAL and/or BRACHYURY; 2) mesoderm and endoderm cell's pedigree can be divided into; With 3) for morphological specificity typical EMPC.

EMPC produces the multipotential cell comprising the cell of mesoderm and endoblastic major cell phenotypes.

Endomesoderm be not yet be divided into mesoderm and entoderm but be doomed to produce described both embryonic blastomeres or cellular layer.The the inside of cellular layer is grown by the blastodisc of the inner cell mass of blastaea.Entoderm produces trachea-bronchial epithelial cell, lung, gi tract, liver, pancreas, bladder, anal tube, pharynx, Tiroidina, tympanum, tonsilla and parathyroid epithelium.Therefore entoderm comprises the tectum of the liner of cavity and the passage of health and most of internal organs.Mesoderm be between ectoderm and entoderm, the centre of three primitive layers of embryo; By its derivative reticular tissue, bone, cartilage, muscle, blood and blood vessel, lymph gland, lymphoid organ, notochord, pleura, pericardium, peritonaeum, kidney and sexual gland.Mesoderm produces hemocyte, endotheliocyte, cardiac muscle and skeletal muscle and adipocyte.

Endomesoderm precursor cell is differentiated by the expression increase detecting endomesoderm precursor cell marker, and endomesoderm precursor cell marker includes but not limited to: MIXL1, NODAL and Brachyury or its arbitrary combination.

T-box gene family is by the member composition sharing unique DNA binding domains.T-box (Tbx) gene of best sign, Brachyury or T are coded in during Early vertebrates is grown the transcription factor played an important role.Tbx gene is have the developmental regulation factor family more than 20 kinds of members recently differentiated in invertebrates and vertebrates.Find that the sudden change in Tbx gene causes several human disease.The understanding of the functional mechanism of Tbx product is mainly from prototype T/Brachyury albumen, and described protein is activating transcription factor.T-structural domain is the feature of the DNA-binding motif of the high conservative be limited at first in Brachyury and the Tbx family for transcription factor.Determine in early days mesoblastic and need Brachyury in breaking up.Brachyury for the back body in all vertebratess formation be absolutely necessary.The sudden change shown in brachyury causes spine malformation.In addition, the imbalance of brachyury can relate in the formation of chordoma (malignant tumour in backbone).

The invention provides for by carrying out to described cell the method that mensuration carrys out derivative endomesoderm precursor cell (EMPC) for endomesoderm precursor cell marker with at least one endomesoderm precursor cell inducing compounds process hPSC.

As used herein, " endomesoderm cell inducing compounds " is the compound that induction hPSC becomes EMPC.Described compound includes but not limited to glycogen synthase kinase 3 inhibitor.

Glycogen synthase kinase 3 (GSK-3) is the serine/threonine protein kitase that mediation phosphate molecule adds on Serine and threonine amino acid residues.Find at the regulation and control kinases as its homonym Glycogensynthase in 1980 at first, after this GSK-3 is identified as more than 40 kinds of different proteins with the kinases of multiple different approaches.In Mammals, GSK-3 is encoded by two kinds of known GSK-3 α (GSK3A) and GSK-3 β (GSK3B).GSK-3 becomes the theme of much research recently, because it is involved in the numerous disease comprising type ii diabetes (diabetes B), Alzheimer, inflammation, cancer and bipolar disorder.GSK-3 has activity in signal transduction path comprising in cell proliferation, migration, inflammation and immunne response, glucose regulation and control and apoptotic many centrocytes.

The classes of compounds of GSK-3 can be suppressed to comprise but do not limit maleimide derivatives, Staurosporine (staurosporine) and organometallic inhibitor, indole derivatives, paillone derivative, pyrazole amide derivatives, pyrimidine and furopyrimidine derivatives, thiazole derivative, Pyrroloazepine (pyrroloazepine), flavones, benzo nitrogen Boom ketone, two indoles, Pyrrolopyrazine, thiadiazolidine ketone, Bi Ding oxadiazole, aminopyrimidine, pyrazolo quinoline quinoline, oxindole (indolone), thiazole, bisindolylmaleimidesfor, azaindolyl maleimide, (hetero) aryl indole maleimide, anilino maleimide, phenyl amino pyrimidine, triazole, pyrrolopyrimidine, pyrazolopyrimidine and chloromethyl thienyl ketone.

