HK1140795A - Early mesoderm cells, a stable population of mesendoderm cells that has utility for generation of endoderm and mesoderm lineages and multipotent migratory cells (mmc) - Google Patents

Description

Early mesodermal cells, a stable population of mesendodermal cells, for use in the production of endodermal and mesodermal cell lines and Multipotent Migratory Cells (MMCs)

Technical Field

The present invention relates to novel methods for the generation of early mesodermal cells from primate pluripotent stem cells (pPSCs), including in particular human embryonic stem cells (hESCs), and for the differentiation of mesodermal cells. These methods can generate key pluripotent precursors with the potential to form cardiomyocyte, smooth muscle cell and endothelial cell lines. These methods can be used for all human embryonic stem cell (hESC) lines including BG01 and BG 02.

The invention also relates to the production of stable pluripotent migratory cells (MMC) capable of differentiating into mesodermal or endodermal cell lineages. MMCs can be passaged at least 20 times (possibly indefinitely), and can be recovered after freezing, reamplified, and differentiated into a variety of cell lines. The methods for generating these cells are directed to the generation of pluripotent cell types (MMCs) from blastocysts (blastocysts) to generate therapeutically useful cell types without going through typical hESC states.

The invention also relates to methods of producing mesendoderm cells from primate pluripotent stem cells (pPSCs), in particular hESCs, methods of producing defined definitive endoderm cells, methods of producing pluripotent migratory cells (MMCs), methods of producing mesoderm precursor cells (IMPs) and cell therapy for cardiovascular diseases.

Priority of prior application US60/898,204 filed on 30/1/2007 and prior application US60/994,354 filed on 19/9/2007, both of which are hereby incorporated by reference in their entireties.

Background

Human embryonic stem cells (hESCs) (markers for hESCs include SSEA3, SSEA4, TRA-1-60 antigen, TRA-1-81 antigen, Nanog, Oct4) are a pluripotent population of cells that can differentiate into cells derived from all three germ and extra-embryonic cell lines. This property of hescs makes them of great interest in cell therapy (e.g., diabetes, heart disease, neurodegenerative diseases), drug development, and development modeling.

Other pluripotent cell types have been identified in mice. Primitive ectoderm-like (EPL; Rathjen et al, 1999, J.CelSci) cells are formed from mESCs and have the ability to dedifferentiate into mESCs. Recently, a new mouse cell, post-implantation ectodermal stem cell (EpiSC; Tesar et al, Nature 448: 196-202; 2007), which has the same properties as hESCs (Nanog +, Sox2+, Oct4+) was identified. All of these mouse pluripotent cell types were able to generate three embryonic stem cell layers in vitro or in teratoma assays.

Ectodermal stem cells (EpiScs) and induced pluripotent stem cells (iPS) conform to a broad pluripotent cell class, and conceptually, the techniques described herein can be applied to these and other pluripotent cell types (i.e., primate pluripotent cells). EpiSc ectodermal stem cells were isolated from embryos early after implantation, expressing Oct4 and having pluripotency (Tesar et al, Nature, VoI. p.19612 July 2007). Adult skin fibroblasts, or other adult cells, are dedifferentiated by retroviral transduction of 4 genes (c-myc, Klf4, Sox2, Oct4) to a pluripotent state to form induced pluripotent stem cells (iPS cells) (Takahashi and Yamanaka, Cell 126, 663-.

The development of other non-ESC self-renewing pluripotent stem cells would help improve the developmental model, promote directed differentiation into adult cells, and be more efficient and economical than traditional methods.

Drawings

FIG. 1: bright field pictures (bright field pictures) of BG02hESCs cells grown on matrigel (matrigel) in defined medium at 10 ×,20 ×, 40 ×.

FIG. 2: schematic representations of possible hESC cell fates of ectodermal, mesodermal, endodermal and extraembryonic cell lines are shown.

FIG. 3: a schematic of the differentiation pathway leading to the formation of T + mesendoderm cells, which can further differentiate into mesoderm cells (Meso) or definitive endoderm cells (DE), is shown.

FIG. 4: shows the formation of mesendoderm cells in defined media with the addition of Wnt3a to BG01 hESCs. (A) Q-PCR analysis of Nanog, T, Eomes and MixL1 transcripts over a period of 3 days after addition of Wnt3a (25 ng/ml). (B) Immunostaining of cells when treated with Wnt3a (25ng/ml) for 2 days (48 hours) and the panel shows staining of E-cadherin (cadherin), Nanog, T, β -catenin (catenin) and Snail in untreated (hESCs) and treated (+ Wnt3a) samples.

FIG. 5: a model of mesendoderm cell formation in the presence of canonical Wnt signaling and the absence of TGF β signaling is shown.

FIG. 6: formation of mesendoderm cells after 48 hours treatment with BIO for BG02hESCs grown on matrigel in defined media. Immunostaining showed staining for T, Nanog, E-cadherin and Snail in untreated hESCs and BIO-treated cells. DNA was stained with DAPI. (B) Q-PCR analysis of Nanog, T, MixL1 transcripts after treating hESCs with BIO for 48 hours.

FIG. 7: pancreatin-passaged BG01hESCs grown on matrigel in MEF-CM were treated with BIO (2. mu.M) for 4 days. Cells were immunostained with (A) T probe and (B) E-cadherin, Oct4 probe. The merged image is also shown. DAPI was used to detect DNA.

FIG. 8: collagenase passaged BG01hESCs grown on matrigel in MEF-CM was treated with BIO (2. mu.M) for 4 days. Cells were immunostained with (A) E-cadherin probe and (B) T and Nanog probes. The merged image is also shown. DAPI was used to detect DNA.

FIG. 9: formation of mesendoderm cells in the presence of BIO and SB 431542. Q-PCR analysis of hESCs cells treated with BIO (2. mu.M) and SB431542 (20. mu.M) for 8 days. The signal levels for Nanog, T, Sox17, CXCR4, FoxF1 and PDGFR α are shown.

FIG. 10: a schematic representation of the formation of mesodermal cells by the mesendoderm intermediate (mesendoderm intermediate) following treatment of hESCs with BMP4 and Wnt3a/BIO is shown.

FIG. 11: hESCs differentiated into Isl1+ pluripotent progenitor cells (IMP) 10 days after treatment with Wnt3a (25ng/ml) and BMP4(100 ng/ml). Transcript analyses of T, Sox17, PDGFR α, KDR, Isl1, Tbx20, GATA4, VE-cadherin and cTNT are shown.

FIG. 12: hESCs undergo a transition in the T + state after 144 hours of treatment with Wnt3a (25ng/ml) and BMP4(100 ng/ml). Immunostaining for T is shown. DNA is represented by DAPI. The representative hierarchical stains of DAPI/T were pooled (staining).

FIG. 13: hESCs differentiated to the Isl1+ state after 144 hours of treatment with Wnt3a (25ng/ml) and BMP4(100 ng/ml). Immunostaining with Isl1, Nanog, Nkx2.5 and Tbx20 is shown. DNA is represented by DAPI. Pooled representations were DAPI stained using Nanog/Isl1 or Nkx2.5/Tbx 20.

FIG. 14: brightfield patterns of hESCs (BG02) and cells treated with Wnt3a (25ng/ml), BMP4(100ng/ml) at the indicated time points. The image magnification is indicated.

FIG. 15: schematic representation of the pathway of hESCs differentiation to mesendoderm cells (MesEnd) and subsequently mesoderm cells (Meso). The first step involved treatment with Wnt3a/BIO for 1-3 days, followed by additional treatment with BMP4 for 2-4 days.

FIG. 16: brightfield images of 5 day hESCs treated with BIO (2. mu.M) and BMP4(100 ng/ml). In this case, hESCs were grown on matrigel in MEF-CM. The magnification is indicated in the figure.

FIG. 17: production of Isl1+ pluripotent progenitor cells (IMPs) 6 days after treatment of hESCs (BG02) with BIO (2. mu.M) and BMP4(100 ng/ml). Q-PCR analysis showed transcript levels of Oct4, Nanog, Lefty A, T, MixL1, Goosecoid, Sox17, CXCR4, FoxF1, PDGFR α, PDGFR β, GATA4, Tbx20 and Isl1 during this period.

FIG. 18: a schematic showing the pathway of hescs differentiation into mesendoderm cells and subsequently into mesoderm cells. hESCs differentiated into mesendoderm cells in the presence of TGF β inhibitors (e.g., SB431542), and Wnt3a/BIO and BMP4(1-4 days). Shows that mesendoderm cells differentiate into mesoderm cells in the presence of Wnt3a/BIO and BMP42-4 days.

FIG. 19: a schematic showing the pathway of hescs differentiation into mesendoderm cells and subsequently into mesoderm cells. hESCs differentiate into mesendoderm cells in the presence of TGF β inhibitors (e.g., SB431542), and Wnt3a/BIO (1-4 days). Mesendoderm cells differentiated into mesoderm cells in the presence of BMP42 for-4 days.

FIG. 20: hESCs were differentiated into IMPs by 6 days of culture on matrigel in medium containing Wnt3a (25ng/ml), BMP4(100 ng/ml). On day 6, cells were passaged 1: 5 and then plated on matrigel in defined medium containing the same concentrations of Wnt3a and BMP4 for 10 more days. Cells were immunostained for the smooth muscle marker calcinin (a calcium binding protein), Smooth Muscle Actin (SMA), smooth muscle myosin heavy chain (SM-MHC), and Caldesmin (a calmodulin binding protein). DNA was stained with DAPI.

FIG. 21: cardiomyocytes and endothelial cells were generated from Isl1+ pluripotent progenitor cells (IMPs). Cells were treated for 6 days with three different methods to generate IMPs. Treatment 1: the first 24 hour hESCs were grown in defined media containing activin A (100ng/ml), Wnt3a (25ng/ml) for 1-4 days, and BMP4(100ng/ml) for 2-6 days. And (3) treatment 2: hESCs were grown for 1-2 days in defined media without IGF-I, Heregulin and EGF2 but with Wnt3a (25ng/ml) and for 2-6 days in defined media with BMP4(100 ng/ml). The cells were then grown in defined media for a further 14 days. Q-PCR analysis was performed on cardiac alpha actin/ACTC 1, cTNT, CD31/PECAM1, and CDH 5/VE-cadherin markers.

FIG. 22: a schematic representation of hESCs differentiation into definitive endoderm cells (DE) via intermediate states of mesendoderm cells (MesEnd) is shown. hESCs in defined media were treated with TGF-beta inhibitors (e.g., SB431542), Wnt3a/BIO for 1-3 days, and then changed to defined media containing high levels of activin A (100ng/ml) but not IGF-I and Heregulin for 1-3 additional days.

FIG. 23: a schematic representation of hESCs differentiation into definitive endoderm cells (DE) via intermediate states of mesendoderm cells (MesEnd) is shown. hESCs in defined media were treated with BIO for 3-5 days under conditions containing high levels of activin A (100ng/ml) but no IGF-I and Heregulin.

FIG. 24: a schematic representation of hESCs differentiating into definitive endoderm cells (DE) via the intermediate state of mesendoderm cells (MesEnd) is shown. hESCs in defined media were treated with BIO for 3-5 days under conditions containing low levels of activin A (10ng/ml) but no IGF-I and Heregulin.

FIG. 25: hESCs grown on matrigel in defined media (BG02) were treated for 4 days with BIO (2 μ M) and SB431542(20 μ M) and then treated for 4 more days with high levels of activin a (100ng/ml) but without Heregulin and IGF-I to form definitive endoderm cells (DE). The Q-PCR analysis of Nanog, T, Sox17, CXCR4, FoxF1 and PDGFR α transcripts is shown.

FIG. 26: formation of Multipotent Mesenchymal Cells (MMCs) by treating hESCs (BG02) with BIO (2 μ M) and SB431542(20 μ M) for 8 days. The Q-PCR analysis of Nanog, T, Sox17, CXCR4, FoxF1 and PDGFR α transcript levels is shown.

FIG. 27 is a schematic view showing: formation of Multipotent Mesenchymal Cells (MMCs) after 4 days treatment of hESCs (BG02) with BIO (2 μ M) and SB431542(20 μ M). Immunostaining for (A) T, (B) Oct4 and Nanog, and (C) E-cadherin are shown. DNA was stained with DAPI.

FIG. 28: multipotent Mesenchymal Cells (MMCs) were grown continuously for 10 passages in defined media containing BIO (2. mu.M) and SB431542 (20. mu.M). Accutase is used every 5 days^TM(Innovative cell technologies) cells were passaged at a ratio of 1: 5. Q-PCR analysis showed transcript levels of Nanog, T, Eomes, FoxF1, Sox17 and Fgf5 in different generations of cells and hESCs (BG 01).

FIG. 29: pluripotent mesenchymal cells (MMCs) derived from BG02hESCs, when grown to passage 7, showed no expression of pluripotent hESC markers such as E-cadherin, Nanog and Oct 4. DAPI staining indicates DNA. The figure shows a merged image of DAPI with Nanog or E-cadherin/Oct 4.

FIG. 30: (A) bright field patterns of growth of pluripotent mesenchymal cells (MMCs) derived from BG02hESCs for 20 passages in defined media containing BIO (2 μ M) and SB431542(20 μ M). Cells 3 days after plating (low density) and 6 days after plating (high density) are shown. The image magnification is indicated. (B) Generation 14 MMCs generated from BG02hESCs were cryopreserved, thawed, and replated under the aforementioned conditions to maintain the MMCs. Bright field images of the MMCs before cryopreservation (passage 14) and the resuscitated MMCs are shown. (C) Generation 14 MMCs were stained with APC-conjugated anti-CXCR 4 antibody and then subjected to FACS (fluorescence activated cell sorting) analysis. CXCR4+ cells recovered with FACS were plated under standard MMC culture conditions, and bright field images 5 days after sorting are shown.

FIG. 31: analysis of SSEA3 and SSEA4 cell surface markers for hESCs (BG02) grown in defined media and pluripotent mesenchymal cells (MMCs) grown in defined media containing BIO (2 μ M) and SB431542(20 μ M). The algebra of the MMC is marked in the figure. The figure shows a flow cytometry analysis in which the MMCs and hESCs were stained with antibodies to SSEA3 and SSEA 4. IC represents isotype control of the antibody.

FIG. 32: multipotent Mesenchymal Cells (MMCs) were grown for 6 passages in defined media supplemented with BIO (2. mu.M) and SB431542 (20. mu.M). MMCs were then plated on matrigel in defined medium without IGF-I and Heregulin but with high levels of activin A (100ng/ml) and cultured for 4 days. Q-PCR analysis showed transcript levels of Nanog, T, Fgf5, Eomes, Sox17 and CXCR 4.

FIG. 33: schematic representation of the formation of pluripotent mesenchymal cells (MMCs) from hESCs and the likely cell types into which they can differentiate is shown.

Disclosure of Invention

In a first aspect, the invention relates to a novel method of producing a population of mesendoderm cells, the method comprising: primate pluripotent stem cells (pPSCs), particularly hESCs, are exposed to differentiation media containing an effective amount of at least one GSK (preferably GSK3) inhibitor (e.g., BIO), or related compounds (as described further herein) including Wnt proteins (Wingless proteins, e.g., Wnt3 a; and other proteins) or related proteins, for a sufficient time (typically about 18 hours to about 72 hours or more) to produce a population of mesendoderm cells that can be isolated and passaged, stored (frozen storage) or further differentiated (as described below) to produce mesoderm (Isl +) precursor cells.

In a second aspect, the present invention relates to a novel method of generating a population of mesodermal (Isl +) cells, the method comprising: the pPSCs, in particular hESCs, are exposed to a differentiation medium containing an effective amount of at least one inhibitor of GSK, preferably GSK3, e.g. BIO, or related compounds including Wnt proteins, e.g. Wnt3a, and other proteins, or related proteins, as described further herein, for a sufficient time, typically about 18 hours to about 36 hours, preferably about 1-2 days, to produce a population of mesendoderm cells, which can optionally be isolated, and subsequently the population of mesendoderm cells produced in the first step is exposed to a medium containing an effective amount of a GSK inhibitor, e.g. BIO, or related compounds including Wnt proteins, e.g. Wnt3a, and other proteins, as described further herein, and containing an effective amount of bone morphogenic proteins (BMP-2, BMP-4, c-h) or related proteins, as described further herein BMP-6, BMP-7) for a sufficient period of time (typically about 2-9 days, about 3-6 days, about 3-5 days, about 72-132 hours, about 120-130 hours) to generate mesodermal Isl1+ cells (islet 1 cardiovascular progenitor cells). It is noted herein that in certain embodiments, the GSK inhibitor may be removed from the differentiation medium such that an effective amount of BMP may be used to differentiate mesendoderm cells into mesoderm cells in the absence of the GSK inhibitor.

