HK1156966B - Compositions for mesoderm derived isl1+ multipotent cells (imps), epicardial progenitor cells (epcs) and multipotent cxcr4+cd56+ cells (c56cs) and methods of use - Google Patents

Description

Compositions of mesoderm-derived ISL1+ pluripotent cells (IMPs), Epicardial Progenitor Cells (EPCs), and pluripotent CXCR4+ CD56+ cells (C56Cs), and methods of use thereof

Technical Field

The present invention relates to methods of producing and maintaining mesoderm-derived ISL1+ multipotent progenitor cells (IMPs) and compositions thereof, related methods of producing a variety of multipotent progenitor cells as otherwise disclosed herein, and others. Methods of using these cells in therapeutic methods are also disclosed. The present invention also relates to the discovery that human pluripotent stem cells (human pluripotent stem cells), including embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can be differentiated into Isl1+ multipotent cardiovascular progenitor cells (IMPs) using a similar method.

The present invention also relates to the efficient transformation of hESCs and hipSC-derived IMPs into Wilm's tumor protein 1 positive (Wt1+) multipotent Epicardial Progenitor Cells (EPCs). EPCs are capable of differentiating into smooth muscle cells, endothelial cells and cardiac fibroblasts and subsequently becoming a component of the coronary vasculature (coronary vascular). As EPC are progenitor cells of the cells that make up the coronary vasculature, they have utility as therapeutic cells, as drug screening tools, and as research tools. These cells may also differentiate into cardiomyocytes and others, as elaborated herein.

The present invention further relates to the discovery of C-Kit + CXCR4+ multipotent progenitor cells (C56C cells) that can be made directly from Multipotent Migratory Cells (MMCs), or from pluripotent stem cells including hESCs and hiPSCs. As described elsewhere herein, C56C cells are pluripotent cells that have the characteristic of homing properties (cells can also be described as homing mesoderm-derived pluripotent cells) that demonstrate their benefit in repairing damaged and/or injured tissues secondary to various pathological and/or disease states. Methods of producing these cells and methods of using these cells in therapy are alternatively described.

Related application and authorization support

The present application claims benefit of priority from the following provisional applications: US61/137,058 entitled "methods and compositions of hESC-derived multipotent progenitor cell material derived from mesoderm" filed on 25/7/2008; US61/198,861 entitled "use of MMCs and C56C in cell therapy" filed 11/10, 2008; and US61/215,621 entitled "generation of pluripotent Epicardial Progenitor Cells (EPCs) from human pluripotent stem cells", filed 5/7/2009, which are incorporated herein by reference in their entirety.

Background

Human embryonic stem cells (hESC's) (markers for hESCs include SSEA3, TRA-1-60, TRA-1-81 antigen, Nanog, Oct4) are pluripotent cell populations capable of differentiating into all cells of triple embryonic germ layer origin and of extraembryonic lineage (linkage) origin. FIG. 33. This feature of hESC's is of great significance in cell therapy (e.g., diabetes, heart disease, neurodegenerative disease), drug discovery, and developmental models.

Other pluripotent cell types have been identified in mice. Primitive ectoderm-like cells (primary ectoderm like cells) (EPL; Rathjen et al, 1999, J.cell Sci) show formation from and have the ability to differentiate into mESC's. Recently, a new mouse cell with hESC's characteristics (Nanog + Sox2+ Oct4+) was identified, namely post-implantation epiblast stem cells (EpiSC; Tesar et al, Nature 448: 196-202; 2007). These mouse-derived pluripotent cell types are all capable of producing three embryonic germ layers in vitro or in teratomassay (teratomassay).

Ectodermal stem cells (EpiScs) and induced pluripotent stem cells (iPS) are suitable for a broad spectrum of pluripotent cell types, and conceptually, the techniques described herein can be applied to these and other pluripotent cell types (i.e., primary pluripotent cells). EpiScs ectodermal stem cells were isolated from early post-implantation stage embryos, and expressed Oct4 and were pluripotent (Tesar et al, Nature, volume 448, p.196, day 12, 7/2007). Induced pluripotent stem cells (iPS cells) are obtained by dedifferentiating adult skin fibroblasts or other adult human cells back to a pluripotent state by retroviral transduction of 4 genes (c-myc, Klf4, Sox2, Oct4) (Takahashi and Yamanaka, Cell 126, 663-.

The advantages of developing other non-ESC, self-renewing, pluripotent/multipotent stem cells would help to improve developmental models, improve directed differentiation into adult cells and enable more efficient and less costly approaches than traditional approaches.

Human pluripotent cells (such as human embryonic stem cells [ hESCs ] and induced pluripotent stem cells [ iPS cells ]) can be differentiated from a bipotent mesendoderm (T +, MixLl +) progenitor cell that can be further differentiated into a wide range of mesoderm lineages, such as bone, blood, muscle, and kidney. See fig. 12. We have developed conditions for differentiation of human pluripotent cells into Multipotent Migratory Cells (MMCs) by adding small molecule compounds to the culture medium. FIG. 13. These compounds are well known inhibitors of GSK3 activity (BIO) and of TGF β/activin A (activin A)/Nodal signaling (Nodal signaling) (SB 431542). By further processing, the MMCs are able to transform into a CXCR4+ CD56+ cell population (C56Cs, CXCR4+/CD56+ cells) that upregulate other cell surface markers. The mesenchymal stem cells express some, but not all, of the markers. In addition to expressing the cytokine receptors CXCR4 and CD56, C56Cs was able to up-regulate the stem cell marker C-Kit. C56C does not express a marker for hematopoietic stem cells, such as CD45, or an endothelial marker, such as CD 31.

Since C56Cs expresses markers common to stem/progenitor cells of mesodermal origin (e.g., skeletal bone marrow-derived mesenchymal stem cells) and receptors for cytokine signaling known to be associated with "homing" of stem cells to ischemic inflammatory tissue, it is likely that these cells will have the ability to reach the site of tissue injury. Systemic administration by intravenous injection will become a method of homing the cellular tissues and participating in the repair process. Once these cells colonize the damaged tissue, they may promote tissue repair by a paracrine mechanism or by transdifferentiation into cells that are directly involved in repair. These cells may also be involved in the suppression of inflammatory responses through immunomodulation (suppression of T-cell, natural killer cell activity).

The epicardium (epicardium) forms the outer surface of the vertebrate heart and develops from the epicardium derived from the diaphragm. Epicardium is composed of a single layer of flat epithelium that is connected to the heart muscle by subendocardial connective tissue (Manner et al, 2001, Cell Tissues Organs (cells Tissues Organs)169, 89-103). In cardiac development, epicardial formation is synchronized with the development of coronary vessels (Olivey et al, Trends in cardiovascular Med 2004,14, 247-. The protoepicardium comes into contact with the developing heart, presumably during its beating, from which it will stretch over the myocardium to form a new membrane, the epicardium. The epicardium then produces a variety of cell types, including smooth muscle cells, endothelial cells, cardiac fibroblasts, which together form the coronary vasculature. See fig. 36. Epicardial cells also have the ability to differentiate into cardiomyocytes (Zhou et al, 2008 Nature 454, 109-113). Immediately after invading the surface of the cardiomyocytes, the epicardial cell subpopulation undergoes a single epithelial-to-mesenchymal transition and migrates into the subendocardial space. Some of these cells then have the ability to migrate further into the tight regions of the myocardium. In the subendocardial space and myocardium, coronary vessels form as the epicardial-derived angioblasts combine to form a native vascular plexus. Eventually, these endothelial vessels elongate to form larger vessels that become the coronary arteries and veins. The cells that make up the complement of the coronary arterial vasculature include smooth muscle and endothelial cells and scattered fibroblasts-all derived from progenitor cells in the protoepicardial/epicardial. Epicardium is typically represented by expression of Wilms' mother tumor protein 1(WT1), T-box factor 18(T-box factor 18, TBx18), epicatechin (epicardin) (Tcf21), and RALDH2 (Zhou et al, 2008; Cai et al, 2008, Nature 454,104-. The epicardium of WT1+ is thought to be formed from Isl1+ Nkx2.5+ progenitor cells (Zhou et al, 2008).

Drawings

FIG. 1: schematic representation of the generation of Isl1+ IMPs after 4-6 days of hESCs treatment with (1) Wnt3a (25ng/ml) + BMP4(l00ng/ml), or (2) BIO (2. mu.M) + BMP4(l00ng/ml) for 4-6 days. IMP cells are able to maintain a stable self-renewal state for at least 10 passages without loss of IMP marker expression and differentiation potential.

FIG. 2: production of self-renewing IMP populations following treatment of IMP's with Bio (2. mu.M) and BMP4(100ng/ml) in medium of known composition (definedmedia). WA09hESCs were passaged every 4-6 days at a ratio of 1:6 and fixed with 4% paraformaldehyde at (P) 0-3. Each of the passaged materials A) Isl1, B) Nkx2.5, C) E-cadherin (E-cadherin), and D) β -catenin (β -catenin) and Nanog were immunostained. Isl1 and Nkx2.5 were expressed in all passages. Initially, β -catenin localizes in the nucleus of P0 and becomes more dispersed during passage. E-cadherin (a marker for epithelial cells) disappeared together with Nanog, a marker for hESCs. The fused image is displayed under DAPI (nuclear stain). The image is at 20 x magnification.

FIG. 3: clonal propagation of Isl1+ pluripotent progenitor cells (IMPs). Accutase grown in BIO (2. mu.M) and BMP4(100ng/ml) for 24-336 hours and treated with methylcellulose (final concentration 0.9%) for the first 72 hours^TMBright field image at 10X magnification of passaged IMP cells.

FIG. 4: cardiomyocyte production from self-renewing IMP's (originally derived from WA09 hESCs). Fifth passage IMP's were grown for fourteen days in media of known composition with activin A and IGF removed and VEGF (10ng/ml) and DKK (150ng/ml) added. Cells were fixed in 4% paraformaldehyde and immunostained for Smooth Muscle Actin (SMA), sarcomeric actin (sarcomeric actin) and cardiac troponin t (ctnt). Confocal images were imaged at 40 x magnification.

FIG. 5: endothelial cells from IMP's (WA09 derived) were produced after treatment with BMP4(10ng/ml) and DKKL (150ng/ml) in medium of known composition with actin A and IGF removed. Cells were fixed in 4% paraformaldehyde and immunostained for VE-cadherin and CD 31. Dapi was used as a nuclear stain. A fusion picture of Dapi/VE-cadherin/CD 31 is shown. The fluorescence images were at 20 and 40 x magnification.

FIG. 6: production of smooth muscle cells from IMP's (WA 09-derived) 14 days after treatment with Wnt3a (25ng/ml) and BMP4(l00 ng/ml). Cells were split at a ratio of 1:4-1:6, fixed in 4% paraformaldehyde, and immunostained positive for a) Smooth Muscle Actin (SMA) and B) smooth muscle calcium regulatory protein and negative for the cardiac cell marker C) sarcomere actin (sarcomeric actin). DNA was stained with dapi. Fusion images of SMA/Dapi, calponin/Dapi, and sarcomeric actin/Dapi are shown. The images were at 20 and 40 x magnification.

FIG. 7: WA09hESCs were differentiated into Isletl + multipotent progenitor (IMP) cells over 4 days in medium containing Wnt3a (25ng/ml) and BMP4(100ng/ml) of known composition. hESCs and IMP cells were stained with SSEA3 or PDGFR α antibody and analyzed by flow cytometry. The percentage content of cells positive for SSEA3 or PDGFR α at each stage is indicated.

FIG. 8: a schematic representation of the differentiation of self-renewing MMCs into c-kit + CXCR4+ progenitor cell types.

FIG. 9: differentiation of BG02 hESC-derived MMCs during 6 days after addition of BMP4, Wnt3a, and sodium butyrate (NB) under conditions of known composition media. Q-PCR transcriptional analysis for BG02ES cells, MMCs passaged 23 (MMC p23), and a6 day period of pdgfra, CXCR4, KDR, c-KIT, CD56(N-CAM), and Isletl transcripts of differentiated MMC p23 on day two (d2), day four (d4), and day six (d6) are shown.

FIG. 10: histograms of flow cytometric analysis of BG 02-derived differentiated MMC 2(a), 4(B) and 6(C) days after addition of BMP4, Wnt3a and sodium butyrate under conditions of known composition of medium. The percentage of positive cells for SSEA3, c-KIT, CXCR4, CD56, CD31, PDGFR α and KDR were calculated relative to the isotype control (isotype control) for each antibody, respectively. (D) Day 2,4 and 6 differentiated MMCs brightfield pictures (C-KIT + CXCR4+) as described in (A-C). 10x, 20 x magnification.

