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    • 专利摘要:
    GENERATION OF CLONAL MESENCHYMAL PROGENITORS AND MESENCHEMICAL STEM CELL LINES UNDER SERUM-FREE CONDITIONS. Method for obtaining side-plate mesoderm cells positive for the Apelin receptor, multipotent mesenchymal stem cells and mesangioblasts under serum-free conditions are disclosed
    公开号:BR112012023537B1
    申请号:R112012023537-0
    申请日:2011-03-16
    公开日:2020-11-03
    发明作者:Maksym A. Vodyanyk；Igor I. Slukvin
    申请人:Wisconsin Alumni Research Foundation；
    IPC主号:
    专利说明:

    CROSS REFERENCE TO RELATED REQUESTS
    [001] This application claims priority to US Patent Application N2 12 / 726,814, filed on March 18, 2010. US Patent Application N2 12 / 726,814 is a partial extension of US Patent Application N2 12 / 554,696, which is an extension of US Patent Application No. 12 / 024,770, which claims the benefit of US Provisional Patent Application No. 60 / 974,980, filed on September 25, 2007; and U.S. Provisional Patent Application No. 60 / 989,058, filed on November 19, 2007, each of which is incorporated in its entirety in this report by reference. FUNDAMENTALS
    [002] The invention relates, in general, to clonal primate mesenchymal progenitors and mesenchymal stem cell lines (MSC) and methods for identifying and generating such cells, and, more particularly, methods for generating mesenchymal progenitors. clonal and MSC strains under serum-free conditions. The invention also relates to a population of shared endothelial and mesenchymal cell precursors and to methods for identifying and generating such cells. The invention, moreover, relates to a population of cells comprising cells of the lateral plate mesoderm and methods for their generation and isolation from cultured pluripotent stem cells.
    [003] During the embryonic development of animals, gastrulation forms three germ layers, that is, endoderm, ectoderm and mesoderm, which give rise to distinct body cells. The mesoderm develops from a primitive line, a transient embryonic structure formed at the beginning of gastrulation. The nascent mesoderm is transitionally differentiated into paraxial mesoderm, intermediate mesoderm and lateral plate mesoderm. The paraxial mesoderm gives rise to the axial skeleton and skeletal muscles. The intermediate mesoderm forms the urogenital system. The lateral plate mesoderm gives rise to the circulatory system, including blood cells, vessels, and heart, and forms the viscera and limbs. The extraembryonic mesoderm is located outside the developing embryo. The evidence suggests that the extraembryonic mesoderm is derived from the primitive line during gastrulation (Boucher and Pedersen, Reprod. Fertile. Dev. 8: 765 (1996)). The extraembryonic mesoderm gives rise to various tissues that provide the embryo with nutrients, a means of eliminating waste and mechanical protection.
    [004] Both the lateral and extraembryonic mesoderm can generate endothelial and blood cells and express F0XF1, HAND1, HAND2, GATA-2, BMP4 and WNT5a, the expression of which is low or undetected in the paraxial and intermediate mesoderm (Mahlapuu et al., Development. 128 (2): 155 (2001); Firulli et al., Nat. Genet. 18 (3): 266 (1998); Morikawa et a!., Circ. Res. 103 (12): 1422 (2008); Kelley et al., Dev. Biol. 165: 193 (1994); Prata et al., Blood 89 (4): 1154 (1997); Fujiwara et al., Proc. Natl. Acad. Sei. 98 (24): 13739 (2001); Takada et al., Genes Dev. 8 (2): 174 (1994)). The distinct markers for the lateral and extraembryonic mesoderm remain to be elucidated. A recent discovery by Bosse et al. suggests that IRX3 is expressed in the lateral plate mesoderm, but not in the extraembryonic mesoderm (Bosse et al., Mech. Dev. 69 (1 - 2): 169 (1997)). For the purposes of this application, the term side plate is used to describe the two fabrics.
    [005] Certain compromised mesodermal progenitors can give rise to cells from more than one strain. Examples of such parents include hemangioblasts, which can give rise to hematopoietic and endothelial cells. Choi K, et al., “A common precursor of hematopoietic and endothelial cells”, Development 125: 725 (1998).
    [006] MSCs can differentiate in at least three me- senquimal cell lines downstream (ie, osteoblasts, chondroblasts and adipocytes). To date, no single MSC marker has been identified. As such, morphological and functional criteria are used to identify these cells. See, Horwitz E, et al., “Clarification of the nomenclature for MSC: the International Society for Cellular Therapy posi-tion statement”, Cytotherapy 7: 393 (2005); and Dominici M, et al., “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement ”, Cytotherapy 8: 315 (2006). Because MSCs can differentiate into several cell types, the technique considers methods to differentiate MSCs for cell-based therapies, for regenerative medicine and for reconstructive medicine.
    [007] Typically, MSCs are isolated from adult bone marrow, fat, cartilage and muscle. Pittenger F, et al., “Multilineage potential of adult human mesenchymal stem cells”, Science 284: 143 - 147 (1999); Zuk P, et al., “Multilin-eage cells from human adipose tissue: implications for cell-based therapies”, Tissue Eng. 7: 211 - 228 (2001); and Young H, etal., “Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors”, Anat. Rec. 264: 51 - 62 (2001). MSCs have also been isolated from human peripheral blood. Kassis I, et al., “Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads”, Bone Marrow Transplant. 37: 967 - 976 (2006). MSCs can also be isolated from human neonatal tissue, such as Wharton's jelly (Wang H, et al., “Mesenchymal stem cells in the Wharton 'jelly of the human umbilical cord”, Stem Cells 22: 1330 - 1337 (2004 )), human placenta (Fukuchi Y, et al., “Human placenta-derived cells have mesenchymal stem / progenitor cell potential”, Stem Cells 22: 649 - 658 (2004)); and umbilical cord blood (Erices A, et al., “Mesenchymal progenitor cells in human umbilical cord blood”, Br. J. Haematol. 109: 235 - 242 (2000)) and human fetal tissues (Campagnoli C, et al. , “Identification of mesenchymal stem / progenitor cells in human first-trimester fetal blood, liver, and bone marrow”, Blood 98: 2396 - 2402 (2001)).
    [008] The technique is limited by an inability to isolate sufficient MSCs for subsequent differentiation and use. Where appropriate donors are available, the invasive procedures required to isolate even a limited number of cells present risks to donors. It also remains difficult to keep MSCs isolated in culture for the long term and to keep such cultures free from bacterial or viral contamination.