The isolated mesendoderm cells are capable of further differentiation into mesoderm Isl1+ cells, which have the potential to differentiate to form cardiomyocytes, smooth muscle cells and endothelial cell lineages or to form endoderm cells. The basic method of forming mesendoderm or mesoderm Isl1+ cells is applicable to virtually all pPSCs, including in particular the hESC cell lines, including the BG01 and BG02 cell lines and other cell lines.

Mesodermal (Isl +) cells can be differentiated into cardiomyocytes (cardiac muscle cells) using standard methods in the art. The cardiomyocytes can be used to treat cardiovascular disease, including myocardial infarction (infarcted heart) and other cardiovascular diseases.

Mesoderm (Isl +) cells can also be differentiated into vascular smooth muscle cells by passaging the cells in a cell differentiation medium containing effective concentrations of a GSK inhibitor (preferably Wnt3a) and a bone morphogenetic protein (BMP4) every 5-6 days. The vascular smooth muscle cells produced by the present invention may be used for treating ischemic vascular conditions and repairing blood vessels.

In alternative embodiments, the invention relates to the generation of stable cell populations of multipotent mesenchymal migratory cells (also known as multipotent migratory cells or MMCs). In this aspect of the invention, pPSCs, and in particular hESCs, are grown in a differentiation medium containing an effective amount of a GSK inhibitor, preferably an inhibitor of GSK3 including BIO or a related GSK3 inhibitor including a Wingless protein as described further herein, e.g., Wnt3a, and an effective amount of an Activin A inhibitor (antagonist), such as SB-431542(Sigma), follistatin (follistatin) gene-related protein (FGRP, available from R and D Systems), BMP and Activin Membrane Binding Inhibitor (BAMBI), anti-BAMBI (monoclonal antibody), Smad7 (Heat agar Decapentaplegic homolog 7), TGF RI inhibitor (Calbiochem), and/or a bone morphogenetic protein antagonist (BMP antagonist), such as Noggin, Sclerostin, Gremlin (Dmr Gremlin), and uterine sensitization-associated gene 1 protein (USAG-1, SOSTl1), and the like. In this aspect of the invention, new multipotent migratory cells are efficiently generated by exposing pPSCs, and in particular hESCs, to a differentiation medium as otherwise described herein containing a GSK inhibitor and an activin A inhibitor and/or a BMP inhibitor for a duration of about 3-12 days, about 4-9 days, about 5-8 days, about 6-8 days, about 7 days. The pluripotent migratory cells are stable MMCs and can be harvested and stored (frozen storage) or passaged multiple times (at least 20 to infinity). The cells may be self-renewing. These MMCs are pluripotent and can be further differentiated into a number of mature cell populations, including endodermal and/or mesodermal cells, using techniques otherwise described herein. MMCs can also be isolated from embryonic or fetal tissue at the inner cell mass stage.

The invention also relates to isolated populations of pluripotent migratory cells (MMCs) that are pluripotent and self-renewing. These cells can grow for long periods (over many generations) while maintaining their marker profile and appear to self-renew. These cells can differentiate into a variety of cell types, including endodermal and mesodermal cells, and thus are pluripotent. These cells therefore have significant developmental plasticity. However, based on the signature profile, these cells were not human embryonic stem cells (hESCs). This represents one example of an alternative pluripotent cell derived from hESCs. These cells were isolated and stored (frozen storage).

The Multipotent Migratory Cells (MMCs) according to the invention have the following characteristics:

they are pluripotent and self-renewing;

they can differentiate into a variety of cell types, including endodermal and mesodermal cells;

they are dynamic cells that can alternate between MMCs (E-cadherin-, Oct4-, Nanog-, SSEA3-, CXCR4+) and alternative cell types with E-cadherin +, Oct4+, Nanog +, SSEA3-, CXCR4+ (high density (epithelial layer)) with significant developmental plasticity;

based on the marker profile, these cells are not hESCs.

MMCs according to the invention are stable, can be passaged at least 20 times without affecting the viability of the cell line, and can be stored using standard cryopreservation techniques well known in the art. MMCs according to the present invention can be stored, handled, and used at a remote location (relative to the location where the cells were originally produced).

Detailed Description

The following terminology is used to describe the invention.

Unless otherwise noted, terms used herein should be understood as having the meaning conventionally used by those of ordinary skill in the relevant art. In addition to the definitions of terms provided below, definitions for general terms in molecular biology can be found in Rieger et al, 1991 gloss of genetics: classical and molecular, 5th ed., Berlin: Springer-Verlag; and in Current Protocols in Molecular Biology, F.M. Ausubel et al, eds., Current Protocols, a joint vector between Greene publishing Associates, Inc. and John Wiley & Sons, Inc. (1998 Supplement). It should be understood that the use of "a" or "an" in the specification and claims refers to one or more, should be read in light of its context. Thus, for example, "a cell" can refer to at least one cell.

The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included in this specification. However, before the present invention is disclosed and described, it is to be understood that this invention is not limited to particular conditions or particular methods, etc., as such conditions and methods may, of course, vary, and that many variations and modifications therein will be apparent to those skilled in the art.

Standard techniques for culturing cells, isolating cells, and the associated cloning, DNA isolation, amplification and purification, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, and the like, as well as various isolation techniques are well known and commonly used by those skilled in the art. Many standard techniques are described in Sambrook et al, 1989 Molecular Cloning, Second Edition, Cold Spring harbor laboratory, Plainview, New York; maniatis et al, 1982 Molecular Cloning, Cold spring Harbor Laboratory, Plainview, New York; wu (Ed.)1993meth.enzymol.218, Part I; wu (Ed.)1979meth. Enzymol.68; wu et al, (Eds.)1983 meth.enzymol.100 and 101; grossman and Moldave (Eds.)1980 meth. enzymol.65; miller (ed.)1972 Experiments in Molecular Genetics, Cold Spring Harbor laboratory, Cold Spring Harbor, New York; old and Primrose, 1981 Principles of Gene management, University of California Press, Berkeley; schleif and Wensink, 1982 Practical Methods in Molecular Biology; glover (Ed.)1985 DNACloning vol.i and II, IRL Press, Oxford, UK; hames and Higgins (Eds.)1985Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollander 1979 Genetic Engineering: principles and Methods, Vois.1-4, Plenum Press, New York. The abbreviations and nomenclature used are standard in the art and are common in those skilled in the art cited herein.

"human embryonic stem cells" or hESCs are a subset of "primate pluripotent stem cells", the term "primate pluripotent stem cells" derived from pre-embryonic, embryonic or fetal tissue at any time after fertilization, characterized by the ability to produce offspring under appropriate conditions derived from several different cell types of 3 germ layer cells (endodermal, mesodermal and ectodermal cells) according to art recognized standard tests, such as the ability to form teratomas in 8-12 week old Severe Combined Immunodeficiency (SCID) mice. The term includes a variety of established stem cell lines, as well as pluripotent cells obtained from a source tissue in the manner described.

Included within the definition of pluripotent or pPS cells (pPSCs) are various types of embryonic cells, including in particular human embryonic stem cells (hESCs), as described by Thomson et al (Science 282: 1145, 1998); other primate embryonic stem cells are also included, such as rhesus monkey stem cells (Thomson et al, Proc. Natl Acad. Sci. USA 92: 7844, 1995). Other types of pluripotent cells are also encompassed by this term. Human pluripotent stem cells include stem cells obtained from human umbilical cord or placental blood and human placental tissue. Also included are any primate-derived cells capable of producing progeny derived from all 3 germ layer cells, whether they are derived from embryonic tissue, fetal or other sources. The pPS cells are preferably not derived from malignant cells. It is preferably (but usually not necessarily) a cell of the normal karyotype.

pPS cell cultures are described as "undifferentiated" when a substantial proportion of the stem cells and their derived cells in the cell population exhibit morphological characteristics of undifferentiated cells that clearly distinguish them from differentiated cells of embryonic or adult origin. One skilled in the art can readily identify undifferentiated pPS cells, which under two-dimensional microscopic observation typically show cell colonies with high nucleus/mass ratio and prominent nuclei. It is understood that undifferentiated cell colonies in a cell population are typically surrounded by nearby differentiated cells.

Pluripotent stem cells can express one or more stage-specific embryonic antigens (SSEA)3 and 4, as well as markers detectable with the designated Tra-1-60 and Tra-1-81 antibodies (Thomson et al, Science 282: 1145, 1998). In vitro differentiation of pluripotent stem cells results in loss of expression of SSEA-4, Tra-1-60 and Tra-1-81 (if present) and increased expression of SSEA-1. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde and then developing with Vector Red as a substrate (Vector Laboratories, Burlingame Calif.) according to the manufacturer's instructions. Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT as detected by RT-PCR.

Another desirable phenotype of passaged pluripotent stem cells has the potential to be able to differentiate into cells of all 3 germ layers (endoderm, mesoderm and ectoderm). The pluripotency of pluripotent stem cells can be determined, for example, by injecting the cells into Severe Combined Immunodeficiency (SCID) mice, fixing the resulting teratomas with 4% paraformaldehyde, and then examining their histological evidence of cell types derived from 3 germ layers. Alternatively, pluripotency can be determined by forming an embryoid body and evaluating a marker associated with the 3 blastodermal cells present in the embryoid body.

The karyotype of the passaged pluripotent stem cell line was determined using standard G-banding techniques and compared to the corresponding karyotype of published primate species. It is desirable to obtain cells with a "normal karyotype," which means that the cells are euploid, in which all human chromosomes are present and not significantly altered.

Types of pluripotent stem cells that may be used include established pluripotent stem cell lines derived from tissues formed after pregnancy, including pre-embryonic tissues (e.g., blastocysts), embryonic tissues, or fetal tissues taken at any time during pregnancy, typically (but not necessarily) during the first 10-12 weeks of pregnancy. Non-limiting examples are established human embryonic stem cell lines or human embryonic stem cells, such as the human embryonic stem cell lines WA01, WA07 and WA099 (WiCell). It is also contemplated to use the present disclosure during initial establishment or stabilization of the cells, in which case the source cells may be primate pluripotent cells obtained directly from the source tissue. Cells obtained from a pluripotent stem cell population that has been cultured in the absence of feeder cells are also suitable. Mutant human embryonic stem cell lines such as BG01v (BresaGen, adhens, Ga.), and normal human embryonic stem cell lines such as WA01, WA07, WA09(WiCell), and BG01, BG02(BresaGen, adhens, Ga.) are also suitable.

Ectodermal stem cells (EpiScs) and induced pluripotent stem cells (iPS) belong to the broad class of pluripotent cells, and conceptually, the techniques described herein can be applied to these and other pluripotent stem cell types (i.e., primate pluripotent cells). The EpiSCs described above were isolated from early post-implantation stage embryos (earlypost-implantation embryos). These cells express Oct4 and are pluripotent. (see Tesar et al, Nature, VoI 448, p.19612 July 2007). Adult cells were dedifferentiated back to a pluripotent state by retroviral transduction of 4 genes (c-myc, Klf4, Sox2, Oct4) to produce iPS cells. (see Takahashi and Yamanaka, Cell 126, 663-676, August 25, 2006).

Human embryonic stem cells can be prepared by the methods described herein as well as by methods known in the art (e.g., as described by Thomson et al). (U.S. Pat. No.5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. biol. 38: 133 ff., 1998; Proa Natl. Acad. ScL. U.S. A.92: 7844, 1995).

The term "embryonic stem cell" refers to a pluripotent cell isolated from a blastocyst, preferably a primate (including human). Human embryonic stem cells refer to stem cells of human origin that are preferably used in aspects of the invention relating to human therapy or diagnosis. Human embryonic stem cells express the following phenotypic markers:

SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, alkaline phosphatase, Oct4, Nanog, Rex1, Sox2, and TERT. (see Ginis et al, Dev. biol. 269(2), 360-. Optional primate pluripotent stem cells (pPSCs), including in particular human embryonic stem cells, can be used in the methods of the invention to produce mesendoderm, mesoderm Isl1+ cells and Multipotent Migratory Cells (MMCs) according to the invention, preferred pPSCs for use in the invention include human embryonic stem cells (including human embryonic stem cells derived from the BG01 and BG02 cell lines) as well as many other available stem cell lines.

The term "differentiation" is used to describe a process of non-specific ("amorphous") or less specific to obtain a more specific cell characteristic for a cell, such as a multipotent migratory cell, a mesendoderm cell, a mesoderm cell, a neural cell, a muscle cell, or other cell. The term "differentiated" includes the process of pluripotent stem cells (including hescs) becoming more specific intermediate cells, including progenitor cells, and the process of more specific intermediate cells (MMC, mesendoderm or mesoderm cells) becoming even more specific cells. Differentiated cells or differentiation-induced cells are cells that occupy a more specific (shaped) position in a cell line. The term "committed" when applied to a differentiation process refers to a cell that is at a point in the differentiation pathway where the cell can continue to differentiate into a specific cell type or subset of cell types under normal conditions, and where the cell is unable to differentiate into a different cell type or revert back to a poorly differentiated cell type under normal conditions. "dedifferentiation" refers to the restoration of cells to a low specificity (or committed) position in the cell lineage (line). Cell lineage as used herein is defined as the inheritance of a cell, i.e., from which cell it originates and from which cell it can produce. The location of the cells in the genetic map of development and differentiation is determined by cell lineage. Lineage specific markers refer to features that are specifically associated with the phenotype of the lineage of the target cell and can be used to assess the differentiation of an amorphous cell into a target cell line.

The terms "pluripotent migratory cells", "pluripotent mesenchymal cells" or "MMC" are used interchangeably to refer to one or more cells produced according to the present invention. MMCs are dynamic pluripotent cells characterized by E-cadherin-, Oct4-, Nanog-, SSEA3-, CXCR4+, which have low matrix density and are free-flowing. They are storage stable and can survive many generations. They have significant developmental plasticity. Based on the signature profiles, they are not hESCs.

MMCs according to the invention can be stably stored in the presence of effective amounts of a GSK inhibitor and an activin A inhibitor. BMP inhibitors (e.g. Noggin) may also be used in combination with GSK inhibitors and activin a inhibitors. These cells can differentiate into mesodermal or definitive endoderm cells, as well as many others.

The Multipotent Mesenchymal Cells (MMC) according to the invention have one or more (at least 4, at least 5, at least 6, at least 10, preferably all) of the following characteristics:

can be cultured as a stable cell population for at least 20 generations

Mesenchymal cells when plated at low density and grown into sheets when cultured at high density

The ability to generate hESC cell lines including BG01, BG02, WA09

MMCs can be frozen and cryopreserved using standard methods

MMCs are capable of recovering from cryopreservation (recovered after cryopreservation), resuscitation (recovered) and differentiation

MMCs can be passaged with high plating efficiency (greater than 50% plating efficiency-50% of the passaged cells successfully seeded and survived)

Absence of SSEA3 and SSEA4 antigens on their cell surface

No expression of hESC markers, e.g. Oct4, Nanog

The surface of MMCs is capable of expressing CXCR4

The following transcripts capable of high expression of MMCs: zic1, HoxA9, HoxD4, HoxA5, HoxC10, HoxD3, Pax6, N-CAM, CXCR4

MMCs are not mesendoderm cells because they do not express the T/mouse short-tail mutant phenotype (brachyury) or Eomesoderm

E-cadherin negative

MMCs do not express Sox17, Isl1, Musashi, Nestin (Nestin) at levels detectable by Q-PCR analysis

Maintenance of normal karyotype during passaging

Display of a wandering, mesenchymal phenotype

Pluripotent differentiation (including mesodermal and endodermal cells)

Non-teratoma formation when injected into SCID mice

Capable of isolation from inner cell line embryos (inner cell mass embryos) and fetal tissue

More complete description of MMC gene expression profiles see microarray data

The terms "mesodermal (Isl1+) cells", mesoderm-derived Isl1+ pluripotent progenitor cells or "IMP" as used herein, are used interchangeably to describe mesodermal Isl1+ cells produced from pPSCs (in particular hESCs), mesendoderm cells or MMCs in accordance with the methods of the invention.