FIG. 11: a general model illustrating the principle that hESCs cultured in media of known composition, in the presence of inhibitors of activin/Nodal signaling and/or inhibitors of BMP signaling (Noggin, e.g., C compounds), produce a variety of multipotent mesenchymal progenitor cells by contact with GSK3 inhibitors (e.g., BIO). These cells are generally referred to as GABi cells-GSK 3, activin/Nodal signaling, BMP signaling inhibitor cells.

FIG. 12 is a diagram showing the differentiation of self-renewing human pluripotent stem cells (hESCs, iPS cells) into mesendoderm (MesEnd) and then into mesoderm (Meso). Markers of pluripotent cells and mesendoderm are indicated by the type of lineage that can be produced in the mesoderm lineage.

FIG. 13 is a schematic diagram showing the differentiation of self-renewing human pluripotent stem cells (hESCs, iPS cells) into mesoderm-derived progenitor cells called Multipotent Migratory Cells (MMC). Small molecule inhibitors such as BIO and SB431542 can be added to hESCs to promote cellular transformation to MMCs. MMCs are able to maintain a stable cell population and thus self-renew.

FIG. 14 is a schematic representation showing the differentiation of self-renewing human pluripotent stem cells (hESCs, iPS cells) into MMCs and then into CXCR4+ CD56+ cells (C56 Cs). The production of MMCs is shown in FIG. 2. MMCs were then converted to C56Cs over a 3-6 day period by removing BIO and SB431542 and adding BMP4, Wnt3a, and sodium butyrate. C56Cs is similar to mesenchymal stem cells, expressing CXCR4 and CD56 but not expressing markers for hematopoietic stem cells (CD45) or endothelial cells (CD 31). C56Cs can be produced by direct differentiation of hESCs into MMCs or by self-renewing MMCs.

Figure 15. strategy for using C56Cs as part of a cell therapy strategy, for example, where systemic administration is by intravenous injection. The cells then "home" to the site of tissue damage, site of inflammation, and bone marrow (for example), where they will stimulate tissue repair/regeneration. This does not prevent direct application of these cells to the site of tissue damage/inflammation.

Fig. 16 after C56Cs "homes" to sites of inflammation, tissue damage, they could potentially be involved in tissue regeneration-repair in several ways. First, C56Cs that is "homing" releases cytokines (cytokines), growth factors (growth factors) and other molecules to stimulate the repair process through a paracrine mechanism. This involves the recruitment of cells in the local environment with some regenerative function. Second, these cells can be transformed into functional cell types that directly promote tissue repair/regeneration.

Figure 17, 18, flow cytometric analysis of WA09hESCs, MMCs produced by WA09hESCs, C56Cs produced by treatment with BMP4, Wnt3a and sodium butyrate for 2,4 and 6 days.

FIG. 19 summary of cell surface markers of MMCs and C56Cs as determined by flow cytometry.

Figure 20 a general schematic of MMCs and C56Cs as they can be used for ischemic heart tissue regeneration. MMCs and C56Cs (both CXCR4+) are administered intravenously, for example, to animals. The cells then home to the site of ischemia and inflammation. The functional recovery of the animals was assessed by the indicated method.

Figure 21¹¹¹In]Oxime-labeled cells "home" to the ischemic heart, bone, and liver-lung. Use 2¹¹¹In]Oxime C56Cs was labeled for 5 minutes, washed with 10% rat serum to remove unbound radiolabel (caveiers et al, 2007 Nuclear medicine and molecular imaging journal (Q J Nucl Med Mol)51:61-66), and then injected (about 2-4X 10)⁶Cells per 0.1ml saline) into the tail vein of a rat with a history of heart ischemia due to surgical ligation of the anterior descending left coronary artery-Dawley (Sprague Dawley). Animals were then "live" nuclear imaged with a gamma camera at 24, 48 and 72 hours post infusion. Marked heart regions are indicated by arrows. Other areas of accumulation are also indicated. Over 72 hours, the signal decreased due to radioactive decay and clearance. An overall two-dimensional image is shown.

FIG. 22. autoradiogram of successive short axis slices of the same rat heart as shown in FIG. 10. After 72 hours of cell infusion, the heart was removed, the tissue fixed (as shown in the lower panel) and exposed to autoradiographic membranes for 8 days (upper panel).

FIG. 23 and 24, Experiment 1¹¹¹In]Oxime-labeled fineCells were "homing" to the ischemic heart, bones and liver, lungs, spleen of 2 rats (FIG. 8-rat # 1; FIG. 9-rat # 2). Use 2¹¹¹In]Oxime labeled C56Cs, then injected (ca. 2X 10)⁶Cells per 0.1ml of saline) into the tail vein of chow-dao-chow rats, followed by 'live' nuclear imaging with gamma camera at 0.1, 2 and 24 hours post infusion. Grey arrows indicate bone resorption: black arrows indicate absorption by the heart (incorporation).

FIG. 25 and FIG. 26 Exp 2¹¹¹In]Oxime-labeled cells "home" to ischemic heart in 2 rats (fig. 21-rat # 1; fig. 22-rat # 2). Use 2¹¹¹In]Oxime C56Cs was labeled and then injected (ca. 2X 10)⁶Cells per 0.1ml saline) to the tail vein of chow-dao-chow rats, followed by 'live' nuclear imaging with a gamma camera 2 hours after infusion. Arrows indicate absorption by the heart.

FIG. 27. transthoracic echocardiography examination of athymic rats with acute myocardial infarction receiving saline (0.1ml) administered to the tail vein. Saline was administered daily during the three days after the infarction. Echocardiography examinations were performed 2 weeks after infusion. The short axis view and the long axis view are as shown in the figure. Thin, beating-free myocardial walls are clearly visible in the ischemic areas.

FIG. 28. receiving C56Cs (one dose is about 2X 10 per 0.1ml saline)⁶Cells) administered to the tail vein of athymic rats with acute myocardial infarction. Three days after the infarction, a dose of cells was administered daily. Echocardiography was performed 2 weeks after infusion. The short axis view and the long axis view are as shown in the figure. In contrast to the rats illustrated in figure 25, thickened beating myocardial walls were observed.

FIG. 29 high resolution MRI scans of athymic rats (as shown in FIGS. 23, 24) 2 weeks after treatment with saline alone (cells; animal 2) or C56Cs (-cells, animal 3). The respective diastolic and systolic views of the 2 animals are shown.

FIG. 30 high resolution MRI scans of athymic rats (3, 4) 2 weeks after treatment with saline alone (-cells; animal 7) or C56Cs (+ cells, animal 5). The respective diastolic and systolic views of the 2 animals are shown.

FIG. 31. 2-photon confocal images of GFP + cells localized to the area of stroke in photothrombosis (photothrombotic). Texas red staining makes vessels red.

Figure 32 immunofluorescence staining of frozen brain sections taken from mice with stroke with photothrombosis. The images show the localization of GFP + fused C56C-derived cells near the limbal zone (penumbra) and choroid plexus. The localization of GPF + cells is indicated by arrows. The cells present in these sections showed multiple courses (observed by real-time 2-photon imaging) indicative of dynamic behavior.

Figure 33 is a graph showing the ability of human pluripotent stem cells (e.g., hESCs and hiPSCs) to differentiate into three embryonic germ layers (ectoderm, mesoderm and definitive endoderm) and extraembryonic lineages. The pluripotent cell is typically Oct4⁺And Nanog⁺. Under appropriate conditions, pluripotent cells can be maintained in a stable, self-renewing state.

FIG. 34 pluripotent cells (Oct 4)⁺，Nanog⁺) Schematic representation of the differentiation pathway for growth into IMP (Isl 1+) cells followed by Wt1+ protoepicardium/epicardium.

Figure 35 Wt1+ protoepicardium/epicardium were able to differentiate into smooth muscle, endothelial cells, cardiac fibroblasts and cardiomyocytes. They are therefore capable of generating the coronary vasculature and the myocardium.

FIG. 36. primary cells are involved in the formation of the coronary vasculature and the major vessels of the coronary vasculature.

FIG. 37 treatment of human iPSCs (Fib-iPS4) with BMP4 and Wnt3a allowed differentiation into Islet1 multipotent progenitor cells (IMPs, Isl 1+) over 4 days. Immunostaining indicated that hiPSCs lost Nanog, Nct4 expression after treatment with BMP4 and Wnt3a, but up-regulated nkx2.5 and Isl 1. As part of this process, iPSCs undergo epithelial to mesenchymal transition, as indicated by down-regulation of E-cadherin and up-regulation of snell (Snail).

FIG. 38 marker transcripts of hipSCs (Fib-iPS4) and hipSCs treated with BMP4 and Wnt3a (Fib-iPS4) for 4 days were analyzed by Q-PCR analysis. During this time, Isl1 and Hand (Hand)2 increased significantly. The experiment was performed in triplicate. Error bars represent standard error of the mean (standard error).

FIG. 39. schematic showing the differentiation pathway of pluripotent cells (hESCs and hipSCs, etc.), initially into IMP (Isl 1+) cells, followed by differentiation into protoepicardial/epicardial-like cells, which we call epicardial progenitor cells (EPCs, Wt1 +). The factors added at each stage in the known composition medium (DM) are shown.

FIG. 40 IMPs cells derived from hESCs (WA09) were treated with BMP4, Wnt3a and all-trans retinoic acid for the indicated times. As IMP cells are transformed into EPCs, they down-regulate Isl1, hande (Hand) l and nkx2.5, but up-regulate other markers such as Raldh2, Tbxl8, Tcf21 (epicardial) and Tbx 5. The q-PCR test was performed in triplicate and is shown as the standard error of the mean.

FIG. 41 immunostaining analysis indicated that EPCs expressed Wt 1. 20 × objective lens.

FIG. 42 IMPs cells from hipsCSs (Fib-hPS4) were treated with BMP4, Wnt3a and all-trans retinoic acid for 16 days. As IMP cells are transformed into EPCs, they down-regulate Isl1, but up-regulate Wt1, Tbxl8 and Tbx 5. The q-PCR assay was performed in triplicate and displayed as standard error of the mean.

Fig. 43.a. shows a schematic of the possible differentiation results of Wt1+ epicardium, such as smooth muscle, endothelial cells, cardiac fibroblasts and cardiomyocytes. Each potential factor treatment scenario is shown. B. Indicating that the epicardium is capable of differentiating to give rise to coronary vasculature lineages (smooth muscle, endothelial cells, cardiac fibroblasts) and cardiomyocytes.

FIG. 44 passage of EPCs derived from hESCs (WA09) (1.25X 10) in DM Medium-Activin + VEGFA (DM-media-Activin + VEGFA)⁵Cells/cm²) For 12 days. The resulting cells stained (a) CD31 and VE-cadherin (CDH5) and (b) procollagen and smooth muscle actin. Images obtained at 40 x and 63 x magnification are shown.

FIG. 45 EPCs from hESCs (WA09) passaged in 10% FBS, DMEM, 1 XPcillin/streptomycin (Pen/Strep), sodium pyruvate, L-glutamine (1.25 xl 0)⁵Cells/cm²) For 12 days. The resulting cultures were stained for procollagen and smooth muscle actin.

FIG. 46, Table 1. microarray maps of IMPs generated by hIPSCs (hFib2-iPS4) (Affymetrix Human Genome U133 upgrade 2.0(Affymetrix Human Genome U133Plus 2.0)) indicate a series of gene upregulations compared to the starting pluripotent cell population>log2³. In the medium of known composition with Wnt3a and BMP4 added, the cells differentiated through the IMPs (Isl 1+) phase (four days).

FIG. 47, Table 2. microarray maps of EPCs generated from hESCs (WA0l, WA07, WA09, BG02) and hIPSCs (hFib2-iPS4) (Affymetrix Humangenome U133 upgrade edition 2.0(Affymetrix Humangenome U133Plus 2.0) show a common set of gene upregulations compared to the starting pluripotent cell population>log2³. Cells differentiated through the IMP (Isl 1+) phase (four days) and then differentiated towards EPCs for another 16 days.

FIG. 48: we used to define the sequence of differentiation steps for hESCs or hiPSCs progression to IMP cells (Isl 1+), followed by EPCs (Wt1+), and then to the vascular tube (CD31 +).

FIG. 49: brightfield images of endothelial tubes formed from Epicardial Progenitor Cells (EPCs). This image is shown at 4 x and 10x magnification.

FIG. 50: confocal images of endothelial tubes from epicardial cells. A. Confocal images of tube staining with CD31 (green) and CDH5 (red) at one focal plane indicated the presence of lumen (lumen). All images were at 40 x magnification. B. Endothelial tube reconstruction of 40 × magnification confocal images from Z-stacks (Z-stacks). Yellow indicates coincidence of CD31 and CDH5 expression.

FIG. 51: spheres were generated from EPCs and plated onto (plated) collagen-based matrices (gill poux). A. Indicating adherence of the sphere at t-0. B. Plated spheres cultured in bFGF + 10% fetal bovine serum (B) or, alternatively, in the absence of serum and bFGF (c). Bright field images taken 24 hours after EPC sphere plating. Similar results were obtained when spheres were plated on collagen I matrix (not shown).