    [009] Efforts to develop methods to differentiate embryonic stem cells (ESCs), including human ESCs (hESCs) into MSCs, have required culturing the cells in a medium containing potentially contaminating serum or have produced cells that retain characteristics of undifferentiated hESCs. For example, Barberi et al. differentiated hESCs in MSCs in mitotically inactivated mouse stromal cell lines (ie, feeder cells) with 20% heat inactivated fetal bovine serum (FBS) in alpha MEM medium for 40 days. Barberi T, et al. “Derivation of multipotent mesenchymal precursors from human embryonic stem cells”, PLoS Med. 2: e161 (2005). The cells were collected and evaluated for CD73, and CD73 + cells were then plated in the absence of feeder cells with 20% FBS in alpha MEM for 7 to 10 days. Barberi et al. Differentiated MSCs into adipogenic cells, chondrogenic cells, osteogenic cells and myogenic cells.
    [0010] Similarly, Olivier et al. differentiated hESCs into MSCs by plating raclures (ie, spontaneously differentiated cells that appear in the hESC culture in the center or at the edges of colonies) with D10 medium (DMEM, 10% FBS, 1% penicillin / streptomycin and non-amino acids essential at 1%) weekly modified until the development of a thick, multilayered epithelium. Olivier E, et al., “Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells”, Stem Cells 24: 1914 - 1922 (2006). After approximately four weeks, the MSCs were isolated by dissociating the epithelium with a mixture of trypsin, collagenase type IV and dispase for four to six hours, followed by replating in D10 medium. The MSCs of Olivier et al. they grew robustly, showed stable karyotypes, were inhibited by contact, matured after twenty passages and differentiated into adipogenic and osteogenic cells. Olivier et al. did not report that the cells were differentiated into chondroblasts. Unlike Barberi et al., Olivier et al. they did not require feeder cells to support the differentiation of hESC into MSCs. However, the MSCs of Olivier et al. were positive for SSEA-4, suggesting that these MSCs expressed cell surface markers characteristic of hESC.
    [0011] Pike & Shevde differentiated hESCs in MSCs by means of embryonic bodies (EBs) incubated for ten to twelve days in a specific mesenchymal medium (MesenCult® medium with 10% FBS; alpha MEM with glutamine and nucleosides; or DMEM with glucose and glutamine, replaced every two days). US Patent Publication N2 2006/0008902. EBs were digested and pre-mesenchymal cells were cultured to 80% confluence. The cells were trypsinized and passed three times in a specific mesenchymal medium.
    [0012] Meuleman et al. reported the culture of MSCs in a serum-free medium; however, it was later discovered that the medium, in fact, contains the animal's serum as a component. Meuleman N, et al., “Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-minimal essential medium (MEM)”, Eur. J. Haematol. 76: 309 - 316 (2006); and Meuleman N, etal., “Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-minimal essential medium (MEM)”, Eur. J. Haematol. 77: 168 (2007); but see, Korhonen M, “Culture of human mesenchymal stem cells in serum-free conditions: no breakthroughs yet”, Eur. J. Haematol. 77: 167 (2007).
    [0013] Such methods cultured and differentiated MSCs in medium containing serum. Serum-free conditions for cultivating and differentiating MSCs, if defined, would reduce variation between batches and eliminate a risk of infection transmitted through xenogenic by-products and pathogens. Sotiropoulou P, et al., “Cell culture medium composition and translational adult bone marrow-derived stem cell research”, Stem Cells 24: 1409 - 1410 (2006).
    [0014] For the reasons set out above, there is a need for new methods for obtaining early mesenchymal progenitors and MSCs, especially when derived under serum-free conditions.
    [0015] Mesoderm and neural crest can give rise to me-senquimal precursors during embryonic development. Dennis, J. E., and P. Charbord, "Origin and differentiation of human and murine stroma", Stem Cells 20: 205 - 214 (2002); Takashima, Y, et al., “Neuroepithelial cells supply an initial transient wave of MSC differentiation”, Cell 129: 1377 - 1388 (2007). Although the conditions for generating MSCs of neural crest origin from embryonic stem cells have been described, Takashima et al., Supra; See, G, et al., “Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells”, Nat Bio-technol 25: 1468 - 1475 (2007), is not known as generating MSCs from the mesoderm.
    [0016] For the reasons set out above, there is a need for new methods to obtain early mesenchymal progenitors and MSCs, particularly under serum-free conditions. In addition, there is a need for the identification and generation of MSCs derived from the mesoderm, as well as early mesodermal progenitors that can give rise to MSCs during the differentiation of pluripotent stem cells into MSCs. BRIEF SUMMARY
    [0017] The invention, in general, refers to a recently identified common mesenchymal and endothelial cell precursor, that is, mesangioblasts, derived from differentiated stem cells in vitro.
    [0018] In a first aspect, the invention is summarized in that a method for generating a clonal population of primate MSCs includes the steps to cultivate a single, heterogeneous cell suspension of primate cells that contains mesenchymal pro-parents in a semi-solid medium serum-free, containing between about 5 and about 100 ng / ml of bFGF until independent colonies form, and grow one of the independent colonies in a serum-free liquid medium, containing between about 5 and about 100 ng / ml, or about 5 ng / ml, or between about 20 and about 100 ng / ml bFGF to obtain a substantially pure clonal population of MSCs.
    [0019] The heterogeneous suspension for use in the method can be obtained, for example, by differentiating pluripotent cells from a primate (eg, human), such as ESCs or induced pluripotent stem cells (iPS), in culture, until the cells in the culture are mesenchymal progenitors. This can be done by co-cultivating pluripotent cells with bone marrow stromal cells in a medium that supports differentiation, as described in this report, for at least two to five days, or dissociation of EBs, which can be obtained by themselves. by culturing pluripotent cells using well-known methods, and then suspending the cells as a single cell suspension. The bone marrow stromal cells can be mouse OP9 cells. A heterogeneous suspension substantially free of some or all non-derived cells through in vitrode differentiation of pluripotent cells (specially co-cultured bone marrow cells) can be obtained by depleting such cells from the suspension. These cells can be depleted from the suspension before use, for example, by non-covalent binding of the cells to be depleted to the paramagnetic monoclonal antibodies specific for the epitopes in the cells to be depleted and then segregation of the cells bound to the antibody with a magnet. Cells in a suspension obtained from pluripotent cells can express at least MIXL1 and T (BRACHYURY).
    [0020] The medium can become semi-solid, including about 1% methylcellulose in the medium. The medium can optionally contain between about 10 and about 20 ng / ml of PDGF-BB. The suspension can be grown for about ten to about twenty days or more to produce colonies.