Mesoderm (Isl1+) cells (islet 1+ multipotent progenitor cells or IMPs) have the following characteristics:

expression of Isl1, Tbx20, Nkx2.5, Fgf10, GATA4, KDR (Flk1), FoxF1, PDGFR α

Karyotype normality

No expression of Oct4, Nanog, T, Eomesoderm

Ability to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells

Microarray analysis was performed on the IMPs formed. hESCs were cultured for 6 days in defined medium supplemented with Wnt3a (25ng/ml) and BMP4(100 ng/ml). Samples were taken at 0, 24, 48, 72, 96, 144 hours to extract mRNA, followed by microarray analysis. This microarray analysis is summarized in the table attached to this document. (IMP microarray)

The terms "differentiation medium", "cell differentiation medium", "culture medium", "basal cell culture medium", or "minimal medium", or "stable medium" are used synonymously herein to describe a cell growth medium in which hESCs, mesenchymal cells, mesodermal cells, or Multipotent Migratory Cells (MMCs) are produced, grown/cultured, or differentiated into more mature cells, depending on the additional components used. It is well known in the art that the differentiation medium is at least a minimal essential medium supplemented with one or more optional components, such as growth factors including Fibroblast Growth Factor (FGF), ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin (noted and not excluded), activin a, transferrin, beta mercaptoethanol, and other agents well known in the art as otherwise described herein. Preferably, the culture medium comprises a basic cell culture medium containing 1-20% (preferably about 2-10%) fetal bovine serum, or (preferably) a defined medium free of fetal bovine serum and KSR but including bovine serum albumin (about 1-5%, preferably about 2%). Preferred differentiation media are defined serum-free media. In certain embodiments, the MMCs are produced and the activin A inhibitor is used to remove or substantially remove activin A from the culture medium.

Other agents that may optionally be added to the medium according to the invention include: such as nicotinamide, TGF-beta family members (including TGF-beta 1, TGF-beta 2 and TGF-beta 3, activin A, Nodal), serum albumin, fibroblast growth factor family members, platelet-derived growth factor-AA and platelet-derived growth factor-BB, platelet-rich plasma, insulin growth factor (IGF-I, II), growth differentiation factor (GDF-5, GDF-6, GDF-8, GDF-10, GDF-11), glucagon-like peptides-I and II (GLP-I and GLP-II), mimetibodies of GLP-1 and GLP-2, Exin-4, parathyroid hormone, insulin, progesterone, antiproteinin, hydrocortisone, ethanolamine, Epidermal Growth Factor (EGF), gastrin I and II, endoglin-4, insulin, and insulin, Copper chelators (e.g., triethylenepentamine), forskolin (forskolin), sodium butyrate, beta cellulose, ITS, Noggin, axon growth factor, Nodal, valproic acid (valporic acid), trichostatin (trichostatin) a, sodium butyrate, Hepatocyte Growth Factor (HGF), sphingosine-1, VEGF, MG132(EMD, CA), N2, and B27 supplements (Gibco, CA), steroidal alkaloids (steroid alkaloids) (e.g., cyclopamine (EMD, CA)), Keratinocyte Growth Factor (KGF), Dickkopf protein family, bovine pituitary extract (bovine pituitary extract), islet neogenesis associated protein (INGAP), IHH protein (Indian hedgehog), SHH protein (sonic hedgehog), proteasome inhibitors, Notch pathway inhibitors, SHH protein inhibitors, Heregulin, or combinations thereof. If included, effective amounts of the various ingredients are included.

As yet another example, a suitable medium may be made of the following components: for example, Dulbecco's Modified Eagle's Medium (DMEM), Gibco # 11965-092; knockdown Dulbecco's modified Eagle's medium (KODMEM), Gibco # 10829-; ham's F12/50% DMEM minimal medium; 200 mML-Glutamine, Gibco # 15039-; a non-essential amino acid solution, Gibco 11140-050; beta-mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256-. Preferred embodiments of the media used in the present invention are described further herein.

A particularly preferred differentiation medium for the growth/culture of pPSCs, in particular hESCs, and for cell differentiation in the context of the present invention is DMEM/F12 (50: 50) containing about 2% prealbumin (albumin; Wikipedia/serology), 1 XPicillin/Strep antibiotic (Pen/Strep), 1 XPEAA, 1 XPmicroelement A, B, C (Mediatech), ascorbic acid (10-100ng/ml, about 25-65ng/ml, about 50ng/ml), about 0.1mM (0.025-0.5mM) beta-mercaptoethanol (Gibco), (about 2-10ng/ml, about 5-9ng/ml, about 8ng/ml) bFGF (Sigma), 200ng/ml (5-500ng/ml) LR-IGF (IGF-I; JRH Biosciences), 10ng/ml activin A (about 1ng/ml to no more than about 20ng/ml) and 10ng/ml Heregulin in ml (about 1-20ng/ml or more). Notably, activin a or activin a signaling is not required for the production of Multipotent Migratory Cells (MMCs) and mesendoderm cells, but is required to be included, particularly in the production of mesoderm (Isl +) cells (when included, it is preferred to include a low concentration of activin a, typically less than about 20 ng/ml). In contrast, about 20-100ng/ml or more of activin A or "high concentration of activin A" is used in the production of definitive endoderm cells. Alternatively, hescs can also be passaged using mouse embryonic fibroblast-conditioned medium (MEF-CM) similar in composition to DMED/F12, and producing mesendoderm cells, mesoderm cells (mesoderm Isl1+ cells), and Multipotent Migratory Cells (MMCs) according to the invention.

Differentiation media used in the present invention are commercially available and may be supplemented with commercially available components, commercially available from Invitrogen Corp. (GIBCO), Cell Applications, inc., and biologica industries, Beth HaEmek, Israel, and many other commercial sources including Calbiochem. In a preferred embodiment, at least one differentiating agent (e.g., Fibroblast Growth Factor (FGF), LR-IGF (insulin-like growth factor analog), and Heregulin) (preferably all three factors added in effective amounts) is added to the cell culture medium in which the stem cells are cultured, in which the stem cells differentiate into multipotent migratory cells, mesendoderm cells, or mesoderm cells (or even differentiate from MMCs into definitive endoderm cells). One of ordinary skill in the art can readily modify the cell culture medium to produce optionally one or more target cells according to the invention. Cell differentiation media is essentially identical to the basic cell culture media, but is used herein for the differentiation process and includes cell differentiation agents that differentiate cells into other cells. A stabilizing medium is a basic cell culture medium used before or after the differentiation step, which serves to stabilize the cell line for further utilization. The medium is essentially the same as a stable medium, but refers to a medium in which pluripotent or other cell lines are grown or cultured prior to differentiation. In general, the cell differentiation media and stabilization media used herein may comprise similar components to the basic cell culture medium, but may comprise slightly different components in different contexts to affect the intended results of the media used. In the case of MMCs, particularly storage-stable MMCs, an effective amount of an activin A signaling inhibitor and an effective amount of a GSK inhibitor are included in the cell culture medium as further disclosed herein to differentiate and stabilize the MMCs, i.e., to prevent them from further differentiation and to storage-stabilize the population of cells. For this purpose, the BMP inhibitor may be used in combination with an activin a inhibitor and a GSK inhibitor.

Pluripotent stem cells can also be cultured on a layer of feeder cells that support the pluripotent stem cells in a variety of ways described in the art. Alternatively, pluripotent stem cells are cultured in a substantially feeder cells-free culture system, but the system still supports proliferation of pluripotent stem cells and renders the pluripotent stem cells substantially undifferentiated. The pluripotent stem cells are maintained undifferentiated by supporting growth in feeder cells-free conditions using conditioned medium, which is a medium previously cultured for another cell type. Alternatively, chemically defined media is used to support the growth of pluripotent stem cells in feeder cells-free conditions without differentiating them. These methods are well known in the art. In a preferred aspect of the invention, the cells are grown in a feeder cells-free medium.

Methods for culturing cells on a feeder cell layer are well known in the art. Pluripotent stem cell lines derived from human blastocysts are cultured with mouse embryonic fibroblast feeder cell layers as disclosed, for example, by Reubinoff et al (Nature Biotechnology 18: 399-404(2000)) and Thompson et al (Science 6 Nov.1998: Vol.282.no.5391, pp.1145-1147). Richards et al (Stem Cells 21: 546) -556, 2003) evaluated the ability of 11 different feeder cell layers of human adults, fetuses and neonates to support human pluripotent Stem cell cultures. Richards et al describe: the human embryonic stem cell line cultured on adult skin fibroblast feeder cells maintains the morphology and pluripotency of human embryonic stem cells. US20020072117 discloses a cell line that produces a medium capable of supporting the growth of primate pluripotent stem cells in the absence of feeder cells. The cell lines are mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. US20020072117 also discloses the use of said cell line for primate feeder cell layers. In another example, Wang et al (Stem Cells 23: 1221-1227, 2005) disclose the growth of human pluripotent Stem Cells on a layer of human embryonic Stem cell-derived feeder Cells. In another example, Stojkovic et al (Stem Cells 200523: 306-314, 2005) disclose a feeder cell system derived from the spontaneous differentiation of human embryonic Stem Cells. In yet another example, Miyamoto et al (+ 22: 433-440, 2004) disclose a source of feeder cells obtained from human placenta. Amit et al (biol. reprod 68: 2150) -2156, 2003) disclose human foreskin-derived feeder cell layers. In another example, Inzunza et al (Stem Cells 23: 544-.

Methods for culturing pPSCs in culture medium, particularly feeder cells-free medium, are well known in the art. U.S. patent No.6,642,048 discloses media that support the culture of primate pluripotent stem cells (pPS) in feeder cells-free conditions, and cell lines for producing such media. U.S. patent No.6,642,048 describes: the invention includes mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods of obtaining such cell lines, methods of processing media, and methods of culturing stem cells with conditioned media are described and illustrated. In another example, WO2005014799 discloses a conditioned medium for the maintenance, proliferation and differentiation of mammalian cells. In another example, Xu et al (Stem Cells 22: 972-980, 2004) disclose conditioned media obtained from human embryonic Stem cell derivatives that are typically modified to overexpress the human telomerase reverse transcriptase. In another example, US20070010011 discloses a chemically defined medium for maintaining pluripotent stem cells.

An alternative culture system uses serum-free media supplemented with growth factors that promote the proliferation of embryonic stem cells. For example, Cheon et al (BioReprodoDOI: 10.1095/bioleprored.105.046870, Oct.19, 2005) disclose a feeder cells-free, serum-free culture system in which embryonic stem cells are maintained in an unlimited Serum Replacement (SR) medium supplemented with different growth factors that initiate self-renewal of the stem cells. In another example, Levenstein et al (Stem Cells 24: 568-574, 2006) disclose methods for long-term culture of human embryonic Stem Cells in the absence of fibroblasts or conditioned medium supplemented with bFGF. In another example, US20050148070 discloses a method of culturing human embryonic stem cells in a defined medium without serum, fibroblast-free feeder cells, the method comprising: culturing stem cells in a medium comprising albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the medium being substantially free of mammalian fetal serum and comprising at least about 100ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the source of the growth factor is other than a fibroblast feeder layer, the medium being capable of supporting proliferation of stem cells in an undifferentiated state in the absence of feeder cells or conditioned medium.

US20050233446 discloses a defined medium for culturing stem cells, including undifferentiated primate naive stem cells. In solution, the medium is substantially isotonic compared to the cultured stem cells. In a given culture, a particular medium includes a minimal medium and amounts of bFGF, insulin, and ascorbic acid necessary to support growth of primate stem cells in a substantially undifferentiated state. In yet another example, WO2005065354 discloses a defined and isotonic culture medium which is substantially feeder cells free and serum free comprising: a. a basal medium; b. a sufficient amount of bFGF to support growth of mammalian stem cells in a substantially undifferentiated state; c. insulin in an amount sufficient to support growth of mammalian stem cells in a substantially undifferentiated state; a sufficient amount of ascorbic acid to support growth of mammalian stem cells in a substantially undifferentiated state.

In another example, WO2005086845 discloses a method of maintaining undifferentiated stem cells, the method comprising: the stem cells are exposed to a sufficient amount of a member of the transforming growth factor-beta (TGF β) family of proteins, a member of the Fibroblast Growth Factor (FGF) family of proteins, or Nicotinamide (NIC) to maintain the cells in an undifferentiated state for a sufficient time to achieve the desired result.

Cells are preferably grown on a cell support or matrix, growing as an attached monolayer, rather than as an embryoid body or suspension. In the present invention, Matrigel (Matrigel) is preferably used as a cell support. The cell support preferably comprises at least one differentiation protein. The term "differentiation protein" or "matrix protein" is used to describe a protein that can be used to culture cells and/or promote differentiation (also preferably attachment) of embryonic stem cells or mesendoderm cells, mesoderm cells or Multipotent Migratory Cells (MMC). Preferred differentiation proteins in the present invention include: for example, extracellular matrix proteins, which are proteins found in extracellular matrices, such as lamin (lamin), tenascin (tenascin), thrombospondin (thrombospondin), and mixtures thereof, which promote growth, and which contain domains having homology to Epidermal Growth Factor (EGF) and which have activities of promoting growth and differentiation. Other differentiation proteins useful in the present invention include: such as collagen, fibronectin, Vibronectin, polylysine (polylysine), polyornithine (polyornithine), and mixtures thereof. Alternatively, gels and other materials, such as methylcellulose from other gels containing effective concentrations of one or more of these embryonic stem cell differentiation proteins, may be used. Examples of the differentiation proteins or materials including these differentiation proteinsIncluding, for example, BDcell-Tak^TMCell and tissue adhesive, BD^TMFibrogen human recombinant collagen I, BD^TMFibrogen human recombinant collagen III, BD Matrigel^TMBase film substrate, BD Matrigel^TMHigh Concentration (HC) substrate film matrix, BD^TM PuraMatrix^TMPeptide hydrogel, collagen I, High Concentration (HC) collagen, collagen II (bovine), collagen III, collagen IV, collagen V, and collagen VI, and the like. Preferred materials for use in the present invention include Matrigel^TMAnd Geltrex^TM。

A preferred component/material containing one or more differentiation or matrix proteins is BDMatrigel^TMA base film matrix. It is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins. The main constituents of the basement membrane matrix are lamins, as well as collagen IV, heparan sulfate, proteoglycans, lactons (entactin) and nidogen (nidogen).

The pluripotent stem cells are preferably plated on a differentiation protein or a matrix protein. The pluripotent stem cells may be plated onto the substrate in a suitable distribution in the presence of a medium that promotes cell survival, proliferation and maintenance of the desired characteristics. Note that the distribution of the inoculation is beneficial for all of these characteristics, and these characteristics can be readily determined by one skilled in the art.

The term "activation" as used herein refers to increased expression of a marker (e.g., Isl), or to upregulation of the activity of Isl or markers associated with blood cells, vascular cells (endothelial cells), renal cells, bone and muscle cells. These cells are useful in the treatment of heart disease, renal degeneration, repair of bone and vascular degeneration.

The term "isolated" as used herein when referring to a cell, cell line, cell culture, or cell population, means substantially isolated from the natural source of the cell, thereby enabling the cell, cell line, cell culture, or cell population to be cultured in vitro. In addition, the term "isolating" is used to refer to the physical selection of one or more cells from two or more cell populations, wherein the cells are screened based on cell morphology and/or expression of various markers.

The term "expression" as used herein refers to transcription of a polynucleotide or translation of a polypeptide (including a marker) in a cell such that the cell expressing the molecule has a detectably higher level of the molecule than a cell not expressing the molecule. Methods for measuring molecular expression are well known to those of ordinary skill in the art and include, but are not limited to, Northern blotting, RT-PCR, in situ hybridization, Western blotting, and immunostaining.

The term "marker" as used herein describes a nucleic acid or polypeptide molecule that is capable of differential expression in a target cell, where differential expression refers to an increased level of positive marker and a decreased level of negative marker. The level of detectable marker nucleic acid or polypeptide in the target cell is sufficiently higher or lower than the level in other cells so that the target cell can be identified and distinguished from other cells by any of a variety of methods, optionally known in the art.

The term "contacting" (i.e., contacting a cell with a compound) as used herein is intended to include co-incubation of the compound and the cell in vitro (e.g., addition of the compound to cultured cells). The term "contacting" does not include situations where a cell is exposed to a differentiating agent in vivo, which can occur in nature (i.e., exposure that may occur as a result of a natural physiological process). The step of contacting the cells with the differentiation medium and one or more growth factors (BMP or others) and inhibitors (GSK, activin a (signal) or inhibitors of BMP (signal)) as further described herein may be guided by optionally suitable means. For example, the cells can be treated in adherent culture, in which the cells act as an adherent layer, an embryoid body, or in suspension culture, with the use of an adherent layer being preferred because an adherent layer can provide an efficient differentiation process that typically provides differentiation into 90% or more of the target cell population (mesendoderm, mesoderm, or multipotent migratory cells). It will be appreciated that the cells contacted with the differentiation agent may be further treated with other cellular differentiation environments to stabilize the cells, or to further differentiate the cells, for example to produce islet cells.