FIG. 52 WA09hESCs were plated on Gill California (Geltrex) and the cytokeratin (red) and vimentin (green) were probed with antibodies. DNA can be detected by DAPI staining. hESCs were positive for the epithelial marker cytokeratin (+ ve) but negative for the mesenchymal marker vimentin.

FIG. 53 EPCs plated on Geltrex as shown in FIG. 4 were fixed with PFA and stained with antibody for cytokeratin (red) and vimentin (green). DNA was detected by DAPI staining. A positive cell for vimentin (green) indicates that it has undergone a process of epithelial to mesenchymal transition, and is mesenchymal-like and migratory.

FIG. 54 EPCs were plated on collagen I matrix. Cells were fixed and stained with antibody for cytokeratin (red) and vimentin (green). DNA was detected by DAPI. A positive vimentin in a cell indicates that it has undergone a process of epithelial to mesenchymal transition, and is mesenchymal-like and migratory.

FIG. 55, these are 2 images of the same heart at different focal planes, showing that three days after implantation of the chick embryo, D14EPC aggregates. Brown clusters of cells (GFP staining) were clearly visible (arrow). Arrows indicate clusters of PE cells that have attached but not invaded.

Figure 56, 57 EPC aggregates were transplanted near developing chicken hearts (figure 8). Tissues were fixed with PFA, paraffin encapsulated and sectioned. The sections were then stained with anti-GFP antibody to detect GFP + EPC cells in the transplant. Immunofluorescent staining indicated that EPCs migrated through the myocardium of chickens and were therefore highly invasive.

FIG. 58.IMP cells were grown into spheres and then co-cultured with mouse heart tissue slices. After 8 days of co-culture, mouse heart tissue was fixed with PFA, paraffin encapsulated and sectioned. Sections were then probed with anti-human β -myosin heavy chain (brown) antibody. The data indicate the presence of human, IMP-derived cardiomyocytes in mouse cardiac tissue, indicating that IMP cells are capable of differentiating into cardiomyocytes.

FIG. 59 GFP + IMP cells were incorporated into the embryonic structure of chicken embryos. Whole-specimen embedded (whole mount) images (a, C, E) and cross sections of embryos (B-G) of HES cells were localized by GFP immunoassay. (A) Stage 12 embryos and corresponding cross sections (B) show the widespread incorporation of HES cells into the endodermal (arrows) coelomic and visceral mesoderm (stars), and perivascular cells (double arrows). (C) Stage 12 and the corresponding cross-section (D) show IMP-derived endoderm (pointed arrow), endothelial cells (arrow) and intermediate mesoderm (white arrow). (E) Stage 12 embryos and corresponding cross sections (F) show IMP-derived endothelial cells in the aorta. (G) Cross sections of stage 13 embryos showed incorporation of IMPs-derived cells into the liver primordia at the level of the anterior gut gate.

Figure 60 Isl1+ cells are labeled by the presence of cadherin 11 and PDGFR β. WA09 cells differentiated on days 4 and 6 in the presence of Wnt3a and BMP4 (as described previously). WA09, day 4 and day 6 cells were treated with akuyasase (Accutase) to form single cell suspensions and stained for cadherin 11 and PDGFR β. At the same time, cells were stained with donkey anti-sheep 488 secondary antibody and IgG2aPE isotype control, respectively. Cells were visualized using a blue (Cyan) flow cytometer (DAKO). Population was visualized with FL4 antibody, red for control population and blue for antibody staining population.

Figure 61. mechanism was studied by migration of C56C into ischemic/damaged tissue, we tested these cells in the Boyden chamber assay. 300,000C56C cells were seeded into the upper chamber of a Boyden (Boyden) chamber. In the lower chamber, these data indicate that C56C cells responded to and migrated to SDFI cytokines (fig. 61). Migration can be blocked with the antagonist AMD3100, indicating that migration is mediated through the CXCR4 receptor.

Disclosure of Invention

Objects of the invention

It is an object of the present invention to provide methods for the long-term maintenance of Islet 1+ multipotent progenitor cells (IMPs) in order to provide a practical means of culturing these cells prior to shipment and/or use.

It is another object of the invention to provide methods for enhancing clonal passaging and expansion of Islet 1+ multipotent progenitor cells (IMPs).

It is a further object of the present invention to provide methods for producing endothelial cells, smooth muscle cells, cardiomyocytes and blood vessels from self-renewing IMPs.

A further object of the invention relates to methods of producing endothelial cells, smooth muscle cells and cardiomyocytes from IMPs derived directly from hPSCs, including hESCs and hipSCs.

Other objects of the invention relate to the fact that IMPs express appreciable amounts of cell surface markers (PDGFR α) that can be used to identify IMPs and to isolate these cells to effective purity.

Still other objects of the invention relate to methods and compositions of matter for the generation of C-kit + CXCR4+ multipotent progenitor cells (C56Cs) from MMCs and conventional methods, wherein a combination of inhibitors of GSK3, activin/Nodal signaling and/or inhibitors of BMP signaling can be used to generate different types of self-renewing progenitor cells.

Further aspects of the invention relate to methods for targeting C56Cs to damaged and/or inflamed tissue in a patient utilizing the unexpected discovery that C56Cs can home to the damaged tissue area and can be used to reconstruct and/or treat such damaged/inflamed tissue.

Still a further object of the present invention relates to methods for generating pluripotent Epicardial Progenitor Cells (EPCs) from hPSCs, including hESCs and hipSCs. Other objects of the invention relate to the pluripotent Epicardial Progenitor Cells (EPCs) produced.

Still other objects of the invention relate to methods of using EPCs, including the production of endothelial cells, smooth muscle and cardiac fibroblasts.

Any one or more of these and/or other objects of the present invention will be readily apparent from the following description of the invention.

Summary of The Invention

The invention provides, inter alia, methods for generating pluripotent migratory cells (MMCs), ISL1+ pluripotent progenitor cells (IMPs), and other aspects from human pluripotent stem cells, including embryonic stem cells (hESCs) and human induced pluripotent stem cells (hipSCs), as described in further detail herein.

In certain aspects, the invention relates to one or more of the following inventive aspects.

1. Methods for long-term maintenance (>10 passages) of Islet 1+ pluripotent progenitor cells (IMPs).

A method for cloning, passaging and amplifying IMPs and application thereof are disclosed.

3. Method for producing endothelial cells, smooth muscle cells and cardiac muscle cells from self-renewing IMPs

4. Methods for producing endothelial cells, smooth muscle cells and cardiomyocytes from IMPs derived directly from hPSCs, including hESCs and hipSCs.

5. Can be used to identify IMPs and isolate these cells to very high purity cell surface markers (PDGFR α) and methods.

Methods and compositions of matter (i) for producing CXCR4+ CD56+ multipotent progenitor cells (C56Cs) from MMCs. (ii) Conventional methods wherein combinations of inhibitors of GSK3, activin/Nodal signaling and/or inhibitors of BMP signaling can be used to generate different types of self-renewing progenitors.

7. Methods for targeting C56Cs to damaged and/or inflamed tissue in a patient utilize the unexpected discovery that C56Cs can home to the damaged tissue area and can be used to reconstruct and/or treat such damaged/inflamed tissue.

8. Methods for generating pluripotent Epicardial Progenitor Cells (EPCs) from hPSCs, including hESCs and hipSCs.

9. A composition of matter for pluripotent Epicardial Progenitor Cells (EPCs).

10. Using EPCs to i) identify secreted epicardial factors that affect cardiomyocyte proliferation, survival function and differentiation; ii) as a source of cells that can be used for cardiovascular drug screening; iii) as a source of cells of therapeutic interest that can be used to repair an ischemic heart, regenerate coronary vasculature; iv) methods for the purpose of tissue engineering to obtain cardiac or coronary components; and v) methods as research tools for the study of cardiovascular development and disease.

11. Methods for producing endothelial cells, smooth muscle and cardiac fibroblasts from epicardial cells (EPCs).

Pharmaceutical compositions represent other aspects of the invention, comprising an effective amount of C56Cs or EPCs together with pharmaceutically acceptable carriers, additives or adjuvants and, optionally, other biologically active agents therapeutically suitable for use in the proposed treatment with C56Cs or EPCs.

The present invention also relates to methods of treating one or more of the following disease states or conditions by administering an effective amount of a population of MMCs, or preferably C56Cs, to a patient in need thereof. The methods of treatment are applicable to the following disease states or conditions: cardiovascular diseases (cardiomyopathy), ischemia), retinomyopathy (retinomypathgy), neuropathy, diabetes (types I and II), stroke, head trauma, autoimmune diseases (lupus, arthritis, multiple sclerosis), immunosuppression, graft-versus-host disease, bone repair, wound repair, inflammatory diseases (arthritis, Crohn's disease, cystic fibrosis) and parkinson's disease, huntington's disease, and the like. Systemic administration of MMCs or C56Cs may be by intravenous administration, or by infusion, directly at the site of injury or focus. Due to the homing properties of MMCs, particularly C56Cs, these cells can be administered away from the site of injury/inflammation and these cells will "home" to the site in the patient for treatment.

As described herein, methods of producing endothelial cells, smooth muscle cells and cardiac fibroblasts and blood or vascular cells from EPC cells in vitro or in vivo represent other aspects of the invention.

Detailed Description

Detailed Description

The following terminology will be used to describe the invention.

Unless otherwise indicated, the terms used herein should be understood in accordance with their conventional usage by those of ordinary skill in the relevant art. In addition to the term definitions provided below, the general term definitions of molecular biology can also be consulted by Rieger et al, 1991, essential to genetics: classical and molecular (scientific of genetics: classical and molecular), fifth edition, berlin: schpringer-varek (Springer-Verlag) and modern experimental methods of Molecular Biology (Current Protocols in Molecular Biology), f.m. ausubel et al, eds., modern experimental methods (Current Protocols), greenish publishing consortium ltd (greene publishing Associates, Inc.) and the union of John Wiley & Sons, Inc. (1998 supplementary). It will be understood that the use of "a" or "an" in the specification and claims may mean one or more, depending on the context of its application. Thus, for example, reference to "an" cell "indicates that at least one cell can be utilized.

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 herein. However, before the present compositions and methods are disclosed and described, it is to be understood that this invention is not limited to particular conditions, or particular methods, etc., as such may, of course, vary, and that numerous modifications and variations will be apparent to those skilled in the art.

Growing cells, isolating cells, and related, cloning, DNA isolation, amplifying and purifying DNA, standard techniques for enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, and the like, as well as a variety of isolation techniques are well known to those skilled in the art and are commonly used. Many standard techniques are described in Sambrook et al, 1989, Molecular Cloning, second edition, Cold Spring Harbor Laboratory, simple edition, New York; maniatis et al, 1982, Molecular Cloning, Cold Spring Harbor Laboratory, simple edition, New York; wu (Ed.), 1993, methods in enzymology (meth.enzymol.)218, part one; wu (Ed.), 1979, methods in enzymology (meth.enzymol.) 68; wu et al, (Eds.), 1983, methods in enzymology (meth. enzymol.)100 and 101; grossman and Moldave (Eds.), 1980, methods in enzymology (meth. enzymol.) 65; miller (ed.), 1972, molecular Genetics Experiments (Experiments in molecular Genetics), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; old and Primrose, 1981, Principles of Gene Manipulation (Principles of Gene management), university of California, Berkeley; schleif and Wensink, 1982, Methods for Practical Molecular Biology (Practical Methods in Molecular Biology); glover (Ed.), 1985, DNA Cloning (DNA Cloning), volumes I and II, IRL press, oxford, uk; hames and Higgins (Eds.), 1985, Nucleic Acid Hybridization (Nucleic Acid Hybridization), IRL press, oxford, uk; and Setlow and Hollaender, 1979, genetic engineering: the Principles and Methods (Genetic Engineering: Principles and Methods), volumes 1-4, Prinermer Press, New York are described. The abbreviations and nomenclature used herein are considered standard in the art and are used extensively in the professional journals cited herein.

The terms "patient" and "subject" throughout the specification refer to an animal, usually a mammal and preferably a human, treated, including prophylactic treatment (prophylaxis), with the cell compositions provided herein. For the treatment of infections, conditions and disease states that are characteristic of a particular animal patient, e.g., a human patient, the term patient refers to that particular animal.

The terms "treat", "treating", and "treatment", and the like, as used herein, refer to any act of providing a benefit to a patient at risk of or afflicted with a disease state, condition or deficiency that is ameliorated by a cellular composition of the invention. Treating a condition includes ameliorating the condition by alleviating or inhibiting at least one symptom, delaying the progression of the effects of the disease state or condition, including preventing or delaying the onset of the disease state or condition, and the like. Treatment as used herein includes prophylactic and therapeutic treatments.