    [0021] Mesenchymal progenitors are identified by presence in the suspension, if mesenchymal colonies are formed during cultivation in serum-free semi-solid medium supplemented with bFGF. An example of such a bFGF-dependent colony-forming assay to detect the mesenchymal parent is described in US Patent No. 7,615,374, integrally incorporated in this report as a reference. The colonies obtained in the colony-forming assay can be identified as mesenchymal by expressing at least a plurality of FOXF1, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2. Colony characteristics include functional, morphological and phenotypic characteristics and gene expression profile. The functional characteristics of colonies include (1) stimulation of growth through factors that promote mesenchymal cell growth (eg, PDGF-BB, EGF and TGF-alpha) and growth suppression through factors involved in mesodermal differentiation (eg, VEGF, TGF-beta and Activin A); (2) differentiation in osteogenic, chondrogenic or adipogenic cell lines; and (3) differentiation in endothelial cells. The morphological characteristics of the colonies include (1) tight packing of cells to form rounded (i.e., spherical) aggregates measuring 100 to 500 pm in diameter; (2) colony formation through the establishment of tightly packed structures (nuclei) that still develop in compact spheroid colonies; and (3) even after prolonged cultivation, lacking a dense outer cell layer and irregular internal structure, which are characteristic of EBs. Colon phenotype characteristics include (1) expression of CD44, CD56, CD105 and CD140a (PDGFRA), CD146, but not the hematoendothelial surface markers (ie, CD31, CD43, CD45 and VE-cadherin); (2) expression of FOXF1, MSX1, MSX2, SNAI1, SNAI2, SOX9 and FUNX2; and (3) vimentin expression, smooth muscle alpha actin and desmin.
    [0022] The mesenchymal colonies formed in the method can also be cultured in the presence of an extracellular matrix protein, such as Matrigel®, collagen, gelatin or fibronectin, as well as combinations thereof.
    [0023] The invention is further summarized as a substantially pure population of clonally derived MSC strains produced using the methods described above that are positive for at least CD44, CD56, CD 73, CD105, CD140a and CD146, but negative for CD31, CD43, CD45 and VE-cadherin.
    [0024] The described modalities have many advantages, including those in which the mesenchymal progenitors and MSCs obtained in the methods can be used to treat diseases associated with bone, cartilage and fat cells.
    [0025] It is also an advantage that a clonal population of MSCs can be obtained from a single mesenchymal colony.
    [0026] It is also an advantage that the cells obtained in the methods can easily be selected for further expansion, due to the fact that the mesenchymal progenitors have a high potential for proliferation and form large colonies.
    [0027] It is yet another advantage that the cells obtained in the methods can be tolerant or tolerogenic to the allo- and autoimmune response in transplantation.
    [0028] It is yet another advantage that the cells obtained in the methods can differentiate in at least osteogenic, chondrogenic and adipogenic strains.
    [0029] It is yet another advantage that the mesenchymal colonies obtained in the methods have angiogenic potential.
    [0030] The invention is further summarized as an in vitro derived cell population of the lateral plate mesoderm receptor for the Apelin receptor (APLNR +). These cells can be isolated from mixed populations of differentiated pluripotent stem cells based on the expression of the Apelin receptor (APLNR). These cells can differentiate into body wall and viscera cells and give rise to mesangiogenic mesenchymal and hemangiogenic blast colonies in cultures in a semi-solid medium in the presence of bFGF. APLNR + cells express transcripts characteristic of the mesoderm, specifically, the lateral plate meoderm.
    [0031] It is yet another advantage of the invention that MSCs obtained by the claimed methods are of mesodermal origin and can be derived from APLNR + cells enriched in cells of the lateral plate mesoderm.
    [0032] These and other characteristics, aspects and advantages of the present invention will become better understood from the description that follows. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims in this report to interpret the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
    [0033] FIG. 1A to E illustrates the properties of two types of hESC-derived colonies, that is, mesenchymal colonies derived from mesangioblasts (MB) and blast colonies derived from hemangioblasts (HB). FIG. 1A represents MB and HB colony morphologies after growth in semi-solid medium for 3, 5, 7, and 12 days. FIG. 1B represents the kinetics of the formation of FGF-dependent colonies. The bars represent the standard deviation of four independent experiments. Depending on whether hESC-derived single cells are initially co-cultured with OP9 cells for 2 days or 3 days, they assume the potential of MB or HB. FIG. 10 illustrates that bFGF, but not PDGF or VEGF alone, supports the formation of MB and HB colonies. The data are represented as mean ± SD (n = 4). The asterisk indicates statistical significance (p <0.01) between cultures containing FGF alone and FGF in combination with PDGF or VEGF. FIG. 1D illustrates the potential for differentiation of colonies derived from MB and HB after coculture with OP9 cells for 4 days. Flow cytometry demonstrated that cells derived from the MB colony collected on day 12 of the clonogenic culture gave rise to CD146 + CD3r and CD3VCD43- endothelial mesenchymal cells, while cells derived from the HB colony gave rise to CD31 + CD43 'and endothelial cells CD43 + hematopoietic lineage cells. Fig.lE illustrates the immunopigmentation analysis of cell clusters developed from a colony of MB (upper part, scale bar, 100 pm) and HB (lower part, scale bar, 50 pm) collected on the day 5 of the clonogenic culture. The cells were identified as CD144 + (also known as VE-cadherin) CD43 ’endothelial, CD43 + hematopoietic, and calponin + CD144‘ mesenchymal. Scale bars represent 100 pm. Colonies developed from cell clusters of a single MB colony generate mesenchymal cells calponin + CD144 (VE-cadherin) and endothelial cells CD144 (VE-cadherin) + calponin '(upper panel). Colonies developed from cell clusters developed from a single HB colony generate CD43 + hematopoietic and CD144 endothelial cells (VE-cadherin) + CD43 '(bottom panel).
    [0034] FIGS. 2A to C illustrate the microarray analysis of gene expression in hESCs co-cultured with OP9 cells from day 0 (H1) to day 7. FIG. 2A represents a heat map for selected gene sets defining the particular germ layers and their subpopulations and derivatives. FIG. 2B represents the expression of the relative gene of MIXL1, T, SNAI1, F0XF1 and S0X17, as determined by the quantitative POR. FIG. 20 represents the double increase in the number of hESC-derived cells after day 1 to 6 of 0P9 co-culture compared to the previous day. The data are represented as means ± SD (n = 3).
    [0035] FIGS. 3A to D illustrate the analysis of APLNR + cells. FIG. 3A re-graphs flow cytometry result points. FIG. 3B represents the effect of mesoderm formation inhibitors (SB431542 (5 pg / ml) and DKK1 (150 pg / ml)) on the generation of APLNR + cells from H1 cells in OP9 cell cultures. FIG. 3C compares the expression of the transcript between APLNR + and APLNR 'cells. FIG. 3D represents the colony-forming potential of APLNR + and APLNR 'cells.