Concrete GSK-3 inhibitor comprises but is limited to lithium chloride, Hymenialdisine, Flavopiridol (Flavopiridol), bank Paro ketone, A Site Paro ketone (Alsterpaullone), azepine Kan Paluo ketone (Azakenpaullone), isatin-30-oxime, 6-bromo-isatin-30-oxime (BIO), 6-bromo-isatin-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 (bisindolylmaleimidesfor I), Ro318220 (bisindolylmaleimidesfor IX), SB216763, SB415286, I5, CGP60474, compound 8b, TWS119, compound 1A, compound 17, lithium atom (competing with Mg2C), beryllium atom (competing with Mg2C and ATP) and zinc atom (noncompetitive).

Chir99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl isophthalic acid H-imidazoles-2-base)-2-pyrimidyl] amino] ethyl] amino]-3-pyridine carbonitrile) (Tocris, Bristol, United Kingdom) for glycogen synthase kinase 3 (GSK-3) effectively and highly selective inhibitor (6.7 nM and 10 nM are respectively for GSK-3 β and GSK-3 α, IC50 value).Relative to closely-related kinases, described compound shows >500 selectivity doubly to GSK-3; Further, also >800 selectivity is doubly shown for 45 kinds of other enzymes and acceptor.Combine with Tranylcypromine (tranylcypromine), the mouse embryo fibroblasts reprogrammed of transduceing by means of only Oct4 with Klf4 can be become iPSC by Chir99021.Described compound also strengthens mouse and people ESC self refresh when combinationally using with PD 0325901.

The present invention includes the Chir99021 (comprising acid salt) in salt form.Suitable salt comprises those salt formed with organic acid and mineral acid.Described acid salt will be pharmaceutically acceptable usually.But acceptable salt can be used for preparation and the purifying of discussed compound in non-pharmaceutical.Also base addition salt can be formed and it is pharmaceutically acceptable.About the preparation of salt and the discussion more comprehensively of selection, with reference to Pharmaceutical Salts:Properties, Selection, and Use (drug salts: character, selection and purposes) (Stahl, P.Heinrich.Wiley-VCHA, Zurich, Switzerland, 2002).

The present invention also comprises the analogue of Chir99021.The term " analogue " used in this article be structurally from another compounds like but compound (as the atomic substitutions atom by different element or under particular functional group exists) slightly different on forming.

In one embodiment, the invention provides a kind of method for generation of endomesoderm cell (EMPC).Described method comprises with glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC) and analyzes described cell for endomesoderm cell marker.On the one hand, by hPSC GSK-3 inhibitor process about 24 hours or 12-36 hour.On the other hand, GSK-3 inhibitor is Chir99021 or its salt or analogue.

On the one hand, endomesoderm precursor cell marker comprises MIXL1, NODAL and Brachyury or its arbitrary combination.

The EMPC derivative from hPSC can use method well-known to those having ordinary skill in the art easily to differentiate.These methods comprise the measurement of use immunohistochemistry, facs analysis and rna expression level to differentiate endomesoderm precursor cell marker.

In one embodiment, the invention provides endomesoderm precursor cell (EMPC).Tested EMPC is by analyzing described cell to produce for endomesoderm cell marker with glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC).On the one hand, by hPSC GSK-3 inhibitor process about 24 hours.On the other hand, GSK-3 inhibitor is Chir99021 and endomesoderm marker is Brachyury.

Once derive EMPC, just cell should be used or freezen protective, thus retain the ability being divided into other mesoderm and endodermal cell types.As shown in Example, GSK-3 inhibitor C hir99021 is identified as induction hPSC and is divided into EMPC.The EMPC colony that hPSC derives is positive to Brachyury, MIXL1 and NODAL cell marker.These EMPC are suitable for further amplification, freezen protective and differentiation, thus make them become the practicality source of EC.So, present invention demonstrates that by the method disclosed in hPSC generation EMPC.

The present invention produces endotheliocyte (EC) by hPSC under being also provided in the electrochemical conditions of restriction.