For the case of definitive endoderm cells produced from mesendoderm cells and/or MMCs, the cells are differentiated in media additionally disclosed herein containing an effective amount of activin A (about 20-100ng/ml or more) and optionally an effective amount of an inhibitor of PI3 kinase signaling. It should be noted that one or more Nodal, TGF β or other TGF members may be used in addition to or in place of activin a. Also, factors affecting/promoting PB kinase signaling (e.g. IGF-I and Heregulin) may be removed from the differentiation medium in addition to or instead of the PD kinase inhibitor.

The term "differentiation agent" as used herein refers to any compound or molecule capable of inducing partial or final differentiation of cells including hESC's, mesendoderm cells, Multipotent Migratory Cells (MMCs) or Isl1+ multipotent progenitor cells (EVIPs), wherein said differentiation is due at least in part to inhibition of GSK, including bone morphogenic proteins (BMP-2, BMP-4, BMP-6, or BMP-7) (e.g., during differentiation of hESCs into mesendoderm Isl1+ cells), or to inhibition of GSK and inhibition of activin a and/or bone morphogenic proteins to produce Multipotent Migratory Cells (MMCs), or to addition of activin a to produce endoderm cells. The differentiation agent may be as described below, but the term is not limited thereto.

The term "differentiation agent" as used herein includes within its scope natural or synthetic molecules or molecules exhibiting similar biological activity.

The term "effective" is used to describe the amount of an ingredient used or contained in a compound or composition herein that is used for a time sufficient to produce the desired effect. For example, an effective amount of a differentiation agent is an amount that, when used in combination with other ingredients, is capable of producing the desired differentiated cells in a differentiation medium.

The term "bone morphogenic protein" or BMP is used to describe a differentiation agent for use in the present invention that, in combination with other components described elsewhere herein, can differentiate hESCs cells or mesendoderm cells into mesoderm Isl1+ cells. An effective amount of any one of BMP-2, BMP-4, BMP-6, or BMP-7 (preferably BMP-2 or BMP-4) may be used to assist in the differentiation process. The amount of BMP is about 1-500ng/ml, about 25-250ng/ml, about 50-150ng/ml, about 75-125ng/ml, about 100 ng/ml.

The term "GSK inhibitor" is used to describe a compound capable of inhibiting GSK (particularly GSK3, including GSK3 α or GSK3 β). Examples of preferred GSK inhibitors for use in the present invention include one or more of the following (all available from Calbiochem):

BIO (2 ' Z, 3 ' E) -6-Bromoindirubin (Bromoindirubin) -3 ' -oxime (oxime) (inhibitor IX of GSK 3);

BIO-acetoxime (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -acetoxime (GSK3 inhibitor X);

(5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine (GSK 3-inhibitor XIII);

pyridocarbazole (Pyridocarbazole) -cyclopentadienyl ruthenium complex (GSK3 inhibitor XV);

TDZD-84-benzyl-2-methyl-1, 2, 4-thiadiazole (thiadiazolidine) -3, 5-dione (GSK3 beta inhibitor I);

2-thio (3-iodobenzyl) -5- (1-pyridinyl) - [1, 3, 4] -oxadiazole (oxadiazole) (GSK3 β inhibitor II);

OTDZT 2, 4-benzhydryl-5-oxothiadiazole (oxothiadiazolidine) -3-thione (GSK3 β inhibitor III);

α -4-Dibromoacetophenone (Dibromoacetophenone) (GSK3 β inhibitor VII);

AR-a014418N- (4-methoxybenzyl) -N' - (5-nitro-1, 3-thiazol-2-yl) urea (GSK-3 β inhibitor VIII);

3- (1- (3-hydroxypropyl) -1H-pyrrolo [2, 3-b ] pyridin-3-yl ] -4-pyrazine (pyrazin) -2-yl-pyrrole (pyrrole) -2, 5-dione (GSK-3 β inhibitor XI);

TWS119 pyrrolopyrimidine (pyrrolopyramidine) compounds (GSK3 β inhibitor XII);

L803H-KEAPPAPPQSpP-NH₂and its Myristoylated (Myristoylated) form (GSK3 β inhibitor XIII); and

2-chloro-1- (4, 5-dibromo-thiophen-2-yl) -ethanone (ethanone) (GSK3 β inhibitor VI).

In addition, many of the Wingless or Wnt proteins function similarly to GSK inhibitors (particularly GSK inhibitors according to the invention). Therefore, they are included within the scope of the term GSK inhibitor. Examples of Wnt proteins that may be used in the present invention include Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt14, Wnt14b, Wnt15 and Wnt16, as well as one or more of the other Wnt proteins. Preferably Wnt3a is used.

Preferred GSK inhibitors for use in the present invention include BIO (GSK-3IX) and Wnt3 a.

GSK inhibitors are useful in all aspects of mesendoderm cells, mesoderm cells, Multipotent Migratory Cells (MMCs) and even definitive endoderm cells contemplated by the present invention. When these GSK inhibitors are used, they are used in effective amounts at concentrations (depending on the molecular weight of the inhibitor used) of about 0.001-100. mu.M or more, about 0.05-75. mu.M, about 0.1-50. mu.M, about 0.25-35. mu.M, about 0.5-25. mu.M. In the case of BIO, the GSK inhibitor is used in the differentiation medium in an amount of about 0.05-50. mu.M, about 0.1-10. mu.M, about 0.5-5. mu.M, about 1-3. mu.M. When a Wnt protein is used, the amount of Wnt used is about 1-100ng/ml, about 5-50ng/ml, about 10-35ng/ml, about 20-30ng/ml, about 25 ng/ml.

The term "activin a inhibitor" is used to describe a compound or composition that is optionally added to the differentiation medium to inhibit the action of activin a during differentiation, which when used results in the production of Multipotent Migratory Cells (MMCs) from hESCs. To generate MMCs from hESCs, the chemo-reagent includes effective amounts of a GSK inhibitor (preferably a GSK3 inhibitor such as BIO, or other GSK3 inhibitor) and an activin A inhibitor, with or without the addition of a Bone Morphogenic Protein (BMP) inhibitor.

Examples of activin a inhibitors useful in the invention include: for example, SB-431542(Sigma), follistatin (follistatin), follistatin gene-related protein (FGRP, obtained from R and DSystems), membrane-bound inhibitors of BMP and Activin (BAMBI), anti-BAMBI (monoclonal antibody), Smad7 (Heat age Decapentaplegic Holog 7), and TGF RI inhibitors (Calbiochem), and the like. An effective amount of the activin A inhibitor used in the present invention is generally about 0.001-100. mu.M or more, about 0.05-75. mu.M, about 0.1-50. mu.M, about 0.25-35. mu.M, about 0.5-25. mu.M.

The term "bone morphogenic protein inhibitor" or "BMP inhibitor" is used to describe a compound or ingredient added to the differentiation medium in an amount effective to inhibit the action of bone morphogenic proteins in the differentiation of hESCs into Multipotent Migratory Cells (MMCs). Examples of BMP inhibitors include: such as Noggin, Sclerostin, Gremlin (Drm/Gremlin), and USAG-I, among others. The BMP inhibitors are used in effective amounts, typically (depending on the molecular weight and potency of the inhibitor used) in the range of about 0.01-500ng/ml or more, about 0.1-350ng/ml, about 0.5-250ng/ml, about 1-500ng/ml, about 5-250ng/ml, about 50-150ng/ml, about 75-125ng/ml, about 100 ng/ml.

The term "PI 3-kinase pathway inhibitor" or "PI 3-kinase signal inhibitor" refers to any molecule or compound that can reduce the activity of a PI 3-kinase or a molecule downstream of at least one PI 3-kinase in a cell contacted with the inhibitor. These inhibitors are preferred inhibitors for the preparation of definitive endoderm cells from mesendoderm cells and/or multipotent migratory cells according to the invention. The term includes compounds that reduce the synthesis and expression of endogenous PI 3-kinases, such as PI 3-kinase antagonists, PI 3-kinase signaling cascade antagonists, and also includes compounds that reduce endogenous PB-kinase release, and compounds that are activators of PI 3-kinase activity. In the specific embodiments described above, the inhibitor is selected from the group consisting of Rapamycin (Rapamycin), LY294002, wortmannin (wortmannin), lithium chloride, Akt inhibitor I, Akt inhibitor II (SH-5), Akt inhibitor III (SH-6), NL-71-101, and mixtures thereof. Akt inhibitor I, Akt inhibitor II, Akt III and NL-71-101 are commercially available from Calbiochem. In other embodiments, the inhibitor is selected from the group consisting of Rapamycin (Rapamycin), LY 294002. In a further preferred embodiment, the inhibitor comprises LY 294002. In another embodiment, the inhibitor may be used to induce a desired differentiation effect. The end result is the production of substantial amounts of endoderm cells that can be used to produce pancreatic endoderm cells and/or hepatic endoderm cells as we disclose in international application No. pct/US2007/01313 (6/4/2007, publication No. WO2008 /), which is incorporated herein by reference in its entirety.

The term "isolated" as used herein when referring to a cell, cell line, cell culture, or cell population, means substantially isolated from the natural source of the cell, thereby enabling the cell, cell line, cell culture, or cell population to be cultured in vitro. Alternatively, depending on the context, the term "isolated" refers to a population of cells isolated from a differentiation medium and culture flask so that the population of cells can be stored (cryopreserved). In addition, the term "isolating" is used to refer to the physical selection of one or more cells from two or more cell populations, wherein the cells are screened based on cell morphology and/or expression of various markers.

The term "passaging" is used to describe the process of dividing cells and transferring them to new culture flasks for further growth/re-growth. Preferred adherent cells (or even embryoid bodies) according to the invention may use an enzyme (Accutase)^TMOr collagenase), artificial passaging (mechanical, e.g., with a spatula or other flexible mechanical device or apparatus), and other non-enzymatic methods (e.g., cell dispersion buffer).

The term "contacting" (i.e., contacting a hESC, mesendoderm, mesoderm, or multipotent migratory cell with a compound) as used herein includes co-incubating the compound and the cell in vitro (e.g., adding the compound to a cell culture). The term "contacting" does not include exposure of the cell in vivo to naturally occurring growth factors and/or other differentiation agents or inhibitors (i.e., exposure to substances that may occur as natural physiological processes). The step of contacting the cells with the growth factors and/or inhibitors in the differentiation medium of the present invention may be directed by any suitable method. For example, the cells may be treated as embryoid bodies in attachment culture or in suspension culture. It will be appreciated that cells contacted with the differentiation agent and/or inhibitor may also be treated with other cellular differentiation environments to stabilize the cells, or to allow the cells to further differentiate, for example to produce definitive endoderm cells, blood cells, vascular cells (endothelial cells), kidney cells, bone and muscle cells (including cardiomyocytes). These cells can be used as regenerative medicine for the treatment of heart disease, renal degeneration, repair of bone and vascular degeneration.

Applicants have demonstrated that culturing hESCs using an effective amount of a GSK inhibitor (specifically BIO) in combination with an effective amount of a bone morphogenic protein (BMP-2, BMP-4, BMP-6, BMP-7) produces mesodermal cells (Isl1 +).

The present invention provides compositions comprising an isolated population of differentiated mammalian cells, in particular human mesendoderm cells, mesoderm (Isl1+) cells and/or Multipotent Migratory Cells (MMCs), wherein the cells are differentiated in vitro from hESCs (derived from mesendoderm cells, in the case of mesoderm (Isl1+) cells), and wherein greater than about 30% of the cells are capable of expressing markers of mesendoderm cells, mesoderm (Isl1+) cells or MMCs. In one embodiment of the invention, more than about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 72%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 90% or more than 90% of the cells are mesendoderm cells. Preferably, at least 50% (even 70-80% or more) of the cell population of cells capable of expressing Pdx1 and/or Isl1 is included in the composition. Mesendoderm cells are capable of expressing one or more of CD48, eomesodermin (eomes), T/mouse Brachyury phenotype (Brachyury), Wnt3a, and GSC markers.

The invention also includes a composition comprising an isolated population of mesodermal Isl1+, nkx2.5, Tbx20, Fgf10 cells, wherein said cells are produced in vitro culture and more than about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 72%, 74%, 75%, 76%, 77%, 78%, 79% or even 80% or 90 +% of said cells are mesodermal (Isl1+) cells.

The invention also includes a composition comprising an isolated population of multipotent migratory cells, wherein the cells are produced in culture in vitro, and more than about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 72%, 74%, 75%, 76%, 77%, 78%, 79% or even 80% or 90 +% of the cells are MMCs.

The invention also includes a method of differentiating hESCs into mesendoderm cells comprising (a) providing hESCs, and (b) contacting the hESCs with an effective amount of a GSK (preferably GSK3) inhibitor in a cell differentiation medium to produce mesendoderm cells, and (c) optionally, isolating the mesendoderm cells. To produce mesendoderm cells, hESCs are differentiated under the conditions described above for about 18-72 hours, preferably about 1-2 days.

The invention also includes a method of differentiating hESCs into mesodermal (Isl1+) cells, the method comprising (a) providing hESCs; and (b) contacting the hESCs with an effective amount of a GSK inhibitor in a cell differentiation medium for about 18-72 hours, preferably for about 1-2 days; then, (c) contacting the cells obtained from step (b) with an effective amount of a bone morphogenetic protein (BMP-2, BMP-4, BMP-6, BMP-7) and optionally a GSK inhibitor as further described herein in a cell differentiation medium for about 2-9 days, about 3-5 days, about 72-132 hours, about 120-130 hours to produce mesodermal (Isl1+) cells; and, optionally, collecting said mesodermal (Isl1+) cells, storing (cryopreserving) said mesodermal cells and further differentiating said mesodermal cells to produce cardiomyocytes and smooth muscle tissue, endothelial cell lineage, or endodermal cells. It should be noted that in step c, it is not necessary to include a GSK inhibitor, but it is preferred to include it.

The invention also includes a method of differentiating a mesendoderm cell into a mesoderm (Isl1+) cell, the method comprising (a) providing a mesendoderm cell; (b) contacting the mesendoderm cells with an effective amount of a bone morphogenetic protein (BMP-2, BMP-4, BMP-6, BMP-7) and optionally a GSK inhibitor in a cell differentiation medium for about 2-9 days, about 3-6 days, about 3-5 days, about 72-132 hours, about 120-130 hours to produce mesoderm (Isl1+) cells; and, optionally, collecting said mesodermal (Isl1+) cells, storing (cryopreserving) said mesodermal cells and further differentiating said mesodermal cells to produce cardiac muscle and smooth muscle tissue, endothelial cell lineage, or endodermal cells. Should optionally and preferably include a GSK inhibitor.

The present invention also relates to a method of differentiating hESCs into pluripotent migratory cells (MMCs), the method comprising (a) providing hESCs, and (b) contacting the hESCs with an effective amount of a GSK inhibitor and an effective amount of an activin a inhibitor and/or a BMP inhibitor for about 4-12 days, about 4-9 days, about 5-8 days, about 6-8 days, about 7 days. The resulting MMCs can be collected and stored (frozen storage), or passaged multiple times to produce stable, self-renewing MMCs. These MMCs can be further differentiated into a number of mature cell populations, including endodermal cells and/or mesodermal cells, using techniques otherwise described herein. The use of MMCs to generate mesodermal (Isl1+) cells is common for mesodermal (Isl1+) cells. In this method, the MMCs are grown for about 2-12 days, 3-9 days, 4-8 days, etc., in a cell differentiation medium in combination with an effective amount of the BMP or an optional GSK inhibitor (while removing the inhibitor of activin A and/or BMP used to produce the MMCs). MMCs can also be used to produce definitive endoderm cells as otherwise described herein.

During further differentiation of the MMCs into mesodermal and/or definitive endodermal cell populations, the activin A inhibitor used and the BMP inhibitor used to differentiate the hESCs into MMCs are removed, and the MMCs are grown under conditions (effective amounts of BMP, e.g., BMP-2, BMP-4, BMP-6, BMP-7, and optionally a GSK inhibitor such as BIO or Wnt3a) capable of producing mesodermal Isl1+ cells, or under conditions (effective amounts of activin A or equivalent compounds (Nodal, TGF, or other TGF members) and optionally a PI3K inhibitor and/or excluding IGF-I members and/or Heregulin) capable of producing definitive endodermal cells, or under other conditions capable of obtaining mesodermal or definitive endodermal cell populations. Methods and conditions for producing definitive endoderm cells from hESCs can be used to produce definitive endoderm cells from MMCs, such methods and conditions are disclosed, for example, in PCT/US2007/013137, publication No. WO 2008/the relevant portions of which are incorporated herein by reference.