    [0036] FIGS. 4A to C illustrate the gene expression profiles of APLNR + cells, APLNR 'cells, nuclei, colonies, and a me-senquimal stem cell line (MSC) (in passages p1 and p5) obtained from H1 differentiated hESCs during 2 (D2) or 3 (D3) days through coculture with OP9 cells. FIG. 4A represents heat maps for selected sets of genes defining the indicated germ layers and their subpopulations / derivatives. The nuclei were collected on day 3 of the clonogenic cultures and colonies completely developed on day 12 of the clonogenic cultures. EMT is epithelial-mesenchymal transition. VSMC is vascular smooth muscle cells. FIG. 4B shows the lack of expression of the neuroepithelial marker SOX1 across all stages of differentiation. Embryonic bodies derived from H1 hESCs that differentiated for 14 days were used as a positive control. Fig. 40 illustrates the quantitative RT-PCR analysis of representative transcripts in the indicated cell subsets. The bars represent gene expression in samples grouped from 3 experiments normalized to RPL13.
    [0037] FIG.5 represents a schematic diagram of mesodermal lineage development and differentiation for MSCs from pluipotent stem cells.
    [0038] FIG.6 represents a schematic diagram of the protocol used for the differentiation of hESC, generation of MB colonies, and clonal MSC strains.
    [0039] FIG.7 represents a schematic diagram of the protocol used to assess the differentiation potential of mesenchymal colonies and blasts. DESCRIPTION OF EXEMPLARY MODALITIES
    [0040] Unless otherwise defined, all technical and scientific terms used in this report have the same meanings, as commonly understood by a person of ordinary skill in the technique to which the invention belongs. Although any methods and materials similar or equivalent to those described in this report can be used in the practice or testing of the present invention, the preferred methods and materials are described in this report. It is commonly understood by a person of ordinary skill in the art that "lack of expression" of a gene or the absence of a certain marker in a cell refers to an inability to detect such expression of the gene or marker using methods known in the art at the time of the deposit. It cannot be ruled out that more sensitive methods can detect low levels of expression of such genes or markers.
    [0041] In the description of the modalities and claim of the invention, the following terminology is used, according to the definitions presented below.
    [0042] As used in this report, "about" means within 5% of an established concentration.
    [0043] As used in this report, “clonal” means a population of cells cultured from a single cell, not from an aggregate of cells. The cells in a "clonal population" exhibit a substantially uniform pattern of cell surface markers and morphology and are substantially and genetically identical.
    [0044] As used in this report, an “embryoid body” or an “EB” is an aggregate of cells derived from pluripotent cells, such as ESCs or iPS cells, where cell aggregation can be initiated through coagulation of the drop, placing plate on plates treated with tissue-free culture or spinner flasks (ie, low binding conditions); and any method that prevents cells from adhering to a surface to form typical colony growth. EBs appear as rounded collections of cells and contain cell types derived from all three germ layers (ie, the ectoderm, mesoderm and endoderm). The methods for generating EBs are well known to a person skilled in the art. See, Itskovitz-Eldor J, et al., “Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers”, Mol. Med. 6:88 - 95 (2000); Odorico J, et al., Stem Cells 19: 193 - 204 (2001); and U.S. Patent No. 6,602,711, each of which is incorporated herein in its entirety by reference.
    [0045] As used in this report, “serum free” means that neither the culture nor the culture medium contains serum or plasma, although purified or synthetic components of serum or plasma (eg FGFs) may be provided in the culture in reproducible quantities, as described below.
    [0046] As used in this report, a "substantially pure population" means a population of derived cells that contains at least 99% of the desired cell type. Cell purification can be performed by any means known to a person of ordinary skill in the art. For example, a substantially pure population of cells can be obtained by growing cells or selecting a less pure population, as described in this report.
    [0047] As used in this report, “pluripotent cells” means a population of cells capable of differentiating into all three germ layers and becoming any cell type in the body. Pluripotent cells express a variety of cell surface markers, have a cell morphology characteristic of non-differentiated cells, and form teratomas when introduced into an immunocompromised animal, such as a SCID mouse. Tera-tomas typically contain cells or tissues characteristic of all three germ layers.
    [0048] As used in this report, “multipotent” cells are more differentiated than pluripotent cells, but are not permanently compromised to a specific cell type. Pluripotent cells, therefore, have a greater potency than multipotent cells.
    [0049] As used in this report, “induced pluripotent stem cells” or “iPS cells” are cells that are differentiated somatic cells reprogrammed as to pluripotency. The cells are substantially and genetically identical to their respective differentiated somatic cell of origin and exhibit characteristics similar to cells of higher potency, such as ES cells. See, Yu J, et al., “Induced pluripotent stem cell lines derived from human somatic cells”, Science 318: 1917 - 1920 (2007), incorporated in this report as a reference.
    [0050] As used in this report, a "mesenchymal stem cell" (MSC) is a cell capable of differentiating into skeletal cell lines (ie, osteoblasts, chondroblasts and adipocytes). As noted above, no single MSC marker was identified. As such, morphological and functional criteria well known to those of ordinary skill in the art are used to identify these cells. See, Horwitz et al., Supra; Dominici et al., Supra; Trivedi P & Hematti P, “Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells”, Exp. Hematol. Jan. 5, 2008 [Epub ahead of print]; Trivedi P & Hematti P, “Simultaneous generation of CD34 + primitive hematopoietic cells and CD56 + mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells”, Exp. Hematol. 35: 146 - 154 (2007); and U.S. Published Patent Application No. 2006/0008902, each of which is incorporated herein in its entirety by reference. The MSCs produced by the methods described in this report can be characterized, according to the phenotypic criteria. For example, MSCs can be recognized for their mononuclear ovoid, star-shaped or axis-shaped features, with a round to oval core. The elongated oval nuclei typically have prominent nucleoli and a mixture of hetero and eukromatin. These cells have little cytoplasm, but many fine processes that seem to extend from the nucleus. MSCs are typically believed to be pigmented by one, two, three or more between the following markers: CD106 (VCAM), CD73, CD146, CD166 (ALCAM), CD29, CD44 and alkaline phosphatase, although they are negative for markers hematopoietic cell lines (for example, CD14 or CD45) and endothelial cell line markers (for example, CD31 and VE-cadherin). MSCs can also express STRO-1 as a marker.
    [0051] As used in this report, a "mesangioblast" is a parent to MSCs, as well as endothelial cells.
    [0052] As used in this report, a “mesenchymal colony” is a colony made up of mesenchymal cells that originate from mesangioblasts.
    [0053] As used in this report, a "hemangioblast" is a precursor to blood cells, as well as endothelial cells.
    [0054] As used in this report, a “blast colony” is a colony made up of predominantly hematopoietic cells that originate from hemangioblasts.