The term "initiating pluripotent stem cells", wherein "human embryonic stem cells" or hESCs and human induced pluripotent stem cells or hiPSCs are a subset thereof, derived from pre-embryonic, fetal tissue or adult stem cells at any time after fertilization (in the case of human induced pluripotent stem cells), and tested according to standard techniques (standard-accepted test), such as the ability to form teratomas in 8-12 week old SCID mice, characterized by the ability to produce progeny of several different cell types, which are developmental products of all three germ layers (endoderm, mesoderm, ectoderm) under appropriate conditions. This term includes the various types of established stem cell lines and cells derived from pluripotent starting tissues in the manner described.

The definition of pluripotent or pPS cells (pPSCs) includes embryonic cells of various types, including in particular human embryonic stem cells (hESCs), described by Thomson et al (Science 282:1145, 1998); and embryonic cells from other primates, 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 obtainable from human umbilical cord or placental blood and human placental tissue. It includes any cell of primate origin that is capable of producing progeny that are the product of development of all three germ layers, whether they are derived from embryonic tissue, fetal or other sources. The pPS cells are preferably not derived from a malignant source. It is desirable (but not always necessary) that the cells be karyotype.

A pPS cell culture is said to be "undifferentiated" if a substantial proportion of the stem cells and their developmental products in the population exhibit morphological characteristics of undifferentiated cells and are clearly distinguishable from differentiated cells of embryonic or adult origin. Undifferentiated pPS cells are readily recognizable to those skilled in the art and typically exhibit a high nuclear/cytoplasmic ratio and a cell population of distinct nucleoli in a two-dimensional microscopic view. It will be appreciated that a population of undifferentiated cells will typically be surrounded by differentiated neighbouring cells in the population.

Pluripotent stem cells express one or more stage-specific embryonic antigens (SSEA)3 and 4, as well as markers detectable using antibodies against Tra-1-60 and Tra-1-81 (Thomson et al, Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (if any) and an increase in SSEA-1 expression. Undifferentiated pluripotent stem cells, which typically have alkaline phosphatase activity, can be detected by fixing the cells with 4% paraformaldehyde, followed by visualization as described by the manufacturer (Vector Laboratories, burlingham, ca) using Vector Red (Vector Red) as a substrate. Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as detected by RT-PCR.

Another desirable phenotype of proliferating pluripotent stem cells is differentiation into all three germ layers: potential of endodermal, mesodermal and ectodermal tissues. For example, the pluripotency of pluripotent stem cells can be confirmed by injecting the cells into Severe Combined Immunodeficiency (SCID) mice, fixing the resultant teratomas with 4% paraformaldehyde, and then histologically examining them as evidence of cell types from three germ layers. Alternatively, pluripotency can be determined by generating embryoid bodies and detecting the presence of markers associated with the three germ layers in the embryoid bodies.

The expanded pluripotent stem cell line was karyotyped using standard G banding techniques and compared to the published karyotype of the corresponding 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 no significant changes are made.

Cells taken from a pluripotent stem cell population cultured in the absence of feeder cells are also suitable. Variant human embryonic stem cell lines, such as BG01v (Bressagen, Asens, Georgia) and normal human embryonic stem cell lines, such as WA0l, WA07, WA09 (WiCeIl) and BG0l, BG02 (Bressagen, Asens, Georgia) are also suitable.

Ectodermal stem cells (EpiScs) and Induced Pluripotent Stem Cells (iPSCs), particularly human induced pluripotent stem cells (hiPSCs), are conceptually within the broad definition of pluripotent cells, and the techniques described herein apply to these and other pluripotent cells (e.g., naive pluripotent cells) as described above. EpiScs were isolated from early post-phyto-stage embryos. They express Oct4 and are pluripotent. See, Tesar et al, Nature, VoI 448, p.196, 12.7.2007. iPS cells are dedifferentiated back to a pluripotent state by adult somatic cells by retroviral transduction of four genes (c-myc, Klf4, S10X2, Oct 4). See, Takahashi and Yamanaka, Cell (Cell)126, 663-.

Human embryonic stem cells (hESCs) can be prepared by methods described herein and described in the art, e.g., Thomson et al (U.S. Pat. No.5,843,780; Science 282:1145, 1998; Current topics in developmental biology (curr. Top. Dev. biol.)38:133ff., 1998; Proc. Natl. Acad. Sci. U.S. A.)92:7844, 1995).

The term "embryonic stem cell" refers to a pluripotent cell, preferably a primate, including a human, which is isolated from a blastocyst stage embryo. Human embryonic stem cells refer to stem cells from humans, preferably for use in the therapeutic or diagnostic aspects of humans to which the present invention relates. The following phenotypic markers were expressed by human embryonic stem cells:

SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, alkaline phosphatase, Oct4, Nanog, Rex 1, Sox2 and TERT, see Ginis et al, developmental biology (Dev. biol), 269(2), 360-; draper et al, anatomical impurities (J.Ant.), 200(Pt.3), 249-258, (2002); carpenter et al, cloned Stem cells (Cloning Stem cells), 5(1), 79-88 (2003); cooper et al, anatomical impurities (J.Ant.), 200(Pt.3), 259-265 (2002); oka et al, molecular cell biology (MoI.biol.cell), 13(4), 1274-81 (2002); and Carpenter et al, developmental biology dynamics (Dev. Dyn.), 229(2), 243-258 (2004). While any primate pluripotent stem cells (pPSCs), including in particular human embryonic stem cells, can be used in the present methods to produce mesendoderm cells, mesoderm Isl1+ cells, Multipotent Migratory Cells (MMCs), multipotent CXCR4+ CD56+ cells (C56Cs), or multipotent Epicardial Progenitor Cells (EPCs) as described herein, the preferred pPSCs for use in the present invention include human embryonic stem cells, including those from the BG01 and BG02 cell lines, as well as many other stem cell lines available, including human induced pluripotent stem cells.

The term "differentiation" is used to describe the process by which non-specialized (unconfined) or less specialized cells acquire characteristics of more specialized (specialized) cells, such as multipotent migratory cells, multipotent CXCR4+ CD56+ cells, multipotent epicardial progenitor cells, nerve cells, muscle cells, cardiac muscle or other cells. The term "differentiation" includes the process by which pluripotent stem cells, including hescs, become more specialized intermediate cells, such as progenitor cells, and also more specialized intermediate cells (MMC, mesendoderm, mesoderm, C56C or EPC) become still more specialized cells. Differentiated or differentiation-induced cells are cells that occupy a specialized ("committed") location within a cell lineage. The term "restricted," when applied to a differentiation process, refers to a cell that has progressed to a point in the differentiation pathway that under normal circumstances will continue to differentiate into a particular cell type or subset of cell types and under normal circumstances will fail to differentiate into a different cell type or revert to a less differentiated cell type. "dedifferentiation" refers to the process of a cell returning to a less specialized (or restricted) location within the cell lineage. As used herein, a cell lineage defines the heritability of a cell, i.e., from which cell it originates and possibly which cell it produces. Cell lineages place cells into a genetic system of development and differentiation. Lineage specific markers refer to features that are specifically associated with the phenotype of the cell lineage of interest and can be used to assess the differentiation of unrestricted cells to the cell lineage of interest.

The terms "multipotent migratory cell" or "MMCs" are used interchangeably to refer to a cell or cells produced according to the present invention. MMCs are dynamic multipotent cells characterized by E-cad-Oct4-Nanog-SSEA3-CXCR4+, which have low to moderate densities and are migratory. They are storage stable and can be passaged for many generations and still remain viable. They have significant developmental plasticity. Based on the marker profile, they are not hESCs.

The MMCs are storage stable in the presence of effective amounts of a GSK inhibitor and an activin A inhibitor. BMP inhibitors, such as Noggin, may also be used in combination with GSK inhibitors and activin a inhibitors. These cells can differentiate into mesodermal cells or definitive endoderm cells, among a variety of other cells. Further methods relating to MMCs will be disclosed herein.

The Multipotent Migratory 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:

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

When plated at low density, the cells appear interstitial, and at high density, they grow into sheets

Can be generated from a series of hESC cell lines including BG0l, BG02, WA09

MMCs can be frozen and cryopreserved by standard methods

MMCs can recover, recover and differentiate after cryogenic storage

MMCs can be passaged with high plating efficiency (greater than 50% plating efficiency-50% cells successfully passaged and viable)

Absence of SSEA3 antigen and SSEA4 antigen on its cell surface

No expression of hESC markers such as Oct4, Nanog

MMCs can express CXCR4 on their surface

MMCs express high levels of the transcription factors Zicl, HoxA9, HoxD4, HoxA5, HoxC10, HoxD3, Pax6, N-CAM, CXCR4

MMCs are not mesendoderm because they do not express T/brachyury or mesoderm proteins (eomesodermin)

Negative for E-cadherin

At levels detectable by Q-PCR analysis, MMCs do not express Sox17, Isl1, Wucang (musashi), nestin (nestin)

Maintenance of normal karyotype during passaging

Exhibit a migratory, mesenchymal phenotype

Having multipotent differentiation capacity (including mesoderm, endoderm)

No teratoma formation when injected into SCID mice

Ability to isolate embryonic and fetal tissues from the inner cell mass

See microarray data, which more fully describes the MMC gene expression profile.

The term "mesodermal (Isl 1+) cell", mesodermally derived Isl1+ multipotent progenitor cell "ISL + multipotent progenitor cell" or "IMP" as used herein is used interchangeably to describe a mesodermal Isl1+ cell, mesendoderm cell or MMCs produced from pPSCs, particularly hESCs, according to the methods of the invention or as otherwise described herein (see examples section).

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

expression of Isl1, Nkx2.5, Fgf10, Gata4, FoxFl, PDGFR α

Optionally expressing Tbx3 and/or Hand (Hand)1

Karyotype normality

No expression of Oct4, Nanog, T, degermed proteins

Expression of PDGFR beta and cadherin 11 on the cell surface

Differentiation into cardiomyocytes, smooth muscle cells and endothelial cells, etc.

The cell surface markers PDGFR β and/or cadherin 11 of IMP represent immunogenic targets that can be used to conjugate monoclonal antibodies specific for the cell surface markers and thereby isolate IMPs from a population of cells. The use of monoclonal antibodies linked to reporter molecules (fluorescent, radioisotope, etc.) can be used to identify the presence and relative number of cells in a cell sample. anti-PDGFR β monoclonal antibodies are disclosed in U.S. patent publication US2009/0053241, which is incorporated herein by reference in its entirety. Other monoclonal antibodies that may be used in the invention in addition to anti-PDGFR β include IMC-2C5 and the like.

The terms "multipotent CXCR4+ CD56+ cells", "CXCR 4+ CD56+ cells" or "C56 Cs" as used herein are used to describe pre-mesenchymal pluripotent cells (pre-mesenchymal cells) that can be produced from hPSCs and MMCs according to the methods herein, as otherwise described herein.

These cells can be used to treat inflamed and/or damaged tissue by injecting an effective amount of the cells into a patient in need of an effective amount of treatment.

According to reports in the literature where bone marrow derived from mesenchymal Stem cells has been applied to disease models (Phenney and Prockop, 2007; Stem cells (Stem Cell)25: 2896-. This is based on their ability to stimulate repair, either by releasing factors that stimulate other cells to repair paracrine effects of damaged tissues or, by direct transdifferentiation into cell types involved in the repair process (fig. 16).

These applications include, but are not limited to, the following treatments:

cardiovascular disease (cardiomyopathy, ischemia)

Myopathy of retina (retinomyyopathy)

Neuropathy

Diabetes mellitus (type I and II)

Middle wind

Head trauma

Autoimmune diseases (lupus, arthritis, multiple sclerosis)

Immunosuppression

Graft versus host disease

Bone repair

Wound repair

Inflammatory diseases (arthritis, Crohn's disease, cystic fibrosis)

Parkinson's disease, Huntington's disease

The C56Cs of the invention has the following characteristics:

they express biomarkers of CXCR4 and CD56 (CXCR4+ and CD56 +);

they express higher levels of CXCR4 than MMCs;

they express appreciable levels of at least 3, at least 4, at least 5, at least 6, preferably all of the following biomarkers:

C-kit，CD166，CD105，CD44，CD133，CD90；

they do not express CD 31;

and in most cases:

they express PDGFR α at low levels;

they may exhibit properties of homing to sites of inflammation and tissue damage, through the SDF-1/CXCR4 signaling axis (see, e.g., Dalton, regenerative medicine (regen. med), 3, 181-;

these cells are smaller in shape than hESCs and hiPSCs, making them useful for intravenous administration.