In addition, optionally, where a PI3K inhibitor is present in cell differentiation media with added components (preferably DMEM/F12), activin a can be used to differentiate MMCs according to the invention into definitive endoderm cell populations. It should be noted that Nodal, TGF, or other TGF members may be used in place of activin a, or concurrently. Also, removal of factors from the differentiation medium that affect/promote PD kinase signaling, including IGF-I and Heregulin, may be used in place of, or in addition to, PB kinase inhibitors.

The definitive endoderm cells can be isolated and stored after they are produced, or can be used to produce pancreatic endoderm cells and/or pancreatic beta cells. Pancreatic endoderm cells are isolated from the definitive endoderm cells by further exposure for one or more days (preferably two days) to DMEM/F12 optionally containing FCS (preferably about 10%) in the presence of retinoic acid and Fgf10(25ng/ml, about 1-75ng/ml, about 5-50ng/ml, about 15-35ng/ml, about 20-30ng/ml) at a concentration of about 0.05-25 μ g/ml, or 0.1-2 μ g/ml. The pancreatic endoderm cells can be treated with an effective concentration of retinoic acid (at the concentrations described above) and Fgf10 for a number of days (about 10-24 days) to further differentiate into pancreatic beta cells, thereby providing pancreatic beta cells.

Hepatic endoderm cells can be produced from definitive endoderm cells by culturing definitive endoderm cells in the absence of retinoic acid but containing an effective amount of fibroblast growth factor (Fgf10) for at least about 2 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 8 days, at least about 10 days, about 10-24 days, allowing them to differentiate to produce hepatic endoderm cells without producing pancreatic endoderm cells, and optionally isolating the hepatic endoderm cells for further use.

In the present invention, prior to differentiation, mesendoderm cells, mesoderm cells (Isl1+) or MMCs, pPSCs (particularly hESCs) are grown/cultured in cell differentiation media and contacted therein with a suitable differentiation agent/inhibitor as described further herein. The hESCs are expected to differentiate by contact with differentiation agents/inhibitors in differentiation media, correspondingly producing mesendoderm cells, mesoderm cells (Isl1+) or MMCs as otherwise described herein. In one embodiment, the cells are dispersed into a substantially single cell culture prior to contacting the cells with the differentiating agent in the basal cell culture medium. Preferably, the cells are grown in an adherent monolayer to allow effective contact of the cells with the differentiation agent/inhibitor, and the adherent cell layer may be dispersed with a protease, such as, but not limited to, Accutase^TM. In one embodiment, the cells are contacted with the differentiation agent/inhibitor after plating for about 12 hours to 10 days or longer, about 12 to 72 hours, about 24 to 72 hours, about 18 to 36 hours, or for a period of time otherwise described herein. In one embodiment, the cells are contacted with the differentiation agent/inhibitor described further herein for more than about 18 hours, more than about 24 hours, more than about 48 hours, more than about 72 hours, more than about 96 hours, more than about 150 hours, more than about 136-152 hours, more than about 144 hours, or for a time further described herein. After exposing the cells to a cell culture medium containing a differentiation agent/inhibitor, the cells can be isolated directly (with Accutase^TMTreating) the obtained cells (usually attached monolayers or embryoid bodies) and then passaged to regenerate the cell population or to further differentiate the cells into another cell population.

In certain embodiments, the concentration of hESCs, mesodermal cells, mesodermal (Isl1+) cells, or MMCs plated to be further differentiated is less than about 2.5 x 10⁶At least about 2.5X 10 cells/35 mm culture dish⁴Individual cells/35 mm culture dish, about 2.5X 10⁵-2×10⁶Individual cells/35 mm culture dish, about 5X 10⁵-2×10⁶Individual cells/35 mm culture dish, less than about 2X 10⁶Individual cells/35 mm culture dish, or density greater than 4X 10⁵Individual cells/35 mm culture dish. In a particularly preferred aspect, the cells to be differentiated are plated at a concentration of about 7.5 × 10 cells/35 mm culture dish.

In the production of mesendoderm cells, mesoderm (Isl1+) cells or MMCs from hESCs as the first step in certain embodiments of the invention, the invention also includes the use of the composition in culturing cells to produce an adherent monolayer of hESCs. The hESCs are grown as adherent monolayers on cell supports (preferably matrigel) in defined cell culture media (serum-free or KSR). The cell culture medium preferably contains, in addition to typical components otherwise described herein, an effective amount of one or more of the following: ascorbic acid, transferrin, beta-mercaptoethanol (Gibco), Fibroblast Growth Factor (FGF), LR-IGF, activin A and Heregulin, and preferably contains all of these components. The cell culture medium used to initiate growth of hESCs adherent monolayer cells (or embryoid bodies) differentiated from the population of cells can vary within the scope of the teachings of the art.

The hESCs produced as described above are then plated onto a cell support and differentiated in differentiation medium (described further herein) containing an effective amount of a differentiation agent and/or inhibitor. The cells are preferably grown in an adherent monolayer. In the case of mesendoderm cells, hESCs are contacted with differentiation medium containing an effective amount of a GSK inhibitor (preferably BIO or Wnt3a) as otherwise described herein for an appropriate period of time (about 18-72 hours as otherwise described herein) to produce a population of mesendoderm cells. In the case of mesodermal cells, hESCs are contacted with a differentiation medium containing effective amounts of a GSK inhibitor (preferably BIO or Wnt3a) and a bone morphogenic protein (BMP-2, BMP-4, BMP-6, BMP-7) as further described herein for a suitable period of time to produce a population of mesodermal (Isl1+) cells (longer differentiation, about 5-10 days, about 4-8 days, about 5-7 days, about 6 days, about 140-150 hours).

In further embodiments, the cell culture medium may be conditioned medium (MEF-CM). The conditioned medium is obtained from a feeder layer. The desired feeder layer may comprise fibroblasts, and in one embodiment, the feeder layer comprises embryonic fibroblasts. Preferably the medium is feeder cells free.

In particularly preferred embodiments, the differentiation medium used to produce mesendoderm cells, mesoderm (Isl1+) cells, or MMCs comprises: DMEM/F12(50/50), about 2% Probumin (albumin), antibody (1x penicillin/streptococcal antibiotic (Pen/Strep)1xNEAA), trace elements A, B, C (e.g. 1x from Mediatech), ascorbic acid (e.g. about 50 μ g/ml), transferrin (e.g. about 10 μ g/ml), β -mercaptoethanol (about 0.1mM), bFGF (e.g. about 8ng/ml), LR-IGF (e.g. about 200ng/ml), activin a (e.g. about 10ng/ml) and Heregulin (e.g. about 10 ng/ml). It should be noted that activin A and Heregulin should be removed when used to generate pluripotent migratory cells (MMCs). Of course, one or more of the above components may not be added to the differentiation medium in the art, but it is preferable to use all the components in the present invention.

The cells of the invention may also be used in bioassays to identify molecules that affect (promote, inhibit or affect) cell differentiation. The first step in cell differentiation of the invention offers a great possibility to study epithelial to mesenchymal transition, especially in the part of tumor metastasis in the development of cancer. Thus, the methods and cell populations according to the invention provide an advantageous system for understanding EMT at the molecular level and identifying new drug targets and screening for small molecules that block EMT under conditions that promote EMT (BIO). Considering that cells can be grown in 96/384-well culture plates, rapid drug screening can be easily performed to identify possible molecules that block or inhibit EMT, and to show possible valuable anticancer agents.

With respect to MMCs, it is a stable population of cells grown in defined media with pluripotent differentiation capacity. These cells are particularly useful in the screening of molecules capable of promoting or inhibiting differentiation or promoting and specifically differentiating into one cell line or another.

Treatment of

The cell populations and/or methods described herein can provide beneficial treatments for the treatment of diseases and/or conditions associated with the cells.

In one aspect, the invention provides a method of treating a patient suffering from a cardiovascular disease. The method comprises the following steps: culturing pluripotent stem cells, differentiating the pluripotent stem cells into cardiovascular muscle cells (cardiomyocytes) in vitro, and implanting an effective amount of the cardiovascular muscle cells into a patient in need thereof. Optionally, the method of treating a patient suffering from cardiovascular disease (including infarction) comprises: an effective amount of mesodermal (Isl1+) cells is administered to the cardiac tissue of a patient in need of treatment.

In another aspect, the present invention provides a method of treating damaged or ischemic vascular tissue (blood vessels) in a patient in need thereof, wherein the method comprises: an effective amount of mesodermal (Isl1+) cells is administered to the vessel to be repaired. In an alternative embodiment, mesodermal (Isl1+) cells are differentiated into smooth muscle cells by passaging the cells in a cell differentiation medium containing effective amounts of a GSK inhibitor, preferably Wnt3a, and BMP (BMP4) for at least 5-6 days, and the smooth muscle cells thus obtained are administered (implanted) to the site of structural vascular injury in a patient for treatment/repair thereof.

In another aspect, the invention provides a method of treating a patient suffering from, or at risk of developing, type 1 diabetes. Such a method comprises: culturing pluripotent stem cells; differentiating the pluripotent stem cells into a beta cell line in vitro; and implanting cells of the beta cell line into the patient.

Another aspect of the invention provides a method of treating a patient suffering from type 2 diabetes or at risk of developing type 2 diabetes. Such a method comprises: culturing pluripotent stem cells; differentiating the pluripotent stem cells into a beta cell line in vitro; and implanting cells of the beta cell line into the patient.

If appropriate, the patient may also be treated with a pharmaceutical agent or bioactive agent that enables the implanted cells to readily survive and function. For example, these pharmaceutical preparations may include: insulin; transforming growth factor-beta (TGF-beta) family members (TGF-beta 1, TGF-beta 2, and TGF-beta 3); bone morphogenic proteins (BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-11, BMP-12, and BMP-13); fibroblast growth factor-1 and fibroblast growth factor-2; platelet-derived growth factor-AA and platelet-derived growth factor-BB; platelet rich plasma; insulin growth factor (IGF-I, IGF-II); growth differentiation factors (GDF-5, GDF-6, GDF-7, GDF-8, GDF-10, GDF-15); vascular Endothelial Growth Factor (VEGF); pleiotrophin (pleiotrophin); endothelin; and other factors. Other pharmaceutical compounds may include: for example, niacinamide; glucagon-like peptide-I (GLP-1) and glucagon-like peptide-II (GLP-2); mimetibodies of GLP-1 and GLP-2 (mimetopy); exendin-4 (incretin analog); tretinoin; thyroid hormone; mitogen-activated protein kinase (MAPK) inhibitors, such as the compounds disclosed in U.S. published application 2004/0209901 and U.S. published application 2004/0132729.

The pluripotent stem cells may be differentiated into insulin-producing cells prior to being transplanted into a recipient. In a specific embodiment, the pluripotent stem cells are fully differentiated into beta-cells prior to being transplanted into a recipient. Alternatively, the pluripotent stem cells may be transplanted into a recipient in an undifferentiated or partially differentiated state. Further differentiation may also occur in the recipient.

Mesodermal (Isl1+) cells and/or cardiomyocytes can be implanted in a dispersed cellular state or form clusters that can be injected directly into the heart or the hepatic portal vein. Alternatively, specific endoderm cells, pancreatic endoderm cells, or β -cells can be implanted in a dispersed cell state, or form clusters that can be injected into the hepatic portal vein. Alternatively, the cells provided may be located in a biocompatible degradable polymer carrier or porous non-degradable device, or the cells may be embedded to protect them from the host's immune response and be capable of implantation at the appropriate site in the recipient. The site of implantation includes: heart, liver, pancreas (natural pancreas), kidney capsule (renal capsule), omentum, peritoneum, subperiosal space (subcutaneous space), intestine, stomach, or subcutaneous pocket (subcutaneous pocket).

To further promote differentiation, survival or activity of the implanted cells, additional factors (e.g., growth factors, antioxidants, immunosuppressive or anti-inflammatory drugs) may be administered prior to, concurrently with, or after implantation of the cells. In particular embodiments, growth factors are used to differentiate the implanted cells in vivo. These factors can be secreted by endogenous cells and exposed to implanted cells in situ. The implanted cells are induced to differentiate by endogenous and exogenous administration of various combinations of growth factors well known in the art.

The amount of cells to be transplanted depends on a number of different factors, including the condition of the patient and the response to treatment, and can be determined by one skilled in the art.

In another aspect, the invention provides a method of treating a patient suffering from or at risk of developing cardiovascular disease or diabetes. Such a method comprises: culturing pluripotent stem cells; differentiating the pluripotent stem cells into a cardiomyocyte lineage or β -cells in vitro; and incorporating the resulting cells into a three-dimensional vector. The cells may be maintained on the carrier in vitro prior to implantation into a patient. Alternatively, the vector containing the cells may be implanted directly into the patient without additional in vitro culture. Optionally, the carrier may have at least one pharmaceutical agent capable of facilitating survival and functioning of the transplanted cells, and in other aspects, the carrier may also be used to treat diabetes, or cardiovascular disease or dysfunction.

Materials suitable for the support object of the present invention include: tissue templates (tissue templates), catheters (conduits), barriers (barriers), and containers (reservoirs) for tissue repair. In particular, foamed, spongy, gelatinous, hydrogel-like, fabric-like, and non-fabric structured synthetic and natural materials for the reconstruction or regeneration of biological tissues and the delivery of chemotactic agents (chemotactic agents) for inducing tissue growth, both in vitro and in vivo, are suitable for use in practicing the methods of the present invention. See, for example, the materials disclosed in the following patents: U.S. patent No.5,770,417, U.S. patent No.6,022,743, U.S. patent No.5,567,612, U.S. patent No.5,759,830, U.S. patent No.6,626,950, U.S. patent No.6,534,084, U.S. patent No.6,306,424, U.S. patent No.6,365,149, U.S. patent No.6,599,323, U.S. patent No.6,656,488, U.S. published application 2004/0062753a1, U.S. patent No.4,557,264, and U.S. patent No.6,333,029.

To form a carrier with a drug formulation, the drug formulation may be mixed with a polymer solution prior to forming the carrier. Optionally, the pharmaceutical formulation is coated on a preformed carrier, preferably in the presence of a pharmaceutically acceptable carrier. The pharmaceutical formulation may be in liquid, solid powder or other suitable physical form. Optionally, excipients may be added to the carrier to modify the release rate of the pharmaceutical formulation. In an alternative embodiment, the carrier is combined with at least one pharmaceutical compound (anti-inflammatory compound), such as the compound disclosed in U.S. patent No.6,509,369.

The carrier may be combined with at least one pharmaceutical compound (anti-apoptotic compound), for example, the compound disclosed in U.S. patent No.6,793,945. The carrier may be combined with at least one pharmaceutical compound (compound that inhibits fibrosis), for example, a compound disclosed in U.S. patent No.6,331,298. The carrier may be associated with at least one pharmaceutical compound (a compound capable of promoting angiogenesis), for example, compounds disclosed in U.S. published application 2004/0220393 and U.S. published application 2004/0209901. The carrier may be combined with at least one pharmaceutical compound (immunosuppressant compound), for example, a compound disclosed in U.S. published application 2004/0171623.

The carrier may be combined with at least one pharmaceutical compound (growth factor), for example, a TGF- β family member (TGF- β 1, TGF- β 2, and TGF- β 3); bone morphogenic proteins (BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-11, BMP-12, and BMP-13); fibroblast growth factor-1 and fibroblast growth factor-2; platelet-derived growth factor-AA and platelet-derived growth factor-BB; platelet rich plasma; insulin growth factor (IGF-I, IGF-II); growth differentiation factors (GDF-5, GDF-6, GDF-7, GDF-8, GDF-10, GDF-15); vascular Endothelial Growth Factor (VEGF); pleiotrophin (pleiotrophin); endothelin; and other factors. Other pharmaceutical compounds may include, for example, nicotinamide; hypoxia inducible factor 1-alpha; glucagon-like peptide-I (GLP-1) and glucagon-like peptide-II (GLP-2); mimetibodies of GLP-1 and GLP-2 (mimetopy); exendin-4 (incretin analog); nodal; noggin; nerve Growth Factor (NGF); tretinoin; thyroid hormone; tenascin-C; a tropoelastin; a thrombin-derived peptide; cathelicidins (an antimicrobial peptide); a defensin; laminin; biological peptides containing cell binding domains of adherent extracellular matrix proteins and heparin binding domains, e.g., fibronectin and vitronectin; mitogen-activated protein kinase (MAPK) inhibitors, such as the compounds disclosed in U.S. published application 2004/0209901 and U.S. published application 2004/0132729.