As used herein, " differentiation " is the change that occurs in phalangeal cell thus causes those cells to present some specific function and lose the ability changing into some other specific functional units.The cell that can break up can be any one in totipotent cell, pluripotent cell or multipotential cell.For the adult cell of maturation, differentiation can be part or completely.

" cell of differentiation " refers to the non-embryonic cell of (i.e. non-embryonic) state having specific differentiation.Three kinds of cell types broken up the earliest are entoderm, mesoderm and ectoderm.

The EMPC derived from hPSC be pluripotency and several cell types comprising endotheliocyte can be divided into.

Endotheliocyte comprises endothelium.Endothelium is liner blood vessel and vasculolymphatic internal surface thus blood circulation in the lumen or form the cell thin at interface between lymph liquid and the remainder of tube wall.The cell forming endothelium is called as endotheliocyte.Be called as vascular endothelial cell with the endotheliocyte that blood directly contacts, and be called as lymphatic endothelium with those endotheliocytes that lymph liquid directly contacts.

The inside of all blood vessels of endotheliocyte liner, thus form endothelium.All endotheliocytes develop organ specificity character all derived from common angioblast precursor and subsequently.Embryonic endothelium cell shows a lot of heterogeneity in organ and between organ.The differentiation of endotheliocyte is by the multifactor impact perhaps comprising direct microenvironment, discharge with the interaction of peripheral cell and the local of cytokine and somatomedin.Human endothelial cells retain significant plasticity-and known response in IL-1, TNF, VEGF and FGF reprogrammed.Because endotheliocyte provides the function of much necessity, so endothelial function disturbance causes vascular disease and can promote the development as atherosclerotic chronic inflammatory condition.

Vascular endothelial cell liner is from heart to the whole recycle system of minimum kapillary.These cells have the very different and function of uniqueness extremely important Vascular Biology.These functions comprise as the filtering flow in the renal glomerulus of kidney, antiotasis (blood vessel tone), hemostasis, neutrophil leucocyte supplement and hormone transport.The endothelium of the internal surface of ventricle is referred to as endocardium.

The invention provides the method for being broken up EC by EMPC.

In another embodiment, the invention provides a kind of method for generation of endotheliocyte (EC).Described method comprises with glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC); Endomesoderm precursor cell (EMPC) is differentiated by the hPSC handled by measuring for endomesoderm cell marker; Described cell is analyzed with EMPC described in the process of at least one somatomedin for endothelial cell marker thing.On the one hand, by hPSC GSK-3 inhibitor process 24 hours.In some aspects, GSK-3 inhibitor is Chir99021 or its salt or analogue.On the other hand, the process of EMPC somatomedin is about 48-96 hour, preferably about 72 hours.On the other hand, somatomedin can be bFGF, VEGF or BMP4 or its arbitrary combination.In preferred at one, somatomedin is bFGF and VEGF.

In the one side of described method, endomesoderm cell marker can be ENDO16, FoxA, GATAE, SMIP, Hex, P19 and Brachyury or its arbitrary combination.

In the another aspect of described method, endothelial cell marker thing can be VE Cadhedrin, PECAM1, ACE/CD143, MCAM/CD146, C1q R1/CD93, Fibronectin-2/CD112, VE-cadherin, PD-ECGF/ thymidine phosphorylase, CC-chemokine receptor D6, foot glycocalicin, CD31/PECAM-1, flatfoot albumen, CD34, S1P1/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)/palatelet-selectin (CD62P), thromboplastin/tissue factor, palatelet-selectin/CD62P, DC-SIGNR/CD299, SLAM/CD150, DCBLD2/ESDN, immobilon-1, EMMPRIN/CD147, immobilon-2, Endoglin/CD105, TEM7/PLXDC1, endothelium Saliva Orthana, 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 α 1, VE-statin, integrin alpha-4/CD49d, VEGF R1/Flt-1, integrin alpha-4 β 1, VEGF R2/KDR/Flk-1, integrin alpha-4 β 7/LPAM-1, VEGF R3/Flt-4, integrin β 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 or its arbitrary combination.In concrete at one, endothelial cell marker thing is VE cadherin and PECAM1 or its arbitrary combination.