C56Cs is prepared by contacting MMCs with an effective amount of a bone morphogenic protein (preferably, BMP4), a Wnt protein (preferably, Wnt3a), and a butyrate salt (preferably, sodium butyrate) in differentiation media for about 1 to 8 days or more, preferably about 2 to 7 days, about 3-6 days, about 4-6 days, as otherwise described herein. In this aspect of the invention, the differentiation of MMCs to C56Cs occurs in the absence of GSK inhibitors (e.g., BIO) and activin A inhibitors (e.g., SB 431542).

The generation pathway of C56Cs is shown in fig. 14. The production of MMCs from hESCs is disclosed herein and previously described (see PCT/US2008/001222, published as WO2008/094597, 8/7/2008, incorporated herein by reference). hPSCs are usually differentiated in the presence of GSK inhibitors (BIO) and activin a inhibitors (SB 431542). Optionally, for the production of MMCs, inhibitors of BMP signaling (noggin, Compound C) may also be included. The method of producing C56Cs is applicable to any human pluripotent cell such as a human induced pluripotent stem cell (hiPS cell) or similar human pluripotent stem cell. To produce MMCs, human pluripotent stem cells, including, inter alia, hESCs or hipSCs, are contacted with a differentiation medium comprising an effective dose of an inhibitor of GSK3, such as BIO (between 0.25 and l 0. mu.M, about 0.5 to about 5. mu.M, about 1 to 4. mu.M, about 1.5 to 3. mu.M, about 2. mu.M), and an activin A inhibitor, such as SB431542 (about 2 to 50. mu.M, about 5 to about 35. mu.M, about 10to 30. mu.M, preferably about 20. mu.M), as otherwise described herein. To produce C56Cs, MMCs are treated with BMP4 (about 10-250ng/ml, preferably about l00ng/ml), Wnt3a (about 5 to about 50ng/ml, about 25ng/ml), sodium butyrate (0.1 to about 5mM, 0.25 to about 1mM, about 0.5mM) in basal medium [ DMEM/F12[50/50] for about 1 to 8 days (preferably 3-6 days). The basal medium (differentiation medium) preferably contains effective amounts of other components as described herein, including approximately 2% prealbumin (probumin), antibiotic [1 x penicillin/streptomycin (Pen/Strep)1 x nonessential amino acid (NEAA) ], trace elements A, B, C [1 x, from media technology (Mediatech) ], ascorbic acid [ about 10to 100. mu.g/mL,. about.50. mu.g/mL ], transferrin [. about.10. mu.g/mL ], beta-mercaptoethanol [ about 0.1mM ], and bFGF [ e.g., about 8ng/mL ], LR-IGF [ e.g., about 200ng/mL ], activin A [ e.g., about 1 to 20ng/mL, 10ng/mL ], hybrid (heregulin) [ e.g., about 1 to 20ng/mL, about 0ng/mL ]). Importantly, GSK inhibitors (as opposed to wingless or Wnt proteins) and activin a inhibitors were absent when MMCs differentiated into C56 Cs. Furthermore, when MMCs are used to produce C56Cs, inhibitors of bone morphogenetic proteins (noggin, Compound C) should also be absent.

A composition useful in therapy as described above, comprising an effective amount of C56C cells for performing the therapy. The composition comprises about 5 xl0⁵To 5X 10⁸Preferably about 10⁶And 10⁸Cells suspended in saline solution. The amount of saline solution is generally in the range from 50. mu.l to about 10ml, preferably about 100. mu.l to 2 ml. The composition may be administered intravenously directly into the site of treatment with the cells of the invention, or by perfusion. The purity of C56Cs cells used for treatment is from at least about 50% to greater than 99.5%, about 75% or higher, about 85% or higher, about 90% or higherHigher, about 95% or higher, about 97.5% or higher, about 98% or higher, about 99% or higher, about 99.5% or higher. Generally, the conditions for differentiating the MMCs to produce C56Cs will result in C56Cs of high purity, thus eliminating the need for further purification. The cells can be administered in the absence of or in addition to the bioactive agent. Pharmaceutical compositions represent other aspects of the invention, comprising an effective amount of C56Cs together with a pharmaceutically acceptable carrier, additive or adjuvant and optionally, other bioactive agents therapeutically suitable for use in the proposed treatment with C56 Cs.

The term "epicardial pluripotent cells" or "EPCs" is used to refer to pluripotent cells derived from human pluripotent cells (hPCs) including hESCs or from Isl1+ pluripotent cells (IMPs) according to the invention by contacting the hPCs with conditions that produce IMPs and then contacting the resulting IMPs with conditions that produce EPCs. As described, EPCs are produced by contacting IMPs with differentiation medium in the presence of an effective amount of a GSK inhibitor (e.g., a Wnt protein such as Wnt3a, or a GSK inhibitor such as BIO, as otherwise described herein), a bone morphogenic protein (e.g., BMP4), and retinoic acid (preferably, all-trans retinoic acid) for a sufficient period of time (e.g., about 8 to 20 days or more, about 10to 18 days, about 15-17 days or more) to convert the IMPs to EPCs. EPCs can be produced directly from hPCs by contacting the cells with an effective amount of a GSK inhibitor (e.g., WNT3a or BIO), a bone morphogenic protein (e.g., BMP4), and optionally an activin a inhibitor (e.g., SB431542), and then (usually over about 2-8 days) further contacting the intermediate cells produced (which are IMPs) with the same conditions described above for the conversion of IMPs to EPCs (e.g., WNT3a or BIO, BMP4, and all-trans retinoic acid for up to about 16 to 20 days or more).

EPCs (protoepicardial/epicardial cells) are characterized by their ability to form an outer layer over the surface of the myocardium and to migrate into the myocardium by means of invasion (Olivey et al, Trends Cardiovasc Med. 14, 247;) 251). The standard test to assess the protoepicardial/epicardial migration properties is to plate the cells on collagen I matrix.

Microarray analysis of EPCs generated from three hESC lines and the human iPSC line showed that EPC cells expressed Wilm's tumor suppressor 1(Wt1), Tcf21 (epicardial element), Raldh2(Aldhla 2). These transcription factors/biomarkers are the initial means of identifying EPCs, protoepicardial/epicardial cell types, produced by pluripotent cells in culture.

In addition to the above, EPCs may also express one or more of Tbxl8, COL3a1, GAT a6, Tbx3, and Tbx 5(2, 3,4, or 5). As shown in fig. 47 and table 2, the genes with the highest up-regulation were extracted from the table.

EPCs have many uses. They can be used to identify epicardial-produced secreted factors that affect cardiomyocyte proliferation, survival, function and differentiation; they provide a source of cells that can be used for cardiovascular drug screening; they provide a source of cells for therapeutic purposes for repairing ischemic heart and/or regenerating coronary vasculature; they can be used for tissue engineering purposes to obtain the vascular component of the heart or coronary arteries; and they can be used as research tools for researching cardiovascular development and diseases.

As described herein, EPCs can further differentiate into endothelial cells (at an effective amount of VEGF)₁₆₅Or VEGF₁₆₅And SB431542, or other activin a inhibitors); smooth muscle and cardiac fibroblasts (in effective amounts of VEGF)₁₆₅Or VEGF₁₆₅And platelet-derived growth factor beta (PDGF beta), or VEGF₁₆₅And hDkk1 in 10% fetal bovine serum) or vascular (in the presence of FGF2, LR-IGF, heterozygote (hetergulin) β and VEGF), as otherwise described herein. These cells may also be used to treat and/or reduce the likelihood of cardiovascular disease/damage to cardiac tissue or vascular disease/damage by administering an effective amount of EPCs to a patient in need of treatment.

As used herein, the terms "differentiation medium", "cell differentiation medium", "culture medium", "basal cell culture medium" or "basal medium" or "stable medium" are synonymous and are used to describe a cell growth medium (depending on the additional components used) in which hESCs, mesoderm ISl1+ pluripotent cells (IMPs), Multipotent Migratory Cells (MMCs), C56Cs, EPC's, or other cells are produced, grown/cultured, or, alternatively, differentiated into more mature cells. Specific examples thereof are shown in the examples section below. Differentiation media are well known in the art and comprise at least one minimal essential medium plus one or more optional components such as growth factors, including Fibroblast Growth Factor (FGF), ascorbic acid, glucose, nonessential amino acids, salts (including trace elements), glutamine, insulin (which is specified and not excluded), activin a, transferrin, beta mercaptoethanol, and other agents well known in the art and described elsewhere herein. Preferred media include basal cell culture media containing between 1% and 20% (preferably, about 2-10%) fetal bovine serum, or media of known composition (preferred) that is free of fetal bovine serum and KSR, and optionally contains bovine serum albumin (about 1-5%, preferably about 2%). Preferred differentiation media are of known composition and serum-free. In certain embodiments in which MMCs are produced and an activin A inhibitor is used, the medium can eliminate or substantially reduce the amount of activin A.

Other factors that may optionally be added to the differentiation medium according to the present invention include, for example, nicotinamide, TGF- β family members including TGF- β 1,2 and 3, activin A, nodal (nodal), serum albumin, Fibroblast Growth Factor (FGF) family members, platelet-derived growth factor-AA, and platelet-derived growth factor-BB, platelet rich plasma, insulin growth factors (IGF-I, II, LR-IGF), growth differentiation factors (GDF-5, -6, -8, -10, -11), glucagon-like peptides-I and II (GLP-I and II), GLP-I and GLP-2 mimetics (mimotobodies), exenatide-4 (Exin-4), parathyroid hormone (parathyroid hormone), insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, Epidermal Growth Factor (EGF), gastrin I and II, copper chelators such as triethylenepentamine, forskolin (forskolin), sodium butyrate, betacellulin (betacellulin), ITS, noggin, neurite growth factor, nodal (nodal), valproic acid (valporic acid), trichostatin A (trichostatin A), sodium butyrate, Hepatocyte Growth Factor (HGF), sphingosine-1, VEGF, MG132(EMD, CA), N2 and B27 supplements (Gibco, CA), sterol alkaloids such as cyclopamine (EMD, CA), Keratinocyte Growth Factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis associated protein (ININ), indian hedgehog (indian hedgehog), sonic hedgehog (sonic hedgehog) protein, sonic hedgehog inhibitor (inhibitor of the sonic pathway, hedgehog inhibitor (bitchinone) protein, a hybrid (heregulin), or a combination thereof. Effective dosages are included for each component.

By way of further example, suitable media may be prepared from, for example, Darlington's modified-Ethich medium (DMEM), Gibco # 11965-; knockdown Darlington's modified Escherchia medium (KODMEM), Gibco # 10829-; hamm's F12/50% DMEM basal medium; 200mM L-glutamine, Gibco # 15039-; a non-essential amino acid solution, Gibco 11140-050; beta-mercaptoethanol, Sigma # M7522; recombinant human basic fibroblast growth factor (bFGF), Gibco # 13256-. Preferred embodiments of the media used in the present invention are described elsewhere herein.

A particularly preferred differentiation medium for growing/culturing pPSCs, in particular hESCs, and differentiated cells in the context of the present invention (depending on the components used) is DMEM/F12(50:50) containing about 2% prealbumin (albumin; Millipre/serology), 1 XPenicillin/streptomycin (Pen/Strep)1 Xnonessential amino acids (NEAA), 1 Xtrace elements A, B, C (Medium technology (Mediatech)), ascorbic acid (10-100ng/ml, about 25-65ng/ml, about 50ng/ml), about 0.lmM (0.025-0.5mM) beta-mercaptoethanol (Gibco), about 2-10ng/ml, about 5-9ng/ml, about 8ng/ml FGF bSigma), 200ng/ml (5-500ng/ml) of IGF-JLR (IGF-I; biological scientific RH), 10ng/ml activin A (from about 1ng/ml to no more than about 20ng/ml, and not included in certain aspects), and l0ng/ml (about l-20ng/ml or more) of a hybrid (heregulin). The components used are effective dosages and the dosage ranges of the individual components, as well as the preferred dosages, are applicable to the medium used in the present invention, and are not limited by the cells produced. It is noted that activin a or activin a signaling is not required for production of multipotent migratory cell MMCs, but may be present (if present, activin a is preferably present at low concentrations, typically less than about 20ng/ml, and in some cases preferably absent), especially when generating mesodermal (Isl +) cells. In contrast, about 20ng/ml to 100ng/ml or more of activin A or "high concentration activin A" can be used to produce other cells, as described herein. Alternatively, mouse embryonic fibroblast conditioned medium (MEF-CM) according to the invention, having a composition similar to DMEM/F12, can also be used to passage hESCs and produce Isl1+ mesodermal cells (IMPS) and Multipotent Migratory Cells (MMCs), as well as CXCR4+ CD56+ (C56Cs) cells and epicardial multipotent cells (EPCs).