The binding of the cells of the invention to the scaffold can be achieved by simply depositing the cells on the scaffold. The cells are able to enter the scaffold by simple diffusion (J.Peditar. Surg.23(1Pt 2): 3-9 (1988)). Various other approaches have been developed to enhance the efficiency of cell seeding. For example, spinner flasks (spinner flash) may be used during seeding of chondrocytes to polyglycolic acid (Biotechnol. prog.14 (2): 193-202 (1998)). Other methods of seeding cells are by centrifugation, which creates minimal pressure on the seeded cells and increases the efficiency of seeding. For example, one method of cell seeding disclosed by Yang et al involves centrifugation of cells to immobilize (CCI) (J.biomed.Mater.Res.55 (3): 379-86 (2001)).

The present invention may be better understood by reference to the following detailed description of preferred embodiments of the invention and the examples included herein. However, before the compositions and methods are disclosed and described, it is to be understood that this invention is not limited to these specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions or specific methods, etc., as various modifications and improvements will be apparent to those skilled in the art.

Examples

The following examples are provided to describe the invention in more detail. It should be noted that the following examples are not to be construed as limiting the invention in any way.

All embodiments can use, but are not limited to, a variety of human embryonic stem cell lines (hescs), including: BG01, BG02, WA 09.

Example 1: culture of human embryonic stem cells

(a) Human embryonic stem cells

Human embryonic stem cells (hESCs; FIG. 1) were isolated from the inner cell line (ICM) at the blastocyst stage or from late-stage pre-implantation embryos (late pre-implantation embryos). Their ability to serve as an experimental tool in developmental biology derives from their ability to self-renew and to differentiate into three embryonic germ layers (ectoderm, endoderm and mesoderm) and extra-embryonic cell lines (figure 2) that respond to specific signals. Since the nature and behavior of human embryonic stem cells (hESCs) recapitulate a number of embryonic processes, they can be used to understand the mechanisms by which pluripotent stem cells develop in ectoderm and differentiate into germ layers during gastrulation. They may also serve as a source of material for the generation of therapeutically useful cell types.

(b) Method of culturing hescs

The method comprises the following steps: expression of hESCs comprising markers such as POU domain transcription factor Oct4 are preferably grown in mouse embryo feeding conditioned medium MEF-CM or in defined medium using matrigel as growth substrate (for example). Typically, 1-1.5X 10 of seed is plated per 60mm dish⁶And (4) cells. Cells were passaged every 4-5 days at a ratio of 1: 4 to 1: 10.

(i) Mouse embryo fibroblast conditioned medium (MEF-CM)

In the presence of Fgf2, hESCs can be grown in matrigel (BD biosciences; dilution of 1: 20 to 1: 200 is preferred) or other matrix that can be used to support the maintenance of hESCs in mouse embryo fibroblast conditioned media (McLean et al. Stem Cells 25: 29). The cells may be cultured by methods including enzymatic methods (pancreatin, Accutase)^TMCollagenase), artificial (mechanical) and non-enzymatic methods. A typical 1.5X 10 plating per 60mm dish⁶And (4) cells. Cells were passaged every 4-5 days at a ratio of 1: 4 to 1: 10.

(ii) Determined Conditions (DC)

(a) A defined medium for routine culture of hESCs was purchased from Invitrogen(Wang et al, Blood 110: 4111). Except Accutase^TMFor passaging cells with single cell suspensions, according to the manufacturer's recommended recipeThe method uses the culture medium. This formulation was prepared routinely on our own, when we needed to change the composition of the medium to perform differentiation experiments. This formulation is summarized below and has the ability to maintain hESCc in a pluripotent state (pluralittorate). Serum-free medium conditions identified below work well, but are not limited to this particular formulation and may include feeder-free culture: DMEM: f12(Gibco), 2% BSA (serum, #82-047-3), 1 XPcillin/streptococcal antibiotic (Pen/Strep) (Gibco), 1 XP nonessential amino acids (Gibco), 1 XP microelements A, B and C (Cellgro; #99-182-Cl, #99-176-Cl, #99-175-Cl), 50 ug/ml ascorbic acid (Sigma, # A4034), 10ug/ml transferrin (Gibco, #11107-018), 0.1nM beta mercaptoethanol, 8ng/ml Fgf2(Sigma, # F0291), 200ng/ml LR-IGF (JRH Biosciences, #85580), 10ng/ml activin A (R)&DSystems, #338-AC), Heregulin-beta at 10ng/ml (Peprotech; # 100-03).

(b) hESCs can also be cultured in additional commercially available defined media formulations (e.g., mTeSRl (BD/Stem Cell Technologies; Ludwig et al, Nat biotechnol.24: 185)) according to manufacturer's recommended procedures. Accutase may be substituted^TMPassages were used in conjunction with this medium.

Differentiation Capacity of hESCs

hESCs have the ability to differentiate into each of the three embryonic germ layer cells (ectodermal, endodermal and mesodermal) and an additional extraembryonic cell line (fig. 2). Thus, they are starting points for the generation of a variety of different therapeutically useful cell types.

Example 2: methods of producing mesendoderm cells

Differentiation of hESCs into mesodermal and endodermal cells involved the transition of the cells through the intermediate cell type of T +/Brachyury + mesendodermal cells by the addition of factors (e.g., Wnt3a and GSK inhibitors) that induce differentiation (fig. 3).

(a) Production of mesendoderm cells from hESCs using Wnt

The method involves differentiation of hESCs into mesendoderm cells by addition of the canonical Wnt signaling molecule Wnt3 a.

(a-i) production of mesendoderm cells using Wnt3a in Medium containing Low levels of activin A

hESCs (BG01) were plated in defined media under the specific conditions of example 1. After 16-24 hours, human Wnt3a (25 ng/ml; R & D Systems) was added to the cultures. Q-PCR analysis showed a decrease in the amount of Nanog transcripts at 48 hours; the mRNA levels of T, Eomes and MixL1 increased during days 1-2, and then decreased on day 5 (fig. 4A).

E-cadherin and Nanog downregulation after 48 hours was determined using immunostaining. Over this time frame, β -catenin (catenin) and Snail accumulated in the nucleus and T/brachyury levels increased (fig. 4B). In combination with the Q-PCR data, these results indicate that Wnt3a caused differentiation of hESCs into mesendoderm cells, including epithelial to mesenchymal transition.

(a-ii) use of Wnt3a to produce mesendoderm cells in the absence of activin A signaling

hESCs were differentiated into mesendoderm cells by addition of Wnt3a (25ng/ml) to defined media supplemented with SB431542(20 μ M). This approach is feasible because hescs differentiate into mesendoderm cells do not require activin a signaling, but rely on activation of the canonical Wnt signaling pathway (fig. 5).

(a-iii) use of Wnt3a in activin A-free media to produce mesendoderm cells

hESCs were differentiated into mesendoderm cells by addition of Wnt3a (25ng/ml) to defined media or MEF-CM lacking activin A. This approach is feasible because hescs differentiate into mesendoderm cells do not require activin a signaling, but rely on activation of the canonical Wnt signaling pathway (fig. 5).

(a-iv) use of Wnt3a to produce mesendoderm cells in the absence of activin A signaling and in the presence of SB431542

hESCs were differentiated into mesendoderm cells by addition of Wnt3a (25ng/ml) to defined media in the absence of activin a and in the presence of activin a signaling inhibitor SB 431542. This approach is feasible because the differentiation of hescs into mesendoderm cells does not require activin a signaling.

(b) Production of mesendoderm cells from hESCs using GSK inhibitors

The method involves differentiation of hESCs into mesendoderm cells by addition of GSK inhibitor BIO ((2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime, GSK3 inhibitor IX, Calbiochem).

(b-i) production of mesendoderm cells from hESCs Using BIO in defined Medium

hESCs (BG01) were plated in defined media under the specific conditions of example 1. After 16-24 hours, BIO (2. mu.M) was added to the culture. Q-PCR analysis showed a decrease in the amount of Nanog transcripts at 48 hours; mRNA levels of T and MixL1 increased and then decreased at the first 24 hours (fig. 6). E-cadherin and Nanog downregulation after 48 hours was determined using immunostaining. Over this time frame, Snail accumulated in the nucleus and T/brachyury levels increased (FIG. 6). These results, combined with the Q-PCR data, indicate that BIO caused hESCs to differentiate into mesendoderm cells, involving an epithelial to mesenchymal transition.

(b-ii) addition of GSK inhibitor BIO to hESCs grown in MEF-CM

hESCs (BG02) were grown in MEF-CM on matrigel-covered dishes and passaged on day 4 in MEF-CM supplemented with BIO (2. mu.M). The first experiment shows cell passaging with pancreatin. In this experiment, BIO treatment caused down-regulation of Oct4 and E-cadherin, and up-regulation of T/Brachyury (FIGS. 7A, 7B). This indicates that BIO treatment promotes epithelial to mesenchymal transition and differentiation into mesendoderm cells. The second experiment was similar to the first experiment, adding BIO to hESCs cultured in MEF-CM, and this experiment was passaged with collagenase. BIO treatment caused down-regulation of Nanog and E-cadherin, and up-regulation of T/Brachyury (FIG. 8A, FIG. 8B). These results again indicate that inhibition of GSK by BIO results in the differentiation of mesendoderm cells and is associated with epithelial to mesenchymal transition.

(b-iii) differentiation of cells into mesendoderm cells by addition of GSK inhibitors (BIO and SB431542)

hESCs (BG02) grown on matrigel in defined medium with Accutase^TMPassage was into defined medium supplemented with BIO (2. mu.M) and SB431542 (20. mu.M). The differentiation of these cells over a period of 8 days was monitored using a Q-PCR assay. This data shows that BIO/SB431542 treatment caused a decrease in the amount of Nanog transcripts within 24-48 hours, indicating a loss of pluripotency (FIG. 9). After 24 hours, T/Brachyury transcript levels increased significantly and remained over this period. The mesodermal (FoxF1, PDGFR α) and endodermal (Swx17, CXCR4) cell markers were not significantly altered during this time (fig. 9). These results indicate that BIO can promote differentiation of hESCs into mesendoderm cells in the absence of activin A signaling due to the inhibitory effect of SB 431542.

(b-iv) differentiation of hESCs into mesendoderm cells by treatment with GSK inhibitors in media lacking activin A

The differentiation of hESCs into mesendoderm cells was performed by adding BIO to hESCs grown in MEF-CM or defined media lacking activin A. This is based on our previous study showing that mesendoderm cell differentiation is dependent on activin a signaling and that BIO can promote differentiation of EMT/mesendoderm cells in MEF-CM and defined media (figure 5).

(b-v) differentiation of hESCs into mesendoderm cells by treatment with GSK inhibitors in media containing high concentrations of activin A

The differentiation of hESCs into mesendoderm cells was performed by adding BIO to hESCs grown in MEF-CM or defined media containing high levels of activin A (100 ng/ml). This is based on our previous study showing that mesendoderm cell differentiation is dependent on activin a signaling and that BIO can promote EMT/mesendoderm cell differentiation in MEF-CM and defined media.

Example 3: methods of generating mesoderm-derived Isl1+ pluripotent progenitor cells (IMPs)

This example describes a method of generating mesoderm-derived Isl1+ pluripotent progenitor cell (IMPs) cell types that have the ability to differentiate into cardiomyocytes, smooth muscle cells, or endothelial cells. The cell type differentiated via a pathway through the mesendoderm cell state and then to mesoderm cells (fig. 10).

(a) Addition of Wnt3a and BMP4 to hESC cultures to generate ISl1+ pluripotent progenitors (IMP)

In thatBG02hESCs grown in medium was determined with Accutase^TMPassaging, removal of media supplemented with BMP4(100ng/ml, R)&D Systems) and human Wnt3a (R)&D Systems) in matrigel-covered Petri dishes (1.0X 10 inoculations per 60mm Petri dish) as described in example 1⁶Individual cells). The medium was changed daily. Q-PCR analysis was performed over 240 hours (10 days) to assess differentiation. This analysis showed that markers of mesendoderm cells (e.g. T) increased at 24 hours post-treatment, and then decreased (figure 11). After 24 hours of treatment, transcript markers indicative of mesodermal cell differentiation were significantly upregulated (Isl1, PDGFR α, KDR, Tbx20, GATA4) (fig. 11).

Immunostaining revealed that most cells stained positive for T within 24-96 hours after treatment, while the number of such cells decreased by 144 hours (fig. 12). More than 90% of the cells stained positive for nkx2.5, Isl1 and Tbx20 after 6 days (144 hours) of treatment with BMP4 and Wnt3a (figure 13). This gene expression profile represents a pluripotent Isl1+ progenitor cell from a second cardiac site (secondary heart field) (Laughitz et al, Development 135: 1933-. Differentiation into Isl1+ cells was accompanied by different morphological changes of the cells (fig. 14).

(b) Isl1+ pluripotent progenitor cells (IMP) were generated by addition of Wnt3a on days 1-3 followed by BMP4

hESCs grown in MEF-CM or defined media were treated with Wnt3a for the first 1-3 days followed by additional treatment with BMP4 for 2-4 days to generate Isl1+ mesodermal cells (fig. 15).

(c) Addition of BMP4 and GSK inhibitors (e.g., BIO) to hESCs in MEF-CM to generate Isl1+ pluripotent progenitor cells (IMP)

BG02hESCs grown on matrigel in MEF-CM were passaged with pancreatin and 1.5X 10 inoculates per 60mm dish⁶The density of individual cells was seeded back onto matrigel in MEF-CM supplemented with BIO (2. mu.M) and BMP4(100 ng/ml). The medium was changed daily. Q-PCR analysis was performed over 240 hours (10 days) to assess differentiation. The treated cells underwent morphological changes indicative of differentiation (fig. 16) compared to hESCs (fig. 1). Q-PCR analysis of transcript levels showed that hESC markers (e.g., Nanog, Oct4, Lefyt a) decreased 48 hours after treatment, while mesendoderm cell markers (T, MixL1) peaked at 48 hours and then declined at 96 hours. When levels of mesendoderm cell markers decreased, early mesoderm cell markers (FoxF1, GATA4, Isl, Tbx20, PDGFR α, PDGFR β) increased from 24-48 hours (fig. 17). These marks indicate IMPs formation.

(d) Addition of BMP4 and GSK inhibitors (e.g., BIO) to hESCs cultured in defined media to generate Isl1+ pluripotent progenitor cells (IMP)

hESCs were differentiated into Isl1+ progenitor cells (IMP) by adding BMP4 and BIO to hESCs cultured in defined media. Treated with BMP4 and BIO for 6 days (fig. 10).

(d) Treatment with GSK inhibitors (e.g. BIO) for 1-3 days, followed by addition of BMP4 to generate Isl1+ pluripotent progenitor cells

hESCs grown in MEF-CM or defined media were treated with GSK inhibitors (e.g., BIO) for 1-3 days followed by BMP4 for 2-4 days to generate Isl1+ mesodermal cells from hESCs (fig. 15).

(e) Adding Wnt3a, BMP4, and a TGF-beta inhibitor (e.g., SB431542) to hESC cultures to produce ISl1+ pluripotent progenitor cells (IMP)

hESCs grown in MEF-CM or defined media were treated for 1-4 days with Wnt3a, BMP4 and TGF β inhibitors (e.g., SB431542) added, followed by removal of TGF β inhibitor and continued culture with Wnt3a and BMP4 for 2-4 days to produce Isl1+ mesodermal cells from hESCs (fig. 18).

(f) Isl1+ pluripotent progenitor cells are generated by treatment with Wnt3a and a TGF-beta inhibitor (e.g., SB431542) for 1-4 days, followed by additional 2-4 days of culture with BMP4

hESCs grown in MEF-CM or defined media were treated with Wnt3a and TGF β inhibitors (e.g., SB431542) for 1-4 days, followed by BMP4 for 2-4 days to generate Isl1+ mesodermal cells from hESCs (fig. 19).

(g) Isl1+ pluripotent progenitor cells were generated by treatment with Wnt3a and SB431542 for 1-3 days followed by additional culture with BMP4 for 2-4 days

hESCs grown in MEF-CM or defined media were treated with Wnt3a and SB431542 additions for 1-3 days followed by BMP4 additions for 2-4 days to generate Isl1+ mesodermal cells from hESCs.

Example 4: IMPs can differentiate into cardiac cell lines, endothelial cells, cardiac myocytes and smooth muscle cells

(a) Generation of smooth muscle cells from IMPs

hESCs were grown for 6 days in defined media in the presence of Wnt3a (25ng/ml) and BMP4(100 ng/ml). The cells were seeded in the same medium at a ratio of 1: 4 to 1: 6 and cultured for 4 days. The cells were then fixed and stained for smooth muscle markers (actin, Calponin (a calcium binding protein), Caldesmin (a calmodulin binding protein) and SM-MHC) (fig. 20). Most cells were able to stain for these smooth muscle markers.