Somatomedin is can the naturally occurring material of stimulate cell growth, propagation and cytodifferentiation.Usually, it is protein and steroid hormone.Somatomedin is important for regulation and control various kinds of cell process.Somatomedin serves as the signal transduction molecule between cell usually.Example is the cytokine that is combined of the specific receptors on its target cells and hormone.They often promote cytodifferentiation and maturation, and they are different between somatomedin.Such as, bone morphogenetic protein stimulates bone cell differentiation, and fibroblast growth factor and vascular endothelial growth factor stimulate blood vessel differentiation (vasculogenesis).The example of somatomedin and growth factor family includes but not limited to: adrenomedullin (AM), angiogenin (Ang), autocrine motility factor, Delicious peptide (BMP), Brain Derived Neurotrophic Factor (BDNF), Urogastron (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line derived neurotrophic sex factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granular leukocyte macrophage colony-stimulating factor (GM-CSF), GDF-9 (GDF9), pHGF (HGF), hepatoma-derived growth factor (HDGF), rhIGF-1 (IGF), migration-stimulating factor, flesh amicine (GDF-8), nerve growth factor (NGF) and other neurotrophin, platelet-derived growth factor (PDGF), thrombopoietin (TPO), α transforming growth factor (TGF-α), β transforming growth factor (TGF-β), cachectin (TNF-α), vascular endothelial growth factor (VEGF), Wnt signal transduction path, placenta growth factor (PlGF), tire Trobest (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, bFGF and VEGF.

In another embodiment, the invention provides endotheliocyte (EC).Tested EC is by using glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC); Endomesoderm precursor cell (EMPC) is differentiated by measuring the handled hPSC crossed for endomesoderm cell marker; Analyze described cell to produce for endothelial cell marker thing with EMPC described in the process of at least one somatomedin.In some aspects, by hPSC with GSK-3 inhibitor process 24 hours and by EMPC somatomedin process 72 hours.In concrete, GSK-3 inhibitor is Chir99021 and somatomedin is bFGF and VEGF.On the other hand, endomesoderm marker is Brachyury and endothelial cell marker thing is VE cadherin and PECAM1.

As shown in Example, disclosed method facilitates the generation of complete function endotheliocyte.EC by with Chir99021 process hPSC, by measure Brachyury express differentiate EMPC, with bFGF and VEGF process EMPC and measure VE cadherin and PECAM1 expression produce.As by measured by RT-PCR and immunofluorescence, the EC cell expressing VE cadherin produced and PE CAM1.In addition, the EC that hPSC matrigel coated panel growing for >=6 generations derives continues to express VE-cadherin and PECAM1.This proves that the method disclosed in use produces endotheliocyte by hPSC.

EMPC and EC can play an important role in the treatment of vascular disease and illness.The loss of endothelial function disturbance or suitably endothelial function is the mark of vascular disease, and is often regarded as the crucial earliest events in progression of atherosclerosis.Hypertension and thrombotic Endothelial dysfunction is caused usually to be seen in the patient suffering from coronary artery disease, diabetes, hypertension, hypercholesterolemia and in smoker.Also show endothelial function disturbance and imply that following disadvantageous cardiovascular event, and it is also present in the inflammatory diseases as rheumatoid arthritis and systemic lupus erythematous.One of main mechanism of endothelial function disturbance is nitrogen oxide minimizing, and this is often owing to high-caliber ADMA, and it disturbs the nitrogen oxide of normal L-arginine-stimulation synthesize and therefore cause hypertension.The topmost mechanism of endothelial function disturbance is that reactive species of oxygen increases, and it produces and activity by several mechanism infringement nitrogen oxide.Signal conductive protein ERK5 is absolutely necessary for maintenance normal endothelial cell function.Another result of infringement endothelium is von Willebrand disease (von Willebrand) factor of release pathology amount, and this promotes platelet aggregation and adheres to subendothelium, and therefore forms potential fatal thrombus.