Differentiation media useful in the present invention are commercially available and can be supplemented with commercially available components, available from the Invitrogen Corp. (GIBCO)), cell applications, Inc. (CellAplications, Inc.) and the Biological industry (Biological Industries), Beth HaEmek, Israel, as well as numerous other commercial sources including Cambridge Biochemical (Calbiochem). In a preferred embodiment, at least one differentiation factor such as Fibroblast Growth Factor (FGF), LR-IGF (an analogue of insulin-like growth factor), hybrid (heregulin) and optionally VEGF (preferably all three in effective amounts) is added to the cell culture medium, in which the stem cells are cultured and differentiated into multipotent migratory cells or endothelial cells (vascular cells). One of ordinary skill in the art can readily modify the cell culture medium to produce any one or more of the target cells of the present invention. Cell differentiation culture is essentially identical to the basal cell culture medium used for the differentiation process and contains cell differentiation factors that differentiate cells into other cells. A stable medium is a basic cell culture medium used before or after the differentiation step to stabilize the cell line for further use. Culture medium is substantially equivalent to a stable medium, but refers to a medium in which a pluripotent or other cell line is grown or cultured prior to differentiation. Generally, as used herein, a cell differentiation medium and a stabilization medium may comprise substantially similar components to the basal cell culture medium, but in different circumstances and possibly slightly different components to achieve the desired results of using the medium. In the case of MMCs, and in particular storage-stable MMCs, cell culture media comprising an effective amount of an activin A signaling inhibitor as disclosed herein and an effective dose of a GSK inhibitor as described herein can be used to differentiate and stabilize MMCs, i.e., prevent their further differentiation, and achieve storage stability of the cell population. BMP inhibitors may be used to couple the activin a inhibitor and GSK inhibitor used for this purpose.

Pluripotent stem cells can also be cultured on feeder cell layers, which support the pluripotent stem cells in a variety of ways as described in the art. Alternatively, the pluripotent stem cells are cultured in a culture system that is substantially free of feeder cells, but still supports proliferation of the pluripotent stem cells without substantial differentiation. The growth of pluripotent stem cells in a culture without differentiation and feeder cells can be supported by a medium conditioned by culturing another cell type. Optionally, growth of pluripotent stem cells in a non-differentiated feeder cells-free culture is supported by chemically known composition media. Such methods are well known in the art. In a preferred aspect of the invention, the cells are grown in feeder cells-free medium.

Methods of culturing cells on a feeder cell layer are well known in the art. For example, Reubinoff et al (Natural Biotechnology)18: 399-. Richards et al, (Stem Cell)21:546-556, 2003) evaluated the ability of a panel of 11 different feeder Cell layers of human adults, fetuses and newborns to support human pluripotent Stem Cell cultures. Richards et al indicate that: "human embryonic stem cell lines cultured on adult skin fibroblast feeder cells retain the morphology of human embryonic stem cells and maintain pluripotency". US20020072117 discloses a cell line producing a medium that supports the growth of primate pluripotent stem cells in feeder cells-free culture. The cell lines used are mesenchymal cell lines and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. US20020072117 also discloses the use of cell lines as an initial feeder cell layer. In another example, Wang et al (Stem Cell)23:1221-1227, 2005) disclose methods for growing human pluripotent Stem cells for extended periods on feeder Cell layers derived from human embryonic Stem cells. In another example, Stojkovic et al (Stem Cell)200523:306-314, 2005) disclose a feeder Cell system derived from the spontaneous differentiation of human embryonic Stem cells. In another example, Miyamoto et al (+22:433-440, 2004) disclose a source of feeder cells obtained from human placenta. Amit et al (reproductive biology (biol. reprod)68: 2150-. In another example, Inzunza et al (Stem Cell)23: 544-.

Methods for culturing pPSCs in culture, particularly feeder cells-free media, are well known in the art. U.S. patent No.6,642,048 discloses media that support the growth of primate pluripotent stem cells (pPS) in feeder cells-free culture, and these cell lines can be used to produce such media. U.S. patent No.6,642,048 states that: the invention includes mesenchymal and fibroblast cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods of producing such cell lines, treating the medium, and growing stem cells using conditioned medium are described and explained herein. In another example, WO2005014799 discloses a conditioned medium for the maintenance, proliferation and differentiation of mammalian cells. In another example, Xu et al (Stem cell)22:972-980, 2004) disclose conditioned media obtained from human embryonic Stem cell developmental products that have been genetically modified to overexpress human telomerase reverse transcriptase. In another example, US20070010011 discloses a chemically known composition medium for maintaining pluripotent stem cells.

Alternative culture systems employ serum-free media supplemented with growth factors that promote proliferation of embryonic stem cells. For example, Cheon et al (reproductive biology (BioReprod) DOI 10.1095/bioleprord.105.046870, 2005, 10/19) disclose a feeder cells-free, serum-free culture system in which embryonic stem cells are maintained in unconditional Serum Replacement (SR) medium supplemented with different growth factors that trigger the self-renewal of embryonic stem cells. In another example, Levenstein et al (Stem cell)24:568-574, 2006) discloses a method for long-term culture of human embryonic Stem cells in the absence of fibroblasts or conditioned medium using bFGF-supplemented medium. In another example, US20050148070 discloses a method of culturing human embryonic stem cells in a medium of known composition free of serum and free of fibroblast 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 having the ability to activate fibroblast growth factor signaling receptors, wherein the growth factor is provided by a source other than a fibroblast feeder cell layer, the medium supporting proliferation of stem cells in an undifferentiated state and being feeder cells free or conditioned medium.

US20050233446 discloses a medium of known composition for culturing stem cells, including undifferentiated primate naive stem cells. In solution, the medium is substantially isotonic compared to the stem cells being cultured. In a given culture, a particular medium contains a basal medium and the amounts of bFGF, insulin, and essential ascorbic acid required to support the substantially undifferentiated growth of the primitive stem cells. In another example, WO2005065354 discloses an isotonic culture medium of known composition substantially feeder and serum free comprising: a. a basal medium; b. bFGF in an amount sufficient to support the growth of substantially undifferentiated mammalian stem cells; c. insulin in an amount sufficient to support the growth of substantially undifferentiated mammalian stem cells; ascorbic acid in an amount sufficient to support the growth of substantially undifferentiated mammalian stem cells.

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

These cells are preferably grown on a cell support or substrate, such as an adherent monolayer, rather than in an embryonic body or suspension. In the present invention, Matrigel (Matrigel) is preferably used as a cell support. The cell support preferably contains at least one differentiation protein. The term "differentiation protein" or "substrate protein" is used to describe a protein that can be used to grow cells and/or promote differentiation (also preferably adherence) of embryonic stem cells or mesendoderm, mesoderm or Multipotent Migratory Cells (MMC). The differentiation protein preferably used in the present invention includes, for example, extracellular matrix proteins, which are proteins found in extracellular matrices, such as laminin (laminin), tenascin (tenascin), thrombospondin (thrombospondin), and mixtures thereof, which exhibit growth promotion and contain a domain homologous to Epidermal Growth Factor (EGF) and exhibit growth-promoting and differentiation activities. Other differentiation proteins that may be used in the present invention include, for example, collagen, fibronectin (fibronectin), vitronectin (vitronectin), polylysine (polylysine), polyornithine (polyornithine), and mixtures thereof. In addition, gels and other materials such as methylcellulose of other gels may also be used, which contain an effective concentration of one or more of these embryonic stem cell differentiation proteins. Exemplary differentiation proteins or materials containing these differentiation proteins include, for example, BD Cell-Tak^TMCell and tissue adhesive, BD^TMFibrogen human recombinant collagen I, BD^TMFibrogen human recombinant collagen III, BD Matrigel^TMBase film matrix, BD Matrigel^TMHigh concentration of substrate film substrate (HC), BD^TMPuraMatrix^TMPeptide hydrogel, collagen I, High Concentration (HC) collagen I, 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 composition/material comprising one or more differentiation or substrate proteins is BD Matrigel^TMA base film matrix. This is a solubilized basement membrane preparation, which is extracted from the sarcomas of mice of the ingelbreth-Holm-swarm (ehs), a tumor rich in ECM proteins. The main components are laminin, followed by collagen IV, heparan sulfate (heparin sulfate), proteoglycans, lactocin (entactin) and nidogen (nidogen).

Pluripotent stem cells are preferably plated on a differentiation or base protein. Pluripotent stem cells can be plated onto a substrate in a suitable distribution in the presence of a medium that promotes survival, propagation, and retention of desired properties of the cells. All of these features benefit from careful attention to the seeding profile and are readily determinable by one skilled in the art.

The term "activation" as used herein refers to an increase in the expression of a marker, such as Isl or up-regulating Isl or the activity of a marker associated with blood cells, vascular cells (endothelial cells), kidney cells, bone and muscle cells. These cells are useful in the treatment of heart disease, renal failure, bone repair and vascular degeneration.

The term "isolated" when used herein with reference to a cell, cell line, cell culture, or cell population, refers to being substantially isolated from a natural cell source such that the cell, cell line, cell culture, or cell population can be cultured in vitro. Furthermore, the term "isolating" is used to refer to the physical selection of one or more cells from a collection of two or more cells, wherein the cells are selected based on cell morphology and/or various markers of expression.

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

The term "marker" or "biomarker" as used herein is a nucleic acid or polypeptide molecule that describes differential expression in a cell of interest. In this context, differential expression refers to an increase in the level of a positive marker and a decrease in the level of a negative marker. The level of marker nucleic acid or polypeptide detectable in the cell of interest is sufficiently high or low compared to other cells, and the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

The term "contacting" (i.e., contacting a cell with a compound) as used herein is meant to include incubating the compound and the cell together in vitro (e.g., adding the compound to the cell in culture). The term "contacting" is not intended to include exposure of a cell to a differentiating agent that is naturally present in a subject ("exposure") in vivo (i.e., exposure is due to a natural physiological process). The step of contacting the cells with the differentiation medium and one or more growth factors (BMP or others) and/or inhibitors (GSK, inhibitors of activin a (signaling) or BMP (signaling, etc.)) can be performed in any suitable manner, as described elsewhere herein. For example, cell treatments can be performed in adherent culture as a parietal layer, as embryo bodies or in suspension culture, although parietal layers are preferred because they provide an efficient differentiation process, typically into 90% or more of the target cell population (mesendoderm, mesoderm or pluripotent migratory cells). It is understood that the cells contacted with the differentiating agent may be further treated with other cellular differentiation environments to stabilize the cells, or to further differentiate the cells, such as to produce islet cells.

The term "differentiation agent" as used herein refers to any compound or molecule that can induce partial or complete differentiation of cells such as hESC's, multipotent migratory cells (mmscs), C56Cs, Isl1+ multipotent progenitor cells (IMPs), EPCs, wherein, for example, the differentiation is: differentiation of hESCs into mesodermal Isl1+ cells (IMPs) due to at least partial inhibition of GSK, at least partially comprising bone morphogenetic protein (BMP-2, BMP-4, BMP-6 or BMP-7); or alternatively, inhibits GSK and inhibin a and/or inhibits bone morphogenetic proteins to produce Multipotent Migratory Cells (MMCs); alternatively, Wnt3a, BMP4 and sodium butyrate activin a were added to produce C56Cs from MMCs; alternatively, Wnt3a, BMP4 and all-trans retinoic acid were added to produce EPCs and the like from IMPs. The differentiating agent may be as follows, and the term is not limited thereto. The term "differentiation agent" as used herein includes within its scope natural or synthetic molecules, or molecules that exhibit similar biological activities.

The term "effective" is used to describe a component, compound or composition that is used or included in an amount and/or for a time sufficient to produce the desired effect in the relevant environment. For example, an effective amount of a differentiation agent is an amount of differentiation agent that, together with other components, produces desired differentiated cells in a differentiation medium over an appropriate period of time, including the sequential number of times that different differentiation agents are contacted with the cells to be differentiated.