(b) Generation of cardiomyocytes and endothelial cells from IMPs

IMPs are generated by three different processes. Treatment 1: hESCs were grown in defined media containing activin A (100ng/ml), Wnt3a (25ng/ml) for 1-4 days, and BMP4(100ng/ml) for 2-6 days at the first 24 hours. And (3) treatment 2: hESCs (BG02) were grown for 1-4 days in defined media containing Wnt3a (25ng/ml) with IGF-I, Heregulin and FGF2 removed, and for 2-6 days in defined media containing BMP4(100 ng/ml). And (3) treatment: hESCs were grown in defined media containing activin A (100ng/ml), Wnt3a (25ng/ml) for 1-2 days, and BMP4(10ng/ml) for 2-6 days at the first 24 hours. At the end of day 6, the cells were placed in defined medium for an additional 14 days. Cells were harvested and then subjected to Q-PCR analysis, which showed that treatment 2 produced endothelial cell markers (CD31/Pecam1 and CDH 5/VE-cadherin) and treatment 3 produced cardiomyocyte markers (ACTC/myocardial alpha actin, and cTNT) (FIG. 21). These results show that IMP cells can differentiate into cardiomyocytes and endothelial cells.

Example 5: combinatorial characterization of mesoderm-derived Isl1+ pluripotent progenitor cells (IMP)

Islet 1+ multipotent progenitor cells (IMPs) have the following characteristics:

expression of IsIl, Tbx20, Nkx2.5, Fgf10, GATA4, KDR (Flk1), FoxF1, PDGFR α

Karyotype normality

No expression of Oct4, Nanog, T, Eomesoderm

Can differentiate into myocardial cells, smooth muscle cells and endothelial cells

No teratoma formation when injected into the hindlimb muscle of SCID mice

Microarray analysis was performed on the IMPs formed. hESCs were cultured for 6 days in defined medium supplemented with Wnt3a (25ng/ml) and BMP4(100 ng/ml). Samples were taken at 0, 24, 48, 72, 96, 144 hours to extract mRNA, followed by microarray analysis. This microarray analysis is summarized in the table attached to this document. (IMP microarray)

Example 6: IMP cells may be used for cellular therapy of cardiovascular disease; heart and vessels

Since IMP cells have a major cell line that differentiates into cardiac vascular cells, IMP cells can be used for cell therapy. For example, they can be used for regeneration of damaged myocardium after their implantation by a person skilled in the art. They may also be used to repair damaged vessels after implantation by those skilled in the art.

Example 7: method for producing definitive endoderm cells (DE)

(a) Treatment of hescs with Wnt and TGF β inhibitors (e.g., SB431542) and subsequently with high concentrations of activin a to produce DE

To defined hESC medium was added Wnt and SB431542 treatment for 1-3 days, followed by removal of Wnt and SB431542 as well as IGF-I and Heregulin, and addition of high concentration activin (50-100ng/ml) for an additional 1-3 days to form DE. Hescs can be passaged onto matrigel-covered petri dishes or equivalents (fig. 22).

(b) Treatment of hescs with GSK inhibitor (BIO) and high concentration of activin a to produce DE

To the hESC-defined medium (without IGF-I and Heregulin) was added BIO (2. mu.M) and high concentration activin (50-100ng/ml) for 3-5 days (FIG. 23). Hescs can be passaged onto matrigel-covered petri dishes or equivalents.

(c) Treatment of hESCs with BIO and Low concentration activin A (10ng/ml) to form DE

To the hESC-defined medium (without IGF-I and Heregulin) BIO (2-5. mu.M) was added for 3-5 days (FIG. 24). Hescs can be passaged onto matrigel-covered petri dishes or equivalents.

(d) DE formation from hESC by addition of BIO for 1-3 days followed by addition of activin A for 1-3 days

The hESC confirmation medium was treated with BIO (2 μ M) for 1-3 days to reach the stage of mesendoderm cells, followed by removal of BIO as well as IGF-I and Heregulin and addition of activin a for 1-3 days (fig. 22). Hescs can be passaged onto matrigel-covered petri dishes or equivalents.

(e) Treatment for 1-4 days by addition of BIO and SB431542 followed by 1-3 days with activin A to yield DE

Hescs were grown for 4 days in defined medium in the presence of BIO (2 μ M) and SB431542(20 μ M). The cells were then grown in 1-4 aliquots in the same medium or in a medium without IGF-I and Heregulin plus activin A (100ng/ml) for 4 additional days (FIG. 22). Samples (BG02) were taken every 2 days up to 8 days for Q-PCR analysis. In the first step BIO/SB431542 differentiated cells into T/brachury positive, Nanog, Sox17 and CXCR4 negative cells on the first 2 days. This cell type was maintained in the presence of BIO/SB 431542. When the cells were transferred to defined medium supplemented with activin a (100ng/ml) (without IGF-I and Heregulin), they further differentiated into DE, as evidenced by upregulation of the DE markers Sox17 and CXCR4, and the lack of the mesodermal cell marker FoxF1 (fig. 25). Hescs can be passaged onto matrigel-covered petri dishes or equivalents.

Example 8: method of generating pluripotent mesenchymal cells (MMCs)

In thatBG02hESCs grown in medium was determined with Accutase^TMSubculture, plating onto matrigel-covered Petri dishes (1.0X 10 per 60mm Petri dish) as described in example 1, except that the medium was supplemented with BIO (2. mu.M) and SB431542 (20. mu.M; Sigma)⁶Individual cells). Changing the culture medium every day, and adding Accutase every 5-6 days^TMAnd (4) carrying out passage, and separating the cells in a ratio of 1: 5-1: 10 for each passage. When cultured under such conditions, the pluripotency marker Nanog decreased and T transcript levels increased in the first generation (P0), while Sox7, FoxF1, CXCR4 and PDGFR α continued to be low (fig. 26). After 4 days of treatment with BIO and SB431542, 90% of the cells stained + ve, indicating that they were transformed at some point via the mesendoderm cell stage (FIG. 27A). Immunostaining showed that during this periodOct4 and E-cad (E-cadherin) were significantly downregulated (FIG. 27B, FIG. 27C). Hours for E-cadherin indicate that the cells have undergone an epithelial to mesenchymal transition, consistent with the differentiation of mesendoderm cells. For the case of continuous passage, T expression decreased during P1-P10 (as determined by Q-PCR) and the pluripotency marker Nanog no longer appeared (FIG. 28). This result was confirmed by immunostaining, in which P7 cells did not express Nanog, Oct4, or E-cadherin, as opposed to hESCs (fig. 29). During this time period, the markers of mesodermal and endodermal cells did not increase. To establish the fate of the BIO/SB431542 treated cells, they were continuously passaged under the same conditions and found to maintain potent proliferative activity and retain morphology within 20 passages (FIG. 30A). MMCs were frozen using standard methods and resuscitated at a plating efficiency (plating efficacy) of greater than 10%. The growth characteristics and morphology of the frozen MMCs differed from those of the MMCs before freezing (FIG. 30B).

Among the MMCs generated from BG02hESCs, CXCR4 antibody was used to enrich the CXCR4+ cell population, demonstrating that the MMCs can be selected, replated and expanded with fluorescence-active cell sorting (FACS) (fig. 30C).

Flow cytometric analysis showed that hESCs treated with BIO/SB431542 lost cell surface markers SSEA3 and SSEA4, indicating a differentiation pathway from pluripotent hESC status (fig. 31). Treated cells maintained from P0-P19 were consistently deficient in SSEA3 and SSEA4 (fig. 31). Thus, the treated cells are not hESCs as demonstrated by this standard.

Although the presence of mesodermal and endodermal cell markers was detected in P0-P3 cells (for example), no signals were observed to differentiate into mesodermal and endodermal cell lineages, suggesting that BIO/SB431542 treatment arrests the developmental stage of the cells before the presence of mesodermal and endodermal cell lineages. These treated cells were considered to be pre-mesodermal and pre-endodermal cells, and no longer hESCs. This increases the likelihood that these cells retain pluripotent differentiation capacity after removal of BIO/SB431542 and addition of cell differentiation factors. To test the possibility that the cells could be further differentiated into endodermal cells in BIO/SB431542 containing medium, the cells were cultured for 6 passages in BIO/SB431542, and then BIO/SB431542 as well as IGF-I and Heregulin (two activators of PI3K activity) were removed from the medium and supplemented with activin A to 100 ng/ml. These are conditions under which late hESCs differentiate into definitive endoderm cells. Within 4 days after altering the differentiation conditions of endoderm cells, an increase in the amount of Sox17 and CXCR4 transcripts was observed, while Nanog, T, and eopolysidermin transcripts remained low (fig. 32). These results indicate that cells passaged in BIO/SB431542 retain the potential to produce endodermal cells and are induced to differentiate towards endodermal cells when cultured under appropriate conditions. If the BIO/SB 431542-treated cells are exposed to the appropriate specific factors, the cells can also differentiate into mesodermal cells and possibly other cell lines (ectodermal cells). For example, if BIO/SB431542 is removed from the medium and BMP4 is added, BIO/SB431542 treated cells can be converted to mesodermal cells.

Treatment of hESCs with BIO/SB431542 results in a self-renewing population of cells with stable characteristics that can differentiate into endodermal and possibly into mesodermal cells. Due to this mesenchymal nature, this cell type is called Multipotent Mesenchymal Cell (MMC) (fig. 33).

Example 9: combinatorial characterization of pluripotent mesenchymal cells (MMCs)

The Multipotent Mesenchymal Cells (MMC) have the following characteristics:

capable of culturing as a stable cell population for at least 20 passages

Mesenchymal cells when plated at low density and grown into sheets when cultured at high density

The ability to generate hESC cell lines including BG01, BG02, WA09

MMCs can be frozen and cryopreserved using standard methods

MMCs are capable of recovering from cryopreservation (recovered after cryopreservation), resuscitation (recovered) and differentiation

MMCs can be passaged with high plating efficiency

Absence of SSEA3 and SSEA4 antigens on their cell surface

No expression of hESC markers, e.g. Oct4, Nanog

The surface of MMCs is capable of expressing CXCR4

MMCs are capable of expressing high levels of the following transcripts: zicl, HoxA9, HoxD4, HoxC6, N-CAM

MMCs are not mesendoderm cells because they do not express the T/mouse short-tail mutant phenotype (brachyury) or Eomesoderm

E-cadherin negative

MMCs do not express high levels of Sox17, Isl1, Musashi, Nestin (Nestin)

Maintenance of normal karyotype during passaging

Display of a wandering, mesenchymal phenotype

Pluripotent differentiation (including mesodermal and endodermal cells)

Non-teratoma formation when injected into SCID mice

More complete description of MMC gene expression profiles see microarray data

Claims (72)

1. A method of differentiating primate pluripotent stem cells into mesendoderm cells comprising (a) providing primate pluripotent stem cells, and (b) contacting the primate pluripotent stem cells with an effective amount of a GSK inhibitor in a cell differentiation medium for at least about 18 hours to produce mesendoderm cells, and (c) optionally, isolating the mesendoderm cells.

2. The method of claim 1, wherein the primate pluripotent stem cells are human embryonic stem cells.

3. The method of claim 1 or 2, wherein the GSK inhibitor is selected from the group consisting of:

BIO (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime (GSK3 inhibitor IX);

BIO-acetoxime (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -acetoxime (GSK3 inhibitor X);

(5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine (GSK 3-inhibitor XIII);

pyridocarbazole-cyclopentadienyl ruthenium complex (GSK3 inhibitor XV);

TDZD-84-benzyl-2-methyl-1, 2, 4-thiadiazole-3, 5-dione (GSK3 beta inhibitor I);

2-thio (3-iodobenzyl) -5- (1-pyridinyl) - [1, 3, 4] -oxadiazole (GSK3 β inhibitor II);

OTDZT 2, 4-benzhydryl-5-oxothiadiazole-3-thione (GSK3 β inhibitor III);

alpha-4-dibromoacetophenone (GSK3 beta inhibitor VII);

AR-a014418N- (4-methoxybenzyl) -N' - (5-nitro-1, 3-thiazol-2-yl) urea (GSK-3 β inhibitor VIII);

3- (1- (3-hydroxypropyl) -1H-pyrrolo [2, 3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-pyrrole-2, 5-dione (GSK-3 β inhibitor XI);

TWS119 pyrrole pyrimidine compounds (GSK3 β inhibitor XII);

L803 H-KEAPPAPPQSpP-NH₂or its myristoyl form (GSK3 β inhibitor XIII);

2-chloro-1- (4, 5-dibromo-thiophen-2-yl) -ethanone (GSK3 β inhibitor VI); and

mixtures thereof.

4. The method of claim 1 or 2, wherein the GSK inhibitor is a Wnt protein.

5. The method of claim 4, wherein the Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt14, Wnt14b, Wnt15, Wnt16, and mixtures thereof.

6. The method of any one of claims 1-3, wherein the GSK inhibitor is BIO (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime (GSK3 inhibitor IX).

7. The method of any one of claims 1-2 and 4-5, wherein the Wnt protein is Wnt3 a.

8. The method of any one of claims 1-7, wherein the contacting step is for a time period ranging from about 18 to 72 hours.

9. The method according to any one of claims 1 to 8, wherein the cell differentiation medium is at least a minimal essential medium supplemented with one or more optional ingredients selected from the group consisting of growth factors, ascorbic acid, glucose, non-essential amino acids, salts including trace elements, glutamine, insulin, activin A, transferrin, and beta mercaptoethanol.

10. The method of any one of claims 1-9, wherein the cell differentiation medium is selected from the group consisting of Dartback Modified Eagle Medium (DMEM), knock-out dartback modified eagle medium (KO DMEM), Ham's F12/DMEM minimal medium (50: 50), and Ham's F12/DMEM minimal medium further comprising albumin, antibiotics, trace minerals, ascorbic acid, β -mercaptoethanol, fibroblast growth factor, and insulin-like growth factor analogs.

11. The method of any one of claims 1 to 9, wherein the cell differentiation medium is DMEM/F12 (50: 50) containing effective amounts of albumin, penicillin/streptococcal antibiotics, non-essential amino acids, trace minerals, ascorbic acid, β -mercaptoethanol, fibroblast growth factor, insulin-like growth factor analogs, activin a and Heregulin.

12. A method of differentiating primate pluripotent stem cells into mesodermal (Isl1+) cells, the method comprising (a) providing primate pluripotent stem cells; (b) contacting the primate pluripotent stem cells with an effective amount of a GSK inhibitor in a cell differentiation medium for at least about 18 hours to produce mesendoderm cells; and (c) contacting said cells obtained from step (b) with an effective amount of bone morphogenetic protein (bmps) and optionally a GSK inhibitor for at least about 2 days to produce said mesodermal cells; and (d) optionally, isolating the mesodermal cell.

13. The method of claim 12, wherein the primate pluripotent stem cells are human embryonic stem cells.

14. The method of claim 12 or 13, wherein the GSK inhibitor is selected from the group consisting of:

BIO (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime (GSK3 inhibitor IX);

BIO-acetoxime (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -acetoxime (GSK3 inhibitor X);

(5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine (GSK 3-inhibitor XIII);

pyridocarbazole-cyclopentadienyl ruthenium complex (GSK3 inhibitor XV);

TDZD-84-benzyl-2-methyl-1, 2, 4-thiadiazole-3, 5-dione (GSK3 beta inhibitor I);

2-thio (3-iodobenzyl) -5- (1-pyridinyl) - [1, 3, 4] -oxadiazole (GSK3 β inhibitor II);

OTDZT 2, 4-benzhydryl-5-oxothiadiazole-3-thione (GSK3 β inhibitor III);

alpha-4-dibromoacetophenone (GSK3 beta inhibitor VII);

AR-a014418N- (4-methoxybenzyl) -N' - (5-nitro-1, 3-thiazol-2-yl) urea (GSK-3 β inhibitor VIII);

3- (1- (3-hydroxypropyl) -1H-pyrrolo [2, 3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-pyrrole-2, 5-dione (GSK-3 β inhibitor XI);

TWS119 pyrrole pyrimidine compounds (GSK3 β inhibitor XII);

L803 H-KEAPPAPPQSpP-NH₂or its myristoyl form (GSK3 β inhibitor XIII);

2-chloro-1- (4, 5-dibromo-thiophen-2-yl) -ethanone (GSK3 β inhibitor VI); and

mixtures thereof.

15. The method of claim 12 or 13, wherein the GSK inhibitor is a Wnt protein.

16. The method of claim 15, wherein the Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt14, Wnt14b, Wnt15, Wnt16, and mixtures thereof.

17. The method of any one of claims 12-14, wherein the GSK inhibitor is BIO (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime (GSK3 inhibitor IX).