    [0055] As used in this report, “mesendoderm” is a tissue that gives rise to the mesoderm and endoderm.
    [0056] As used in this report, “mesoderm” is a cell subset that expresses KDFl and PDGFRa at a much higher level than POU4F1, SOX1, and PAX6 (neural crest and neuroectoderm), LAMA3, KRT14, and KRT10 (surface ectoderm) , CGA and PLAC1 (tropectoderm) F0XA1, F0XA2, APOA1, TMPRSS2, TTR1, and AFP (endoderm) and SOX2e DPPA2 (non-differentiated hESCs).
    [0057] As used in this report, “side plate mesoderm” is a subset of mesoderm that expresses at least F0XF1 and HAND1, but lacking the expression of ME0X1 and TCF15 (paraxial mesoderm), PAX2 and PAX8 (intermediate mesoderm), and is capable of at least endothelial and hematopoietic differentiation.
    [0058] It is considered that Matrigel®, laminin, collagen (especially type I collagen), fibronectin and glycosaminoglycans may be suitable as an extracellular matrix, alone, or in various combinations.
    [0059] The invention will be more fully understood under consideration of the following non-limiting Examples. EXAMPLES Example 1: Generation of MSCs from pluripotent stem cells under serum-free conditions.
    [0060] HESCs (H1; WiCell; Madison, Wl) were maintained in mouse embryonic fibroblasts irradiated in serum-free medium, such as DMEM / F12 medium supplemented with 20% Knockout ™ serum substitute, L-glutamine 2 mM, 1x non-essential amino acids (100 pM), 100 pM 2-mercaptoethanol and 4 ng / ml bFGF (all by Gibco-lnvitrogen; Carlsbad, CA). See, Amit M, et al., “Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture”, Dev. Biol. 227: 271 - 278 (2000), incorporated in this report as a reference. The mouse 0P9 bone marrow stromal cells (kindly supplied by Dr. Toru Nakano and available from ATCC, catalog # CRL-2749) were maintained in a four-day subculture on gelatin-coated plates in alpha MEM medium (Gibco-lnvitrogen ) with 20% fetal calf serum (FCS; HyClone; Logan, UT).
    [0061] HESCs were induced to differentiate themselves through coculture with stromal cells from the mouse bone marrow OP9, as previously described. Vodyanik M, et al., “Human embryonic stem cell-derived CD34 + cells: efficient production in the coculture with OP9 stromal cells and analysis of lympho- hematopoietic potential”, Blood 105: 617 - 626 (2005), fully incorporated in this report as reference. Briefly, small hESCs aggregates were added to OP9 cells in alpha MEM supplemented with 10% FCS and 100 pM MTG (Sigma; St. Louis, MO). On the next day (day 1) of culture, the medium was modified, and the cultures were collected on the days indicated below.
    [0062] On day two of the co-culture of hESC (H1) with OP9 stromal cells, the peak expression of transcription factors for the primitive line population (me- sendoderm) (GSC, EOMES, MIXL1 and T (BRACHYURY)) and Early mesoderm (EVX1, LHX1 and TBX6) was detected with NimbleGen® microarrays (Madison, Wl).
    [0063] On days 3 to 5 of coculture, the culture contained the mesenchymal progenitors, as well as the cells that express characteristic genes of the endoderm and mesoderm. Among the characteristic genes for mesoderm, only the characteristic genes for the side plate mesoderm, such as FOXF1, HAND1, NKX2-5 and GATA2, have been consistently expressed. On the contrary, the characteristic genes for the axial (CHRD, SHH), paraxial (MEOX1, TCF15) or intermediate (PAX2, PAX8) mesoderm have not been consistently expressed. Thus, hESCs co-cultured with OP9 cells for 3 to 5 days gave rise to cells that express genes characteristic of the lateral / extraembryonic plaque mesoderm. On days 3 to 5 of the hESC (H1) / OP9 coculture, cells were also characterized by maximum cell proliferation and sustained expression of genes involved in the epithelial-mesenchymal transition (EMT, SNAI1, and SNAI2) and cell expansion (H0XB2 , H0XB3).
    [0064] On days 5 to 7 of the hESC (H1) / OP9 coculture, differentiation in specific mesodermal and endodermal lines was observed, when the markers of development of endoderm cells (AFPe SERPINA1), me-senquimals (S0X9, RUNX2, and PPARG2) and hematoendothelial (CDH5 and GATA1) were detected. Neither the muscle-inducing factors (MY0D1, MYF5, and MYF6) nor the neuroectoderm markers (SOX1, PAX6 and NEFL) or tropectoderm (CGB and PLAC) were expressed during the seven days of coculture, indicating that OP9 cells provided a effective inductive environment for the differentiation of hESC directed to the mesendodermal pathway.
    [0065] On day 2 of the hESC (H1) / OP9 coculture, a single cell suspension was collected from the coculture by successive enzymatic treatment with collagenase IV (Gibco-lnvitrogen) at 1 mg / ml in DMEM / F12 medium during 15 minutes at 37 ° C and 0.05% Trypsin-0.5 mM EDTA (Gibco-lnvitrogen) for 10 minutes at 37 ° C. The cells were washed 3 times with 5% PBS-FBS, filtered through 70 pM and 30 pM cell sieves (BD Labware; Bedford, MA) and stained with anti-mouse CD29-PE (AbD Serotec; Raleigh, NO) and paramagnetic anti-PE monoclonal antibodies (Miltenyi Biotec; Auburn, CA). The cell suspension was purified with a magnet activated cell separator (MACS) passing it through an LD magnetic column connected to a Midi-MACS separation unit (Miltenyi Biotech) to obtain a negative fraction of hESC-derived cells depleted in OP9. Purity was verified using a mixture of anti-human TRA-1- 85 monoclonal antibodies (R&D Systems; Mineápolis, MN).
    [0066] The purified single cell suspension was plated at a density of 0.5 to 2 x 104 cells / ml in a semi-solid serum-free medium composed of StemLine ™ serum-free medium (Sigma; St. Louis, MO) supplemented with 5 to 100 ng / ml bFGF (PeproTech; Rocky Hill, NJ) and 1% methylcellulose (Stem Cell Technologies; Vancouver, Canada) with or without 10 to 20 ng / ml PDGF-BB (PeproTech). PDGF-BB improved the growth of mesenchymal cells, however it was not essential for the formation of colonies. Alternatively, single cell suspensions were plated in a semi-solid colony-forming serum-free medium containing 40% ES-Cult M3120 methylcellulose, 25% serum-free expansion medium (SFEM, Stem Cell Technologies), endothelial serum-free medium 25% (E-SFM, Invotrogen), 10% BIT 9500 (Stem Cell Technologies), GlutaMAX (diluted 1: 100), Ex-Cyte (diluted 1: 1000, Millipore), 100 pM monothioglycerol (MTG), acid ascorbic 50 pg / ml and bFGF 20 ng / ml.