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

The term "GSK inhibitor" is used to describe compounds that inhibit GSK (particularly GSK3, including GSK3 α or GSK3 β). Examples of GSK inhibitors preferably used in the present invention include one or more of the following, all available from cambridge biochemistry (Calbiochem):

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

BIO-Acetoxime (Acetoxime) (2' Z,3' E) -6-bromoindirubin-3' -Acetoxime (GSK3 inhibits X);

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

pyridine carbazole-cyclopentadienyl ruthenium complex (GSK3 inhibits XV);

TDZD-84-Benzyl-2-methyl-1, 2, 4-thiazoline-3, 5-dione (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) (GSK3 beta inhibitor I)

2-Thio (3-iodobenzyl) -5- (1-pyridyl) - [1,3,4] -oxadiazole (2-Thio (3-iodobenzyl) -5- (1-pyridil) - [1,3,4] -oxadiazole) (GSK3 β inhibitor II);

OTTDT 2, 4-Dibenzyl-5-oxothiazoline-3-thione (2,4-Dibenzyl-5-oxothiadiazolidine-3-thione) (GSK3 beta inhibitor III);

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

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

3- (1- (3-Hydroxypropyl) -lH-pyrrolo [2,3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-pyrrole-2,5-dione (3- (1- (3-hydroxypypyl) -lH-pyroro [2,3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-pyrorol-2, 5-dione) (GSK-3 β inhibitor XI);

TWSl19 pyrrole pyrimidine compounds (pyrropyrinidine compounds) (GSK3 β inhibitor XII);

L803H-KEAPPAPPQSpP-NH₂or a tetradecanoyl form (Myristoylate form) thereof (GSK3 β inhibitor XIII); and

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

In addition, many wingless or Wnt proteins function similarly to GSK inhibitors, particularly the GSK inhibitors described herein. Thus, they fall under the term GSK inhibitor, but are not included herein and in the context of GSK inhibitors described above, e.g., where C56Cs is formed from MMCs, as otherwise described herein, and may be used to denote specifically wingless or Wnt proteins. Exemplary Wnt proteins useful in the invention include one or more of Wntl, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt14, Wnt14b, Wntl5, and Wnt16, among others. 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 the invention, which are involved in the differentiation and formation of pluripotent cells (MMCs) and Epicardial Pluripotent Cells (EPCs). When used, they are used in effective amounts at concentrations (depending on the molecular weight of the inhibitor used) of from about 0.001 to about l 00. mu.M or higher, from about 0.05 to about 75. mu.M, from about 0.1 to about 50. mu.M, from about 0.25 to about 35. mu.M, from about 0.5 to about 25. mu.M. For the case of BIO, the GSK inhibitor is used in the differentiation medium in an amount ranging from about 0.05 to about 50. mu.M, from about 0.1 to about l 0. mu.M, from about 0.5 to about 5. mu.M, about l-3. mu.M. When a Wnt protein is used, Wnt is used in an amount ranging from about 1 to about 100ng/ml, about 5 to about 50ng/ml, about 10to about 35ng/ml, about 20 to about 30ng/ml, about 25 ng/ml.

The term "activin a inhibitor" is used to describe a compound or component that is optionally added to the differentiation medium to inhibit the activin a effect (TGF β signaling inhibitor) during differentiation, and when used, to produce Multipotent Migratory Cells (MMCs) from hESCs or endothelial EPCs. To produce MMCs from hESCs, the differentiating agent contains effective amounts of a GSK inhibitor (preferably, a GSK3 inhibitor, such as BIO or other GSK3 inhibitor) and an activin A inhibitor plus or minus a Bone Morphogenic Protein (BMP) inhibitor.

Exemplary activin a inhibitors useful in the invention include, for example, SB431542(Sigma), follistatin (follistatin), follistatin gene-related protein (FGRP, available from R and DSystems), BMP and activin membrane-binding inhibitor (BAMBI), anti-BAMBI (monoclonal antibody), Smad7 (maternal anti-drosophila Homolog)7) and TGF RI inhibitor (cambium), among others. The activin A inhibitor used in the invention is in an effective amount, generally in the range of about 0.001 to about l00 μ M or more, about 0.05 to about 75 μ M, about 0.1 to about 50 μ M, about 0.25 to about 35 μ M, about 0.5 to about 25 μ M.

The term "bone morphogenic protein inhibitor" or "BMP inhibitor" is used to describe a compound or composition that, when added to a differentiation medium in an effective amount, inhibits the action of bone morphogenic proteins (inhibits BMP signaling) in the differentiation of hESCs into Multipotent Mesenchymal Cells (MMCs). Exemplary BMP inhibitors include, for example, noggin, Compound C, sclerostin (sclerostin), Grimlin (Drm/Gremlin), and USAG-I, among others. The amount of BMP inhibitor used is an effective amount, typically (depending on the molecular weight and efficiency of the inhibitor used) falls within the range of about 0.01ng/ml to about 500ng/ml or more, about 0.1 to about 350ng/ml, about 0.5 to about 250ng/ml, about 1 to about 500ng/ml, about 5 to about 250ng/ml, about 50 to about 150ng/ml, about 75 to about 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 reduces the activity of PI3 kinase or at least one molecule downstream of 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, for example, PI3 kinase antagonists, antagonists of the PI 3-kinase signal transduction cascade, compounds that decrease synthesis or expression of endogenous PI3 kinase, compounds that decrease release of endogenous PI3 kinase, and compounds that inhibit activators of PI3 kinase activity. In certain embodiments described above, the inhibitor is selected from the group consisting of: rapamycin (Rapamycin), LY 294002, wortmannin (wortmannin), lithium chloride, Akt inhibitor I, Akt inhibitor II (SH-5), Akt inhibitor III (SH-6), NL-71-101, and mixtures of the foregoing. Akt inhibitors I, II, Akt III, and NL-71-101 are available from Cambridge Biochemical (Calbiochem). In other embodiments, the inhibitor is selected from the group consisting of rapamycin and LY 294002. In a further preferred embodiment, such an inhibitor comprises LY 294002. In another embodiment, the inhibitor comprises Akt 1-II. It will be appreciated that combinations of inhibitors may be used to give the desired differentiation effect. The end result is the production of large quantities of definitive endoderm cells that can be used to produce pancreatic endoderm cells and/or liver endoderm cells, as disclosed in international application No. PCT/US2005/028829, published as 2005-8-15, publication No. WO 2006/020919 (published as 2006-2-23), and PCT/US2008/001222, published as 2008-1-30, 2008-8-7, publication No. WO2008/094597, which are filed 2005-1-30, relevant portions of which are incorporated herein by reference.

As used herein, the term "isolated" when referring to a cell, cell line, cell culture, or cell population refers to substantial isolation from a natural cell source such that the cell, cell line, cell culture, or cell population can be cultured in vitro. Alternatively, depending on the context of the article, the term "isolation" refers to a population of cells isolated from a differentiation medium and culture flask so that the population of cells can be preserved (cryopreserved). Furthermore, the term "isolated" is used to refer to the physical selection of one or more cells from a collection of two or more cells, wherein the cells are selected based on cell morphology and/or various markers of expression.

The term "passaging" is used to describe the process by which cells divide and migrate to new cell flasks for further growth/regrowth. According to the invention, preferably adherent cells (even embryoid bodies) can be passaged using an enzyme (Accutase)^TMOr collagenase), artificial passaging (mechanical, e.g., spatula (spatula) or other flexible mechanical device or apparatus) and other non-enzymatic methods, such as cell diffusion buffers.

The term "contacting" (i.e., contacting hescs, multipotent migratory cells, C56Cs, IMPs, or EPCs with a compound) or "exposing" as used herein is meant to include incubating the compound with the cells in vitro (e.g., adding the compound to the cells in culture). The term "contacting" is not intended to include exposure of a cell to a differentiating agent or inhibitor ("exposure") that naturally occurs in a subject in vivo (i.e., exposure is due to a natural physiological process). The step of contacting the cells with the growth factors and/or inhibitors in the differentiation medium may be carried out in any suitable manner, as described herein. For example, cells can be processed in adherent culture, as embryoid bodies, or in suspension culture. It will be appreciated that cells contacted with the differentiation agent and/or inhibitor may be further treated with other cellular differentiation environments to stabilize the cells, or to further differentiate the cells, such as to produce endothelial cells, muscle cells, including cardiomyocytes and vascular cells, including blood vessels. These cells are useful as restorative agents for the treatment of heart disease, renal failure, bone repair and vascular degeneration.

In certain embodiments, hESCs to be further differentiated, (Isl 1+) pluripotent progenitor cells (IMPs), EPCs, or MMCs are plated at a plating concentration of less than about 2.5 x 10⁶Cells/35 mm dish, at least about 2.5X 10⁴Individual cells/35 mm plate at about 2.5X 10⁵To about 2X 10⁶Cells/35 mm plate, at about 5X 10⁵To 2X 10⁶Cells/35 mm dish, less than about 2X 10⁶Cells/35 mm dish, or density greater than 4X 10⁵Cells/35 mm dish. In certain preferred aspects, the cells to be differentiated are at about 7.5X 10⁵Cells were plated at a concentration of 35mm dishes.

In the production of MMCs (Isl 1+) multipotent progenitor cells (IMPs) from hESCs, as a first step in a particular embodiment of the invention, the invention also encompasses the use of the composition to culture cells to produce adherent monolayers of hESCs. hESC's are grown as adherent monolayers on cell support, preferably Matrigel (Matrigel), in known component cell culture media (serum-free or KSR). In addition to the typical components described herein, the cell culture medium also preferably contains an effective amount of one or more of the following components: ascorbic acid, transferrin, beta-mercaptoethanol (Gibco), Fibroblast Growth Factor (FGF), LR-IGF, activin a and heterozygosity (hetergulin), preferably all of these components. The growth of hESCs parietal (or embryoid) into cell culture media for use as the initial population of differentiated cells can vary within the scope of the teachings in the art.

The hESCs produced above are then plated on a cell support and differentiated in a differentiation medium (as otherwise specified) under an effective amount of a differentiating agent and/or inhibitor. The cells are preferably grown as adherent monolayers. In the case of ISL l + multipotent progenitor cells (IMPs), hESCs are contacted with differentiation medium containing an effective amount of a GSK inhibitor (preferably BIO or Wnt3a) as otherwise described herein for a suitable time to produce a stable population of IMPs. In the case of IMPs production, hESCs are contacted with differentiation media containing an effective amount of a GSK inhibitor (preferably BIO or Wnt3a) in combination with a bone morphogenic protein (BMP-2, BMP-4, BMP-6, BMP-7) as otherwise described herein for a suitable time to produce (Isl 1+) pluripotent progenitor cell populations (IMPs).

IMPs can be cloned and/or expanded and passaged (Accutase, others) in a medium of known composition in the presence of GSK inhibitors (e.g., BIO at 0.5-10. mu.M, 2. mu.M) and BMP 4. These cells can then be brought to low density (20-200 cells/mm)²) The plates were maintained on matrix protein (Metrigel) in methylcellulose (0.9% final concentration) or other thickeners (e.g. cellulose) for several days (3), after which the medium was changed daily. Approximately two weeks later (14 days), individual populations can be isolated and sub-cultured to generate stable, clonable IMP cell lines.

IMPs can be used in the absence of kinetin A +/-IGF, in the presence of an effective amount of BMP (1-25ng/ml, about 10 ng/ml); BMP (1-25ng/ml, about 10ng/ml) + DKK (25-500ng/ml, 150 ng/ml); BMP (1-25ng/ml, about 10ng/ml) + DKK (25-500ng/ml, 150ng/ml) + VEGF (1-25ng/ml, l0 ng/ml); DKKl (25-500ng/ml, 150ng/ml) and VEGF (1-25ng/ml, 10ng/ml) for a period of about two weeks (about 10-20+ days), directly produced cardiomyocytes.

IMPs can be used to generate smooth muscle cells, cardiomyocytes, and endothelial cells in vitro and in vivo, as described herein. IMPs can be directly injected/applied to sites of myocardial tissue damage and can participate in the repair process by differentiating into functional cardiomyocytes, endothelial cells and smooth muscle cells. Furthermore, IMPs can differentiate into cardiomyocytes when cultured with cardiomyocytes.

In the medium of known composition described elsewhere herein, IMPs can be differentiated into EPCs using effective doses of wingless protein (Wnt3a), bone morphogenic protein (BMP4) and all-trans retinoic acid. The endothelial cells produced according to the methods of the invention can be used to generate endothelial cells, smooth muscle cells and cardiac fibroblasts.

IMPs can be directly injected/applied to damaged sites of myocardial tissue and can participate in the repair process by differentiating into functional cardiomyocytes, endothelial cells and smooth muscle cells. In addition, IMPs can differentiate into cardiomyocytes.

EPCs, such as IMPs, can be used to produce smooth muscle cells, cardiomyocytes, and endothelial cells in vitro and in vivo, as described elsewhere herein. IMPs can be injected/directly applied to damaged sites of myocardial tissue and can participate in the repair process by differentiating into functional cardiomyocytes, endothelial cells and smooth muscle cells. EPCs are also thought to be able to incorporate endodermal vascular tissue (chick embryo grafts). Thus, EPCs are thought to be capable of regenerating organs associated with endoderm, such as the gut, which has an inner layer derived from serosal mesothelium (the origin of the original epicardium). Thus, EPCs are believed to have utility in repairing endoderm-derived organs in vivo. According to the present invention, EPCs thus have the ability to colonize the intestinal vasculature and have an extracardiac repair effect (stroke, diabetic complications, etc.) in the context of a variety of tissues where regenerative vascularization is desired. See WiIm et al, Development, 132(23)5317-28, 2005.

In a further embodiment, the cell culture medium may be conditioned medium (MEF-CM). Conditioned media can be obtained from a feeder cell layer. It is contemplated that the feeder cell layer may contain fibroblasts, and in one embodiment, embryonic fibroblasts. Preferred media are feeder-free.