Vascular disease are the pathological state of large-scale and medium-sized muscular artery and trigger because of endothelial dysfunction.Due to the factor of such as pathogenic agent, LDL particle and other inflammatory stimulus endotheliocyte of oxidation are activated.This causes their characteristic changing: endotheliocyte starts secrete cytokines and chemokine and expression of adhesion molecules in its surface.This so cause white corpuscle (monocyte and lymphocyte) to raise, described white corpuscle can penetration rate of blood tube wall.Irritate layer of smooth muscle cells by the cytokine produced by endotheliocyte and the white corpuscle raised cause smooth muscle cell proliferation and move to intravascular space.Described process causes tube wall thickening, thus forms the patch be made up of propagation smooth muscle cell, scavenger cell and dissimilar lymphocyte.Described patch causes blood flow to be obstructed, thus causes the amount of the oxygen and nutrient substance arriving target organ to reduce.In the in the end stage, described patch also may break, thus causes forming grumeleuse, and result causes apoplexy.

In another embodiment, the invention provides a kind of test kit for derivative EMPC.Described test kit can comprise glycogen synthase kinase 3 (GSK-3) inhibitor, for differentiating the reagent of endomesoderm precursor cell marker and the guidance for being produced EMPC by hPSC.On the one hand, GSK-3 inhibitor is Chir99021 or its salt or analogue.Described test kit can comprise the reagent for differentiating the following: ENDO16, FoxA, GATAE, SMIP, Hex, P19 and Brachyury endomesoderm precursor cell marker or its arbitrary combination.

In another embodiment, the invention provides a kind of test kit for generation of endotheliocyte (EC).Described test kit comprises glycogen synthase kinase 3 (GSK-3) inhibitor, for differentiating the reagent of endomesoderm precursor cell marker, somatomedin, for differentiating the reagent of endothelial cell marker thing and the guidance for being produced endotheliocyte by hPSC.On the one hand, GSK-3 inhibitor is Chir99021 or its salt or analogue.On the other hand, somatomedin is bFGF and VEGF.Described test kit can comprise the reagent for differentiating the following: ENDO16, FoxA, GATAE, SMIP, Hex, P19 and Brachyury endomesoderm precursor cell marker or its arbitrary combination and VE cadherin, PECAM1, ACE/CD143, MCAM/CD146, C1q R1/CD93, Fibronectin-2/CD112, VE-cadherin, PD-ECGF/ thymidine phosphorylase, CC-chemokine receptor D6, foot glycocalicin, CD31/PECAM-1, flatfoot albumen, CD34, S1P1/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)/palatelet-selectin (CD62P), thromboplastin/tissue factor, palatelet-selectin/CD62P, DC-SIGNR/CD299, SLAM/CD150, DCBLD2/ESDN, immobilon-1, EMMPRIN/CD147, immobilon-2, Endoglin/CD105, TEM7/PLXDC1, endothelium Saliva Orthana, 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 α 1, VE-statin, integrin alpha-4/CD49d, VEGFR1/Flt-1, integrin alpha-4 β 1, VEGF R2/KDR/Flk-1, integrin alpha-4 β 7/LPAM-1, VEGF R3/Flt-4, integrin β 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 endothelial cell marker thing or its arbitrary combination.

Following examples are intended to illustrate instead of restriction the present invention.

Embodiment 1

the raising thing growth of hPSC

HPSC is maintained on mouse embryo fibroblasts (Millipore) feeder layer of the Mitomycin-C inactivation in following embryonic stem cell medium: knock out DMEM/F12 (Life Technologies), 2 mML-glutamine (GlutaMax-I, Invitrogen), 0.1mMMEM non-essential amino acid (Life Technology), 0.1mM beta-mercaptoethanol (Life Technologies), penicillin/streptomycin/amphotericin B (100 U/100 μ g/250ng) (MP Biomedicals) and 5ng/ml bFGF (Peprotech).Every 5 to 7 days is passage with Dispase or collagenase IV (both are all from Life Technologies), and wherein splitting ratio is 1:4 or 1:6.

Embodiment 2

hPSC grows without raising thing

Then hPSC be transferred to Matrigel (BD Biosciences) coated panel and grow with following StemPro hESC SFM substratum (Invitrogen): there is the DMEM/F12 that GlutaMAX, 1XSTEMPRO hESC SFM grows fill-in, 1.8% bovine serum albumin, 8 ng/mL bFGF and 0.1 mM 2 mercapto ethanol.