    [0067] After 10 to 20 days of culture, large and compact mesenchymal colonies formed such similar embryonic bodies (EBs). Although these mesenchymal colonies were detected as early as possible, 7, 10 to 20 days of culture were required to reveal actively growing colonies. Non-differentiated hESCs or cells collected on day 1 or 6 of the coculture do not form these mesenchymal colonies when grown under the same conditions.
    [0068] Mesenchymal colonies, which resembled embryonic bodies, were distinguished from EBs through several characteristics: (1) formation and growth under serum-free conditions supplemented with bFGF and stimulation through factors promoting mesenchymal cell growth ( for example, PDGF-BB, EGF and TGF-a), but suppression through factors involved in mesodermal differentiation (for example, VEGF, TGF-β and Activin A) in me-senquimal colonies; (2) lack of a dense outer cell layer and irregular cavity structure characteristic of EBs, even after prolonged cultivation in mesenchymal colonies; (3) presence of morphological homogeneity in cells including mesenchymal colonies; and (4) colony formation through the establishment of tightly packed structures (nuclei) that still develop in compact spheroid colonies.
    [0069] To demonstrate that single cell suspensions do not form aggregates under plating in a semi-solid medium, the clonality of the mesenchymal colonies obtained in the culture methods was tested and confirmed using chimeric hESC lines established from retroviral cells markedly marked with a reporter gene, for example, enhanced fluorescent green protein (EGFP) or histone 2B- (H2BB) mOrange fluorescent protein. The expression of a reporter gene product indicated clonality. The chimeric hESC strains were generated from two lentiviral constructs: (1) the EGFP protein constitutively expressed from an elongation factor 1 alpha (EFIalfa) promoter, and (2) the H2BB-mOrange protein constitutively expressed from of the EFIalfa promoter. The two constructs were packaged in 293FT cells, and lentiviruses were used to transfer H1 hESCs to produce stable H1 hESC strains that expressed green EGFP protein or orange H2BB-mOrange protein. The mesenchymal colonies derived from the described methods were single colors, green or orange, thus indicating the clonal origin (ie, single cell) of the MSCs.
    [0070] In addition, the expected phenotypic analysis demonstrated a positive correlation between mesenchymal colony-forming cell frequency (CFC) and KDR expression (VEGFR2), through the KDRh'9hCD34 + population of the earliest hemangiogenic precursors was devoid of of mesenchymal CFCs. Analysis of cells within the mesenchymal colonies revealed a homogeneous population of early mesenchymal cells defined by high expression of CD90, CD140a and CD166, low CD44, CD56 and CD105 expression and lack of CD24, CD31, CD43, CD45, CD144 ( VE-cadherin), and lack of expression of SSEA4. In addition, mesenchymal colonies expressed vimentin, smooth muscle alpha actin and desmin. In addition, mesenchymal colonies expressed specific genes for the strain of MSC, such as FOXF1, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2.
    [0071] The individual mesenchymal colonies were transferred to the wells of a 96-well plate coated with collagen or fibronectin, pre-filled with 0.2 ml / well of serum-free medium StemLine ™ supplemented with 5 to 100 ng / ml of bFGF or serum-free expansion medium consisting of 50% StemLine II serum-free HSC expansion medium (H-SFEM, Sigma) and 50% E-SFM supplemented with GlutaMAX (diluted 1: 100), ExCyte (diluted 1: 2000), 100 pM MTG and 10 ng / ml bFGF. After 3 to 4 days of culture, adherent cells from individual wells were collected through trypsin treatment and expanded into plates coated with collagen or fibronectin in serum-free medium StemLine ™ with 5 to 100 ng / ml of bFGF or serum-free expansion medium (M-SFEM) containing 50% StemLine ™ II serum-free HSC expansion medium (HSFEM; Sigma), 50% E-SFM, GlutaMAX ™ (1/100 dilution), Ex supplement -Cyte® (1/2000 dilution), 100 pM MTG and 5 to 100 ng bFGF.
    [0072] MSCs have been expanded to many passages. When the individual colonies were plated on plates coated with collagen or fibronectin, immediate binding and vigorous growth of fibroblast-type cells were observed. During subsequent passages, cells grew intensely during the first 10 passages; however, the growth rate was attenuated in passages 10 to 15 and gradual senescence was observed during passages 15 to 20. Cultures derived from single MB-CFC accumulated up to 1022 total cells in the observed period of time. Since it is assumed that each colony originated from a single cell, the number corresponds to the expansion potential of a single hESC-derived mesenchymal precursor.
    [0073] Cell lines established from individual colonies were maintained in serum-free medium with bFGF for 10 to 15 passages at a high proliferation rate. All cell lines exhibited a mesenchymal phenotype, characterized by the expression of CD44, CD56, CD73, CD105, CD146 and CD140a (PDGFRA) and are devoid of hematoendothelial markers (ie, CD31, CD43, CD45 and VE-cadherin). When tested under conditions that reveal the potential for mesenchymal differentiation, cell lines were capable of osteogenic, chondrogenic and adipogenic differentiation. Interestingly, these cells resembled bone marrow MSCs, but they expanded and proliferated better than bone marrow MSCs. These expanded mesenchymal cells can be differentiated into cells of the chondro-, osteo- and adi-pogenic lineage. However, these cells cannot give rise to hematopoietic or endothelial cells when grown with OP9 cells, or when grown in feeder-free cultures with hematoendothelial growth factors (VEGF, bFGF, SCF, TPO, IL3, IL6), indicating a limited differentiation potential of these mesenchymal cells.
    [0074] Mesenchymal colonies were also generated from several induced pluripotent stem cells (iPS), such as iPS (IMR90) -1, iPS (SK) -46, and iPS (FSK) -1 reprogrammed using a lentiviral vector ( Yu et al., Science 318: 1917 - 1920 (2007)), or iPS-5 4-3-7T and iPS-1 19-9-7T transgene-free (Yu et al., Science 324: 797 - 801 ( 2009)). Mesenchymal colonies derived from iPS cells containing transgene exhibited irregular or looser morphology. Transgene-free iPSC produced typical spheroid mesenchymal colonies. Example 2. In vitro generation and characterization of mesangioblasts.