18. The method of any one of claims 12-13 and 16-17, wherein the Wnt protein is Wnt3 a.

19. The method of any one of claims 12-18, wherein the contacting step (b) is for a time period ranging from about 18 to 72 hours.

20. The method according to any one of claims 12-19, wherein said bone morphogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7, and mixtures thereof.

21. The method of any one of claims 12-20, wherein the contacting step (c) is for a time period ranging from about 3 to 9 days.

22. The method according to any one of claims 12-21, wherein the cell differentiation medium is at least a minimal essential medium supplemented with one or more optional ingredients selected from the group consisting of growth factors, ascorbic acid, glucose, non-essential amino acids, salts including trace elements, glutamine, insulin, activin a, transferrin, and beta mercaptoethanol.

23. The method of any one of claims 12-21, wherein the cell differentiation medium is selected from the group consisting of Dartback Modified Eagle Medium (DMEM), knock-out dartback modified eagle medium (KO DMEM), Ham's F12/DMEM minimal medium (50: 50), and Ham's F12/DMEM minimal medium further comprises albumin, antibiotics, trace minerals, ascorbic acid, β -mercaptoethanol, fibroblast growth factor, and insulin-like growth factor analogs.

24. The method of any one of claims 12 to 21, wherein the cell differentiation medium is DMEM/F12 (50: 50) containing effective amounts of albumin, penicillin/streptococcal antibiotics, non-essential amino acids, trace minerals, ascorbic acid, β -mercaptoethanol, fibroblast growth factor, insulin-like growth factor analogs, activin a and Heregulin.

25. A method of producing pluripotent migratory cells (MMCs), the method comprising (a) providing primate pluripotent stem cells; and (b) contacting the primate pluripotent stem cells with an effective amount of a GSK inhibitor in a cell differentiation medium in combination with at least one additional agent selected from the group consisting of an activin a signaling inhibitor, a bone morphogenetic protein inhibitor, and mixtures thereof, for at least about 3 days to produce pluripotent migratory cells; and (d) optionally, isolating the pluripotent migratory cells.

26. The method of claim 25, wherein the primate pluripotent stem cells are human embryonic stem cells.

27. The method of claim 25 or 26, wherein the GSK inhibitor is selected from the group consisting of:

BIO (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime (GSK3 inhibitor IX);

BIO-acetoxime (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -acetoxime (GSK3 inhibitor X);

(5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine (GSK 3-inhibitor XIII);

pyridocarbazole-cyclopentadienyl ruthenium complex (GSK3 inhibitor XV);

TDZD-84-benzyl-2-methyl-1, 2, 4-thiadiazole-3, 5-dione (GSK3 beta inhibitor I);

2-thio (3-iodobenzyl) -5- (1-pyridinyl) - [1, 3, 4] -oxadiazole (GSK3 β inhibitor II);

OTDZT 2, 4-benzhydryl-5-oxothiadiazole-3-thione (GSK3 β inhibitor III);

alpha-4-dibromoacetophenone (GSK3 beta inhibitor VII);

AR-a014418N- (4-methoxybenzyl) -N' - (5-nitro-1, 3-thiazol-2-yl) urea (GSK-3 β inhibitor VIII);

3- (1- (3-hydroxypropyl) -1H-pyrrolo [2, 3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-pyrrole-2, 5-dione (GSK-3 β inhibitor XI);

TWS119 pyrrole pyrimidine compounds (GSK3 β inhibitor XII);

L803 H-KEAPPAPPQSpP-NH₂or its myristoyl form (GSK3 β inhibitor XIII);

2-chloro-1- (4, 5-dibromo-thiophen-2-yl) -ethanone (GSK3 β inhibitor VI); and

mixtures thereof.

28. The method of claim 25 or 26, wherein the GSK inhibitor is a Wnt protein.

29. The method of claim 28, wherein the Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt14, Wnt14b, Wnt15, Wnt16, and mixtures thereof.

30. The method of any one of claims 25-27, wherein the GSK inhibitor is BIO (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime (GSK3 inhibitor IX).

31. The method of any one of claims 25-26 and 28-29, wherein the Wnt protein is Wnt3 a.

32. The method of any one of claims 25-31, wherein the contacting step (b) is for a time period ranging from about 4 to 12 days.

33. The method of any one of claims 25-32, wherein said activin a signaling inhibitor is selected from the group consisting of SB-431542, follistatin gene-related protein, bone morphogenic protein, and membrane-bound inhibitor of activin, anti-BAMBI monoclonal antibody, Smad7(Mothers Against Decapentaplegic homolog 7), TGF RI inhibitor, and mixtures thereof.

34. The method of any one of claims 25 to 33, wherein the bone morphogenic protein inhibitor is selected from the group consisting of Noggin, sclerostin, Gremlin (Drm/Gremlin), USAG-1, and mixtures thereof.

35. The method of any one of claims 25-34, wherein the contacting step (b) is for a time period ranging from about 4 to 9 days.

36. The method according to any one of claims 25-35, wherein the cell differentiation medium is at least a minimal essential medium supplemented with one or more optional ingredients selected from the group consisting of growth factors, ascorbic acid, glucose, non-essential amino acids, salts including trace elements, glutamine, insulin, activin a, transferrin, and beta mercaptoethanol.

37. The method of any one of claims 25-36, wherein the cell differentiation medium is selected from the group consisting of Dartback Modified Eagle Medium (DMEM), knock-out dartback modified eagle medium (KO DMEM), Ham's F12/DMEM minimal medium (50: 50), and Ham's F12/DMEM minimal medium further comprises albumin, antibiotics, trace minerals, ascorbic acid, β -mercaptoethanol, fibroblast growth factor, and insulin-like growth factor analogs.

38. The method of any one of claims 25 to 37, wherein the cell differentiation medium is DMEM/F12 (50: 50) containing effective amounts of albumin, penicillin/streptococcal antibiotics, non-essential amino acids, trace minerals, ascorbic acid, β -mercaptoethanol, fibroblast growth factor, insulin-like growth factor analogs, activin a and Heregulin.

39. A method of producing mesodermal (ISl1+) cells from pluripotent migratory cells, the method comprising (a) providing a population of pluripotent migratory cells; and (b) contacting the multipotent migratory cells with an effective amount of a bone-forming factor and optionally in combination with a GSK inhibitor in a cell differentiation medium for at least about 2 days to produce mesodermal (ISl1+) cells; and (c) optionally, isolating the mesoderm (ISl1+) cells.

40. The method of claim 39, wherein said cell differentiation medium is free of activin A inhibitors and bone morphogenetic protein inhibitors.

41. The method of claim 39 or 40, wherein said multipotent migratory cells are obtained from human embryonic stem cells contacted with a cell differentiation medium comprising an effective amount of a GSK inhibitor in combination with an activin A inhibitor and/or a bone morphogenic protein inhibitor.

42. The method of any one of claims 39 to 41, wherein the GSK inhibitor is selected from the group consisting of:

BIO (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime (GSK3 inhibitor IX);

BIO-acetoxime (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -acetoxime (GSK3 inhibitor X);

(5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine (GSK 3-inhibitor XIII);

pyridocarbazole-cyclopentadienyl ruthenium complex (GSK3 inhibitor XV);

TDZD-84-benzyl-2-methyl-1, 2, 4-thiadiazole-3, 5-dione (GSK3 beta inhibitor I);

2-thio (3-iodobenzyl) -5- (1-pyridinyl) - [1, 3, 4] -oxadiazole (GSK3 β inhibitor II);

OTDZT 2, 4-benzhydryl-5-oxothiadiazole-3-thione (GSK3 β inhibitor III);

alpha-4-dibromoacetophenone (GSK3 beta inhibitor VII);

AR-a014418N- (4-methoxybenzyl) -N' - (5-nitro-1, 3-thiazol-2-yl) urea (GSK-3 β inhibitor VIII);

3- (1- (3-hydroxypropyl) -1H-pyrrolo [2, 3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-pyrrole-2, 5-dione (GSK-3 β inhibitor XI);

TWS119 pyrrole pyrimidine compounds (GSK3 β inhibitor XII);

L803 H-KEAPPAPPQSpP-NH₂or its myristoyl form (GSK3 β inhibitor XIII);

2-chloro-1- (4, 5-dibromo-thiophen-2-yl) -ethanone (GSK3 β inhibitor VI); and

mixtures thereof.

43. The method of any one of claims 39-41, wherein the GSK inhibitor is a Wnt protein.

44. The method of claim 43, wherein said Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt14, Wnt14b, Wnt15, Wnt16, and mixtures thereof.

45. The method of any one of claims 39-42, wherein said GSK inhibitor is BIO (2 ' Z, 3 ' E) -6-bromoindirubin-3 ' -oxime (GSK3 inhibitor IX).

46. The method of any one of claims 39-40 and 43-44, wherein the Wnt protein is Wnt3 a.

47. The method of any one of claims 39-46, wherein the contacting step is for a time period ranging from about 3 to 9 days.

48. The method of any one of claims 39-47, wherein the contacting step is for a time period ranging from about 4 to 7 days.

49. The method according to any one of claims 39 to 47, wherein the cell differentiation medium is at least a minimal essential medium supplemented with one or more optional ingredients selected from the group consisting of growth factors, ascorbic acid, glucose, non-essential amino acids, salts including trace elements, glutamine, insulin, activin A, transferrin, and beta mercaptoethanol.

50. The method of any one of claims 39-47, wherein said cell differentiation medium is selected from the group consisting of Darber Modified Eagle Medium (DMEM), knock-out Darber modified eagle medium (KO DMEM), Ham's F12/DMEM minimal medium (50: 50), said Ham's F12/DMEM minimal medium further comprising albumin, antibiotics, trace minerals, ascorbic acid, beta-mercaptoethanol, fibroblast growth factor, and insulin-like growth factor analogs.

51. The method of any one of claims 39 to 47, wherein the cell differentiation medium is DMEM/F12 (50: 50) containing effective amounts of albumin, penicillin/streptococcal antibiotics, non-essential amino acids, trace minerals, ascorbic acid, beta-mercaptoethanol, fibroblast growth factor, insulin-like growth factor analogues, activin A and Heregulin.

52. A method of producing definitive endoderm cells from pluripotent migratory cells, the method comprising (a) providing a population of pluripotent migratory cells; (b) contacting said multipotent migratory cells with an effective amount of activin a and optionally an inhibitor of PI3 kinase signaling for at least about 2 days to produce definitive endoderm cells; and (c) optionally isolating the definitive endoderm cells.

53. The method of claim 52, wherein said contacting step is for a time period ranging from about 4 to 5 days.

54. The method of claim 52 or 53, wherein said PI3 kinase signal inhibitor is selected from the group consisting of rapamycin, LY294002, wortmannin, lithium chloride, Akt inhibitor I, Akt inhibitor II (SH-5), Akt inhibitor III (SH-6), NL-71-101, and mixtures thereof.

55. A method of producing pancreatic endoderm cells from pluripotent migratory cells, the method comprising (a) providing a population of pluripotent migratory cells; (b) contacting the multipotent migratory cells with an effective amount of activin a and optionally an inhibitor of PI3 kinase signaling in a cell differentiation medium for at least about 2 days to produce definitive endoderm cells; (c) optionally isolating the definitive endoderm cells; (d) contacting the definitive endoderm cells with an effective amount of retinoic acid and FGF10 in a cell differentiation medium for at least about 24 hours to produce pancreatic endoderm cells; and (e) optionally isolating the pancreatic endoderm cells.

56. The method of claim 55, wherein said pancreatic endoderm cells are further differentiated into pancreatic β cells by exposing said pancreatic endoderm cells to a cell differentiation medium comprising an effective amount of retinoic acid and FGF10 for about 10-24 days, and optionally isolating said pancreatic β cells.

57. The method of claim 55, wherein said pancreatic endoderm cells are further differentiated into hepatic endoderm cells by exposing said pancreatic endoderm cells to a cell differentiation medium comprising an effective amount of FGF10 without retinoic acid for at least about 2 days, and optionally isolating said hepatic endoderm cells.

58. The method of claim 57, wherein the exposing step is performed for a period ranging from about 10-24 days.

59. The method of any one of claims 57-58, wherein the PI3 kinase signal inhibitor is selected from the group consisting of rapamycin, LY294002, wortmannin, lithium chloride, inhibitor Akt I, Akt II (SH-5), inhibitor Akt III (SH-6), NL-71-101, and mixtures thereof.

60. A pluripotent, migratory cell population that has the following characteristics:

a) the cells are capable of being cultured as a stable population of cells for at least 10 passages;

b) when plated at low density, the cells behave as mesenchymal cells, growing into sheets when cultured at high density;

c) the cell can be produced from human embryonic stem cell lines including BG01, BG02, WA 09;

d) the cells can be frozen and cryopreserved using standard methods;

e) the cells can be recovered, revived and differentiated after being stored at low temperature;

f) the cells can be passaged with high plating efficiency;

g) the cell is free of SSEA3 and SSEA4 antigens on the cell surface;

h) the cells do not express the human embryonic stem cell marker Oct4 or Nanog;

i) the surface of the cell is capable of expressing CXCR 4;

j) the cell is capable of expressing at high levels the following transcripts: zic1, Sox1, Sox2, HoxA9, HoxD4, HoxA5, HoxC10, HoxD3, Pax6, N-CAM, CXCR 4;

k) the cells are not mesendoderm cells, but they express the T/mouse short tail mutant phenotype or Eomesodermin;

l) the cell is E-cadherin negative;

m) the cell does not express Sox17, Isl1, Musashi, nestin at levels detectable by Q-PCR analysis;

n) the cell maintains a normal karyotype during passaging;

o) the cell exhibits a migratory, mesenchymal phenotype;

p) the cells have multipotent differentiation capacity (including mesodermal cells, endodermal cells);

q) no teratoma formation when the cells are injected into severe combined immunodeficiency mice.

61. A pluripotent migratory cell population produced by the method of any one of claims 25-38, the pluripotent migratory cell population having the following characteristics:

a) the cell is pluripotent and self-renewing;

b) the cells are capable of differentiating into a variety of cell types including endodermal and mesodermal cells;

c) the cells are dynamic cells, able to alternate between multipotent migratory cells (E-cadherin-, Oct4-, Nanog-, SSEA3-, CXCR4+) and alternative cell types whose E-cadherin +, Oct4+, Nanog +, SSEA3-, CXCR4+ (high density (epithelial layer)) have significant developmental plasticity;

d) based on the signature profile, the cell is not a human embryonic stem cell.

62. Cryopreserved pluripotent migratory cell populations.

63. A cryopreserved pluripotent migratory cell population produced by the method of any one of claims 25-38.

64. A cell population consisting essentially of isolated mesodermal (Isl1+) cells (mesodermal-derived Isl1+ pluripotent progenitor cells), the cell population having the following characteristics:

a) the cell population expresses Isl1, Tbx20, Nkx2.5, Fgf10, GATA4, KDR (Flk1), FoxF1, PDGFR α;

b) the karyotype of the cell population is normal;

c) the cell population does not express Oct4, Nanog, T, Eomesoderm min;

d) the cell population is capable of differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.

65. An isolated mesodermal (Isl +) cell population obtained by the method of any one of claims 12-24 and 39-51, the cell population having the following characteristics:

a) the cell population expresses Isl1, Tbx20, Nkx2.5, Fgf10, GATA4, KDR (Flk1), FoxF1, PDGFR α;

b) the karyotype of the cell population is normal;

c) the cell population does not express Oct4, Nanog, T, Eomesoderm min;

d) the cell population is capable of differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.

66. A cryopreserved mesodermal (Isl +) cell population obtained by the method of any one of claims 12-24 and 39-51.

67. A cardiomyocyte population obtained by differentiating mesodermal (Isl +) cells obtained according to the method of any one of claims 12-24 and 39-51.

68. A method of treating myocardial infarction in a patient in need thereof, which comprises administering to said patient an effective amount of mesodermal (Isl +) cells or cardiomyocytes.

69. The method of claim 68, wherein the cells are administered directly to the heart of the patient.

70. A method of treating damaged or atrophic vascular tissue in a patient in need of treatment, the method comprising administering an effective amount of mesodermal (Isl1+) cells at a blood vessel in need of repair in said patient.

71. A method of treating damaged or atrophic vascular tissue in a patient in need of treatment, the method comprising differentiating mesodermal (Isl1+) cells into smooth muscle cells by contacting the mesodermal cells with an effective amount of a GSK inhibitor and a bone morphogenic protein in a cell differentiation medium for at least about 4 days to produce smooth muscle cells; isolating the smooth muscle cells and administering the smooth muscle cells to the injured or atrophic vascular tissue of the patient.

72. The method of claim 71, wherein the contacting step is for a time period ranging from about 5 to 6 days.