Embodiment 3

endotheliocyte is derived by hPSC

By dissociating without the hPSC cell Accutase (Sigma) growing for 4 generations under raising thing culture condition and being seeded in Matrigel coated panel and growing with N2/B27 substratum [there is the DMEM/F12 (Invitrogen) of GlutaMAX, 1X N2/B27 fill-in], wherein carry out the process in 6 kinds of differences (stage 1): (1) bFGF+BMP4; (2) bFGF+BMP4+VEGF, two days; (3) Chir99021+bFGF+BMP4+VEGF, two days; (4) Chir99021, one day; (5) Chir99021, two days; (6) Stempro hESC substratum, two days, then uses bFGF (50ng/mL)+VEGF (50ng/mL) (stage 2) to process 3 days [Fig. 1].Process hPSCS cell 1 day with Chir99021 (10 μMs), induce them to make it be divided into the endomesoderm precursor of expressing Brachyury.As passed through measured by RT-PCR [Fig. 2] and immunofluorescence, the hPSC (the endomesoderm precursor that hPSC is derivative) using Chir99021 (10 μMs) to process 1 day is processed 3 days further with N2B27+bFGF (50ng/ml)+VEGF (50ng/ml), makes them be divided into the endotheliocyte of expressing VE-CAM and PECAM1.

Embodiment 4

from the amplification of the endotheliocyte that hPSC derives

Can dissociate with Accutase (Sigma) from the endotheliocyte that hPSC is derivative and make it on Matrigel coated panel, in N2B27 substratum 50ng/ml bFGF and 50ng/ml VEGF, grow for >=6 generations.As passed through measured by immunofluorescence, the hPSC-EC in the 6th generation expresses VE-cadherin and PECAM.

rT-PCR analyzes.Total serum IgE from least three increment product separately with about 100 ten thousand cells uses the automatic purification system of QIAsymphony or RNeasy Plus mini kit to be separated according to the specification sheets (Qiagen) of manufacturers.Total serum IgE is used for use iScript cDNA synthetic agent box (Biorad) and Px2 thermal cycler (Thermo Scientific) reverse transcription.In order to analyzing gene is expressed, the cDNA/ of 1/25-th reaction and QuantiTect primer mensuration and the main mixture of Quantitest SYBR Green (Qiagen) is used to carry out PCR reaction in duplicate.Rotor-Gene Q (Qiagen) is used at 95 DEG C, to carry out qPCR 5 minutes, at 92 DEG C, carry out qPCR 5 seconds and at 60 DEG C, carry out qPCR 20 seconds, circulate 37 times, then melting is to check that amplicon raises 1 DEG C of specificity risen to 99 DEG C of situations from 50 DEG C in every step.Relative quantification is carried out for typical curve, and by quantized value for the input value stdn measured by PPIG.Primer for described analysis is listed in supplementary table S1.Result is rendered as mean value ± s.e.m., and use the two tail Student t-test of the confidence level of 95% (α=0.05) to carry out statistical study for comparing two groups or use one-way ANOVA Deng Nite (Dunnett) inspection to carry out statistical study with for comparing many groups, and P<0.05 is regarded as significantly.

immunocytochemistry.By each sample about 100,000 cell at room temperature fixes 10 minutes with 4% paraformaldehyde, with PBS washing, and closes 1 hour in 0.3%Triton X-100,5% normal donkey serum and the 1%BSA of saturatingization also at room temperature in PBS.First antibody overnight incubation is used by 0.3%Triton X-100,2%BSA at 4 DEG C in PBS of cell.1 hour is hatched by second antibody in 0.3%TritonX-100,5% normal donkey serum and 1%BSA by cells rinsed with PBS three times and at room temperature in PBS.Nucleus DAPI is dyeed.

Although with reference to embodiment above describes the present invention, it should be understood that, modifications and variations will contain within the spirit and scope of the present invention.Therefore, the present invention only limits by following claims.

Claims

1. produce a method for endomesoderm precursor cell, it comprises:

A () uses glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC); And

B () measures described cell for endomesoderm cell marker.

2. method according to claim 1, wherein by described hPSC described GSK-3 inhibitor process 24 hours.

3. method according to claim 2, wherein said GSK-3 inhibitor is Chir99021 or its salt or analogue.

4. method according to claim 1, wherein said endomesoderm precursor cell marker is selected from the group be made up of the following: MIXL1, NODAL and Brachyury or its arbitrary combination.