    [0075] To isolate and characterize a population of mesodermal progenitors that can give rise to the mesodermal cell line with hematopoietic, endothelial and mesenchymal stem cell potentials, H1 hES cells were co-cultured with OP9 cells, as described in Example 1. Then two or three days of coculture, when genes representative of the primitive population (mesendoderm) (MIXL1, T, EOMES) were expressed, cells derived from hESC depleted of OP9 cells using anti-mouse CD29 antibody were plated in semi-solid serum-free medium, essentially, as described in Example 1, with 20 ng / ml bFGF (PeproTech; Rocky Hill, NJ). The number of colony-forming cells (CFCs) was calculated per 1000 H1-derived TRA-1-85 + cells plated.
    [0076] After 2 to 3 days in a semi-solid medium, the cells formed tightly packed structures (nuclei). The nuclei derived from hESCs that were differentiated in coculture with OP9 cells for 2 days still grew in spheroid mesenchymal colonies. The nuclei derived from hESCs that were differentiated in the coculture with OP9 cells for 3 days still grew in dispersed blast colonies with hematopoietic and endothelial potential.
    [0077] The bFGF is necessary and sufficient for the formation of the two colonies from the hESCs. The bFGF supported the formation of mesenchymal colony and blasts. In contrast, in the absence of bFGF, neither VEGF, nor PDGF-BB (FIG. 1A), SCF, IGF1, or HGF (data not shown), alone or in combination, supported the formation of any colony. Although PDGF-BB (10 ng / ml) alone does not support colony formation, PDGF-BB in combination with bFGF significantly increased the yield and size of mesenchymal colonies compared to bFGF alone (FIG. 1A). VEGF alone (20 ng / ml) did not support colony formation, however its addition to bFGF cultures slightly increased the number of blast colonies, but inhibited the formation of mesenchymal colonies (FIG. 1A). The cells that gave rise to each type of colony constituted approximately 2 to 3% of total cells derived from hESC (FIG. 1B).
    [0078] To determine whether cells within the mesenchymal colonies could give rise to hematovascular cells, the individual me-senquimal colonies were selected from methylcellulose on days 5 to 7 and plated in 0P9 cells in alpha-MEM medium with 10% FBS, and the cytokines SCF (50 ng / ml), TPO (50 ng / ml), IL-3 (10 ng / ml), and IL-6 (20 ng / ml). After 4 days of culture, cells were collected and analyzed by flow cytometry or pigmented in situ with rabbit anti-human CD144 (VE-cadherin; 1 pg / ml; eBioscience, San Diego, CA) in combination with anti CD43 - mouse human (0.5 pg / ml; BD Bioscience) or primary mouse anti-human Calponin antibodies (0.5 pg / ml; Thermo Fisher Scientific), followed by a mixture of anti-mouse IgG-DyLight 594 antibodies donkey cross-absorbed and IgG-DyLight-488 donkey anti-rabbit (both at 2 pg / ml; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
    [0079] Mesenchymal colonies originated from precursors that gave origin to endothelial and mesenchymal cells, ie mesangioblasts. As explained in Example 1, MSCs that expanded from mesenchymal colonies in adherent cultures did not give rise to hematopoietic or endothelial cells when co-cultured with OP9 cells. In contrast, approximately 70% of mesenchymal colonies isolated from day 5 to 7 of colony-forming cultures in a semi-solid medium gave rise to endothelial cells CD31 + CD144 (VE-cadherin) + when co-cultured with OP9 cells. (FIG. 1D and E, upper panels). Mesenchymal colonies, therefore, originated from common precursors for endothelial and mesenchymal lines, that is, mesangioblasts. In contrast, blast colonies contained CD31 + CD43 + hematopoietic cells and can give rise to endothelial cells (FIG. 1 D and E, lower panels).
    [0080] The endothelial potential of mesenchymal colonies can be significantly enhanced with the addition of bone morphogenetic protein 4 (BMP4) to the clonogenic assay medium (3.2 ± 2.4% of CD31 + CD43_ cells without BMP4 vs. 11 , 6 ± 0.5 with 5 ng / ml of BMP4). Example 3: Generation and isolation of a population of cells substantially enriched in lateral / extraembryonic mesodermal cells.
    [0081] The genetic profile of H1 hESCs differentiated in OP9 cocultures demonstrated selective impairment for mesodermal and endodermal lines without detectable ectoderm (tropho-, neuro-, or surface ectoderm) (FIG. 2). The cells become compromised to the mesendoderm on day 2 of culture, when the simultaneous expression of primitive line genes (MIXL1, T and EOMES) was detected. In the subsequent days of the culture, specific genes from the mesoderm and endoderm and, eventually, specific genes derived from the endoderm and mesoderm, were expressed. Among the mesodermal genes, those characteristic of the lateral / extraembryonic mesodermal subset (FOXF1, HAND1, NKX2-5 GATA2) were systematically expressed, while the expression of genes from the axial (CHRD, SHH), paraxial (MEOX1, TCF15) or intermediates (PAX2, PAX8) was not compatible. The expression of the Apelin receptor (APLNR) is strongly induced and overloaded on days 2 to 3 of differentiation, concurrently, with mesodermal impairment.
    [0082] To characterize the expression of APLNR and cells expressing it, differentiated hESCs in OP9 cocultures were pigmented with monoclonal antibodies specific for the Apelin receptor (APLNR) (R&D Systems) in combination with the antibodies against CD30 , KDR, PDGFRA, T and FOXA2. Non-differentiated hESCs and hESC-derived cells on day 1 of OP9 coculture were negative for APLNR (FIG. 3A, Day 1 panels). APLNR expression was strongly overloaded in cells co-cultured with OP9 cells for 2 to 3 days (FIG. 3A, Day 2 and 3). On day 2, 15 to 20% of the cells were APLNR + and on day 3, 60 to 70% of cells were APLNR +. This overload coincided with the mesodermal compromise, as evidenced by the overload of mesodermal markers, such as KDR (VEGFR2), T and PDGFRA (FIG. 3A, panels 2 and 3). The number of APLNR + cells gradually decreased in the subsequent days (FIG. 3B). Conversely, hESC markers (for example, CD30) were successively unregulated.
    [0083] Although PDGFRA is expressed only at low levels on day 2 of the cocultures, APLNR is expressed in high density as early as possible on day 2 of the coculture allowing the separation of positive APLNR cells from negative APLNR cells. On days 2, 2.5 and 3 of H1 / OP9 cell coculture, the APLNR + and APLNR- cells were separated by a magnetic separator and the gene expression was analyzed by microarray analysis.
    [0084] MIXL1, T and EOMES, indicative of primitive line cells (mesoderm), were expressed in APLNR + cells, while the transcripts associated with neural crest / neuroectoderm (POU4F1, SOX1, SOX2, SOX3, SOX10) did not can be detected (FIG. 4A and 4B). As expected, APLNR + cells were enriched in transcripts specific to the TCF21 mesoderm, whereas transcripts labeling the pan-endoderm (FOXA2, APOA1), definitive endoderm (FOXA1, TMPRSS2) and visceral (TTR, AFP) were discovered in cells APLNR- (FIG. 4AeC).
    [0085] Interestingly, the APLNR + cells expressed F0XF1, IRX3, BMP4, WNT5A, NKX2.5, HAND1 and HAND2 representative of the lateral / extraembryonic mesoderm plate, but no paraxal / myogenic mesoderm marker (ME0X1, TCF15, PAX3, PAX7) and intermediate (PAX2, PAX8) in the embryo. These data indicate that instead of being a total population of cells committed to mesendodermal development, APLNR + cells represent the mesoderm, or probably its subpopulation similar to the lateral / extraembryonic plate mesoderm (FIG. 3C and FIG. 4).
    [0086] To further confirm the mesodermal identity, APLNR + cells were analyzed for expression of T, an early mesoderm marker, and FOXA2, an endoderm marker. As shown in FIG. 3A, APLNR + cells are T + and maintain T expression, until it decreases on day 4. In contrast, FOXA2 + cells, which comprised less than 5% of total cells in the culture, do not express APLNR. Thus, APLNR + cells are mesodermal precursors T + FOXA2- on day 2 to 3 of the culture.
    [0087] To further support the notion that APLNR + cells are mesodermal precursors, H1 / OP9 cell cultures were supplemented with mesoderm formation inhibitors SB431542 (5 pg / ml) or DKK1 (150 pg / ml) . APLNR + cells cannot be detected in cultures that have received mesoderm formation inhibitors (FIG. 3B), confirming that APLNR + cells are mesodermal. In addition, the potential for forming mesenchymal colonies and blasts was discovered exclusively within the APLNR + cell population (FIG. 3D), further confirming that mesangiogenic mesenchymal and hemangiogenic colonies are formed by APLNR + mesodermal precursors. Example 4. Enrichment of hESCs-derived mesanqioblasts under serum-free conditions by isolating APLNR + side plate / extraembryonic mesoderm cells.
    [0088] To identify the origin of mesenchymal colonies and obtain a population of cells enriched in mesangioblasts, pluripotent stem cells were co-cultured with OP9 for 2 to 3 days to induce the formation of meso-dermis. After depletion of OP9 cells with mouse-specific CD29 antibodies, APLNR + and APLNR-cells were isolated using a magnetic separator. Colony formation assays in a semi-solid medium in the presence of bFGF demonstrated that the potential of mesangioblasts and hemangioblasts was restricted only to the APLNR + fraction (FIG. 3D). Approximately 1 to 5% of cells within the APLNR + fraction showed mesangioblast activity.
    [0089] The invention has been described in combination with those that are now considered more practical and preferred modalities. However, the present invention has been presented by way of illustration and is not intended to be limited to the disclosed modalities. Consequently, those skilled in the art will understand that the invention is intended to cover all modifications and alternative arrangements within the spirit and scope of the invention, as presented in the attached claims.
    权利要求:
    Claims (9)
    [0001]
    1. Method to generate primate mesoderm cells that express Apelin's receptor (APLNR +), the method CHARACTERIZED by the fact that it comprises the steps of: cultivating pluripotent primate stem cells in co-culture with stromal cells bone marrow OP9 for at least 2 days until the Apelin receptor and the alpha-type receptor for platelet-derived growth factor (PDGFRA) are expressed and detectable in the co-cultured cells; and separating co-cultured cells expressing the Apelin receptor and PDGFRA from cells in the co-culture that do not express the Apelin receptor.
    [0002]
    2. Method, according to claim 1, CHARACTERIZED by the fact that the primate pluripotent stem cells are co-cultured with the OP9 bone marrow stromal cells for two to three days.
    [0003]
    3. Method, according to claim 1, CHARACTERIZED by the fact that 1% to 5% of the selected APLNR + cells are able to differentiate in culture in mesenchymal stem cells and endothelial cells.
    [0004]
    4. Method to generate mesangioblasts that are precursors of common me-senquimal and endothelial cells, the method CHARACTERIZED by the fact that it comprises the steps of: cultivating pluripotent primate stem cells in co-culture with OP9 bone marrow stromal cells by at least 2 days until the Apelin receptor and PDGFRA are expressed and detectable in the co-cultured cells; separating co-cultured cells expressing Apelin receptor and PDGFRA from cells in the co-culture that do not express Apelin receptor; and culturing the Apelin + / PDGFRA + receptor cells in a serum-free semi-solid culture medium comprising bFGF for 2 days until a subset differentiation to form mesangioblast colonies.
    [0005]
    5. Method, according to claim 4, CHARACTERIZED by the fact that it further comprises cultivating mesangioblasts in serum-free culture medium comprising bFGF until the mesangioblasts are differentiated in endo-telial cells.
    [0006]
    6. Method, according to claim 4, CHARACTERIZED by the fact that the semi-solid culture medium without serum also comprises bone morphogenetic protein 4 (BMP4).
    [0007]
    7. Method, according to claim 4, CHARACTERIZED by the fact that it also comprises the step of: cultivating mesangioblasts in the presence of bFGF until mesangioblasts are differentiated into mesenchymal stem cells.
    [0008]
    8. Method, according to claim 1, CHARACTERIZED by the fact that Apelin + / PDGFRA + receptor cells still express the kinase domain (KDR) region.
    [0009]
    9. Method, according to claim 4, CHARACTERIZED by the fact that Apelin + / PDGFRA + receptor cells still express KDR.
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    同族专利:
    公开号 | 公开日
    AU2011227274A1|2012-10-04|
    US20150175971A1|2015-06-25|
    US20100261274A1|2010-10-14|
    CA2793380A1|2011-09-22|
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    AU2011227274B2|2014-10-16|
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    WO2011116117A3|2011-11-10|
    MX2012010721A|2012-10-05|
    WO2011116117A2|2011-09-22|
    EP2547763B1|2018-08-22|
    BR112012023537A2|2017-05-23|
    JP5944885B2|2016-07-05|
    US9771561B2|2017-09-26|
    DK2547763T3|2018-11-12|
    US20110236971A2|2011-09-29|
    JP2013521806A|2013-06-13|
    CA2793380C|2020-01-21|
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    申请号 | 申请日 | 专利标题
    US12/726,814|US20110236971A2|2007-09-25|2010-03-18|Generation of Clonal Mesenchymal Progenitors and Mesenchymal Stem Cell Lines Under Serum-Free Conditions|
    US12/726,814|2010-03-18|
    PCT/US2011/028700|WO2011116117A2|2010-03-18|2011-03-16|Generation of clonal mesenchymal progenitors and mesenchymal stem cell lines under serum-free conditions|
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