5. method according to claim 4, wherein said endomesoderm precursor cell marker is Brachyury.

6. method according to claim 1, wherein said hPSC is selected from the group be made up of the following: human embryo stem cell (hESC), people's parthenogenesis stem cell (hpSC) or the multipotential stem cell (iPSC) of inducing or the clone derived from it.

7. an endomesoderm precursor cell, it is derived by method according to claim 1.

8. endomesoderm precursor cell according to claim 7, wherein said GSK-3 inhibitor is Chir99021 or its salt or analogue.

9. endomesoderm precursor cell according to claim 7, wherein said endomesoderm precursor cell marker is Brachyury.

10. produce a method for endotheliocyte, it comprises:

A () uses glycogen synthase kinase 3 (GSK-3) inhibitor handler's multipotential stem cell (hPSC);

B () is by differentiating that endomesoderm cell marker differentiates endomesoderm precursor cell (EMPC);

C () uses EMPC described in the process of at least one somatomedin; And

D () is analyzed described cell for endothelial cell marker thing.

11. methods according to claim 10, wherein by described hPSC described GSK-3 inhibitor process 24 hours.

12. methods according to claim 11, wherein said GSK-3 inhibitor is Chir99021 or its salt or analogue.

13. methods according to claim 10, wherein said endomesoderm precursor cell marker is selected from the group be made up of the following: MIXL1, NODAL and Brachyury or its arbitrary combination.

14. methods according to claim 13, wherein said endomesoderm precursor cell marker is Brachyury.

15. methods according to claim 10, wherein by described EMPC described somatomedin process 72 hours.

16. methods according to claim 10, wherein said somatomedin is selected from the group be made up of the following: bFGF, VEGF and BMP4 or its arbitrary combination.

17. methods according to claim 16, wherein said somatomedin is bFGF and VEGF.

18. methods according to claim 10, wherein said endoderm cell's marker is selected from: VE Cadhedrin, PECAM1, ACE/CD143, MCAM/CD146, C1q R1/CD93, Fibronectin-2/CD112, VE-cadherin, PD-ECGF/ thymidine phosphorylase, CC-chemokine receptor D6, foot glycocalicin, CD31/PECAM-1, flatfoot albumen, CD34, S1P1/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)/palatelet-selectin (CD62P), thromboplastin/tissue factor, palatelet-selectin/CD62P, DC-SIGNR/CD299, SLAM/CD150, DCBLD2/ESDN, immobilon-1, EM MPRIN/CD147, immobilon-2, Endoglin/CD105, TEM7/PLXDC1, endothelium Saliva Orthana, TEM8/ANTXR1, endothelium liquid saliva acid albumin/CD248, thrombomodulin/BDCA-3, EPCR, THSD1, erythropoietin R, Tie-2, ESAM, TN F 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-1RI, VCAM-1/CD106, IL-13R α 1, VE-statin, integrin alpha-4/CD49d, VEGF R1/Flt-1, integrin alpha-4 β 1, VEGF R2/KDR/Flk-1, integrin alpha-4 β 7/LPAM-1, VEGF R3/Flt-4, integrin β 2/CD18, VG5Q, KLF4, vWF-A2 and LYVE-1 or its arbitrary combination.

19. methods according to claim 18, wherein said endothelial cell marker thing is VE cadherin and PECAM1.

20. an endotheliocyte, it is produced by method according to claim 10.

21. endotheliocytes according to claim 20, wherein said GSK-3 inhibitor is Chir99021 and described somatomedin is bFGF and VEGF.

22. endotheliocytes according to claim 20, wherein said endomesoderm precursor cell marker is Brachyury and described endothelial cell marker thing is VE cadherin and PECAM1.

23. 1 kinds of test kits for generation of endomesoderm precursor, it comprises:

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

B () is for measuring the reagent of endomesoderm precursor cell marker; And

C () is for being derived the guidance of endomesoderm precursor cell by hPSC.

24. 1 kinds of test kits for generation of endotheliocyte, it comprises:

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

B () is for measuring the reagent of endomesoderm precursor cell marker;

(c) at least one somatomedin;

D () is for measuring the reagent of endothelial cell marker thing; And

E () is for being produced the guidance of endotheliocyte by hPSC.