In a particularly preferred embodiment, the differentiation medium used to produce mesodermal (Isl 1+) cells (IMPs) or MMCs comprises DMEM/F12(50/50), about 2% proprotein (probumin) (albumin), antibiotic (1 XPenicillin/streptomycin (Pen/Strep)1 Xnonessential amino acids (NEAA)), trace elements A, B, C (e.g., 1X, from media technology (Mediatech)), ascorbic acid (e.g., about 50. mu.g/ml), transferrin (e.g., about l 0. mu.g/ml), beta-mercaptoethanol (about 0.lmM), bFGF (e.g., about 8ng/ml), LR-IGF (e.g., about 200ng/ml), activin A (e.g., about 10ng/ml) and neurotrophin (e.g., about 10 ng/ml). It is noted that activin a and nerve growth factor can be removed in the production of Multipotent Migratory Cells (MMCs). Of course, one or more of the above components may be omitted from the differentiation medium as taught in the art, but it is preferred to use all of the listed components in the present invention.

The present cells also provide potential uses for biological assays to identify molecules that affect (promote, inhibit or affect) cell differentiation. The first step in differentiation of the present cells provides a good opportunity to study epithelial to mesenchymal transition, particularly in cancer progression, as part of tumor metastasis. Thus, the methods and cell populations described herein provide a unique system that allows both the molecular understanding of EMT and the identification of new drug targets, and the screening of small molecules (BIO) that block EMT under EMT-promoting conditions. Since stem cell growth on 96/384 well plates can be easily achieved, rapid drug screening can be used to identify potential molecules that block or inhibit EMT and represent potentially valuable anticancer agents.

With respect to MMCs, they are stable populations of cells with pluripotent differentiation capacity grown in media of known composition. These cells are particularly useful for screening for molecules that promote or inhibit differentiation or promote and specifically differentiate into one or another lineage.

Treatment of

The cell populations and/or methods described herein can provide effective treatments for the management of cell-associated diseases and/or conditions.

In a first aspect, the present invention provides a method for treating a patient suffering from a cardiovascular disease. Such methods comprise 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. Alternatively, a method of treating a cardiovascular disorder, including infarction, in a patient comprises administering to cardiac tissue of the patient in need thereof an effective amount of Epicardial Progenitor Cells (EPCs) or Isl1+ multipotent progenitor cells (IMPs), either systemically, directly to the cardiac tissue or into the cardiac tissue. These cells are also useful in treating stroke and diabetic complications, particularly vascular complications and to repair endodermal organs (liver, pancreas, digestive tract, etc.) of patients.

In another aspect, the present invention provides a method of treating damaged or ischemic vascular tissue (blood vessels) in a patient in need thereof, comprising administering to the blood vessels to be repaired an effective amount of EPCs or IMPs. In an alternative embodiment, EPCs are differentiated into smooth muscle cells by cell passaging for a period of at least about 5-6 days in a cell differentiation medium containing an effective amount of a combination of a GSK inhibitor, preferably Wnt3a, and BMP (BMP4), and the obtained smooth muscle cells are administered (implanted) to the site of structural vascular damage in a patient to treat/repair the same.

The treatment method may utilize MMCs or preferably C56Cs produced as described herein to home to the site of damaged/inflamed tissue. In this regard, an effective amount of C56Cs is administered to a patient in need thereof to treat a disease state or condition selected from the group consisting of: cardiovascular diseases (cardiomyopathy, ischemia), retinomyopathies (retinomyopathy), neuropathies, diabetes (types I and II), stroke, head trauma, autoimmune diseases (lupus, arthritis, multiple sclerosis), immunosuppression, graft versus host disease, bone repair, wound repair, inflammatory diseases (arthritis, crohn's disease, cystic fibrosis) and parkinson's disease, huntington's disease and the like. Systemic administration of MMCs or C56Cs can be by direct intravenous administration or by injection to the site of injury or disease, including cardiovascular tissue (particularly ischemic heart) and skeletal tissue. Because of the homing properties of MMCs and, more importantly, C56Cs, these cells can be administered away from the site of injury/inflammation and the cells will "home" to those sites in the patient's body where treatment is to be administered.

If appropriate, the patient may be further treated with an agent or bioactive agent that is beneficial for the survival and function of the transplanted cells. Such agents may include, for example, insulin, members of the TGF- β family (TGF- β 1,2, and 3), bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and-13), fibroblast growth factors 1 and-2, platelet-derived growth factors-AA, and-BB, platelet rich plasma, insulin growth factors (IGF-I, II), growth differentiation factors (GDF-5, -6, -7, -8, -10, -15), vascular endothelial cell-derived growth factors (VEGF), pleiotrophin (pleiotrophin), endothelin (endonexin), and the like. Other pharmaceutical compositions may include, for example, nicotinamide, glucagon-like peptide-I (GLP-I) and II, GLP-1 and 2 mimetibodies (mimetopy), exenatide-4 (Exendin-4), retinoic acid, parathyroid hormone, MAPK inhibitors, e.g., the compounds disclosed in published U.S. application 2004/0209901 and published U.S. application 2004/0132729.

The Epicardial Pluripotent Cells (EPCs) of the present invention are useful for producing endothelial cells, smooth muscle cells and cardiac fibroblasts, or for producing vascular cells in a patient. These cells are useful for treating cardiovascular diseases and for repairing or treating damaged tissues, including liver, pancreas, and gastrointestinal tissues. The method comprises systemically administering to the subject EPCs in an amount effective to affect and enhance cardiomyocyte proliferation, survival function and differentiation. In addition, EPCs can be used to treat ischemic or damaged heart by regenerating coronary tissue, particularly including the coronary vasculature.

To further enhance differentiation, survival or therapeutic activity of the implanted cells, additional factors, such as growth factors, antioxidants, immunosuppressive or anti-inflammatory agents, are administered prior to, concurrently with or after administration of the cells. In certain embodiments, the growth factor is used to differentiate the administered cells in vivo. These factors may be secreted by endogenous cells and contacted with the cells to be administered in situ. The implanted cells may be induced to differentiate by any combination of endogenously and exogenously administered growth factors known in the art.

The number of cells used for implantation depends on many 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, a vascular disease, or suffering from ischemia. The method involves culturing pluripotent stem cells, differentiating the cultured cells into a population of epicardial pluripotent stem cells (EPCs) in vitro, and injecting the cells into a patient in need thereof, or alternatively, incorporating the cells into a three-dimensional support to produce endothelial tissue, cardiac myocytes, and smooth muscle tissue. The cells may be maintained on the support in vitro prior to implantation in a patient. Alternatively, the support containing the cells can be implanted directly into the patient without additional in vitro culturing. The support may optionally be combined with at least one pharmaceutical agent that facilitates survival and function of the transplanted cells, or may additionally be used to treat diabetes, or cardiovascular disease, or dysfunction.

Support materials suitable for the purposes of the present invention include tissue templates (templates), catheters (conduits), barriers (barriers) and reservoirs (reservoirs) for tissue repair. In particular, biological tissues that have been used in vitro and in vivo for reconstruction or regeneration, and for delivery of tissue growth inducing chemokines, both in the form of foams, sponges, gels, hydrogels, textiles and non-woven structures, synthetic and natural materials, are suitable for use in carrying out the methods of the invention. See, for example, the materials disclosed in 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, published U.S. application No. 2004/0062753a1, U.S. patent No.4,557,264, and U.S. patent No.6,333,029.

To form a support incorporating an agent, the agent and polymer solution may be mixed prior to support formation. Alternatively, the pharmaceutical agent may be coated on a preformed support, preferably in the presence of a pharmaceutical carrier. The medicament may be presented as a liquid, a finely divided solid (fine powdered) or any other suitable physical form. Optionally, adjuvants may be added to the support to modify the release rate of the agent. In an alternative embodiment, the support may be combined with at least one pharmaceutical compound that is an anti-inflammatory compound, such as the compound disclosed in U.S. patent No.6,509,369.

The support may be combined with at least one pharmaceutical compound that is an anti-apoptotic compound, such as the compound disclosed in U.S. patent No.6,793,945. The support may also be combined with at least one fibrosis inhibitor drug compound, such as the compound disclosed in U.S. patent No.6,331,298. The support may also be combined with at least one pharmaceutical compound that increases angiogenesis, such as the compounds disclosed in published U.S. application 2004/0220393 and U.S. published application 2004/0209901. The support may also be combined with at least one pharmaceutical compound of an immunosuppressive compound, such as the compound disclosed in published U.S. application 2004/0171623.

The support may also be combined with at least one growth factor drug compound, such as members of the TGF- β family, including TGF- β 1,2 and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12 and-13), fibroblast growth factors-1 and-2, platelet-derived growth factors-AA, and-BB, platelet rich plasma, insulin growth factors (IGF-I, II), growth differentiation factors (GDF-5, -6, -8, -10, -15), vascular endothelial cell-derived growth factors (VEGF), LDkkl, platelet-derived growth factor β (PDGF β), pleiotrophins, endothelin, and the like. Other pharmaceutical compounds may include, for example, nicotinamide, hypoxia inducible factor 1-alpha, glucagon-like peptide I (GLP-I), GLP-1 and GLP-2 mimetics, and II, exenatide-4 (Exendin-4), nodal (nodal), noggin, NGF, retinoic acid, parathyroid hormone, myosin-C, tropoelastin, thrombin derivative peptides, antimicrobial peptides (cathelicidins), defensins (defensens), laminins, biological polypeptides containing cells of viscous extracellular matrix proteins and heparin binding domains, such as fibronectin (fibronectin) and vitronectin (vitronectin), MAPK preparations, e.g., the compounds disclosed in published application 2004/0209901 and published U.S. application 2004/0132729.

Incorporation of the cells of the invention into a scaffold (scaffold) can be accomplished by simply depositing the cells on top of the scaffold. Cells can enter the scaffold by simple diffusion (J.Pediatr.Surg.: 23(1Pt 2):3-9 (1988)). Several other methods have been developed to increase the efficiency of cell seeding. For example, spinner flasks have been used to seed chondrocytes (chondrocytes) onto polyglycolic acid (polyglycolic acid) scaffolds (Biotech advances (Biotechnol. prog.)14(2):193-202 (1998)). Another method of seeding the cells is by centrifugation, which places minimal stress on the seeding cells and increases the efficiency of seeding. For example, Yang et al developed a cell seeding method (J.biomed.Mater.Res.)55(3):379-86(2001)), known as centrifugal cell fixation (CCI).

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

Examples

All components are used in effective amounts

1. Methods of generating and maintaining mesoderm-derived IsLl + pluripotent progenitor cells (IMPs)

This example describes a method of generating and maintaining multi-passaged mesoderm-derived Isl1+ multipotent progenitor (IMPs) cell types that have the ability to differentiate into cardiomyocytes, smooth muscle cells, or endothelial cells (fig. 1).

a) Generation and maintenance of IMP' s

Production of IMP's from WA09hESCs is shown in example 3(PCT/US2008/001222, publication WO 2008/094597). See below.

Example 3(PCT/US2008/001222, publication No. WO 2008/094597): methods of generating mesoderm-derived Isl1+ multipotent progenitor cells (IMPs)

This example describes a method of generating mesoderm-derived Isl1+ multipotent progenitor (IMPs) cell types that have the ability to differentiate into cardiomyocytes, smooth muscle cells, or endothelial cells. The cell type differentiates along a pathway through the mesendoderm state followed by the mesoderm.

(a) Generation of Isl1+ multipotent progenitor cells (IMP) by addition of Wnt3a and BMP4 into hESC cultures.

Growing in StemAccutase for BG02hESCs in medium of known composition^TMPassages were performed and plated on Matrigel (Matrigel) coated plates (1.0X 10 per 60mm plate) as described in example 1⁶Cells), in addition the medium was supplemented with BMP4(l00ng/ml, development System (R)&D Systems)) and human Wnt3a (R development system (R)&D Systems)). The medium was changed once a day. Within 240 hours (10 days), Q-PCR analysis was performed to assess differentiation. This analysis indicated that mesendoderm markers such as T increased at 24 hours post-treatment, but subsequently decreased (figure 11). After 24 hours of treatment, transcriptional markers indicative of mesodermal differentiation were significantly upregulated (Isl 1, PDGFR α, KDR, Tbx20, GATA4) (fig. 11). Immunostaining showed that most cells stained positive for T during the 24-96 hour period after treatment, but this index was small at 144And decreases in time. After 6 days (144 hours) of treatment with BMP4 and Wnt3a,>90% of the cells stained positive for Nkx2.5, Isl1 and Tbx 20. The expression profile of this gene is indicative of pluripotent Isl1+ progenitor cells in the secondary cardiac region (Laughitz et al, Development 135:193- "205). With unique cell morphology changes, Isl1+ cell differentiation is complete.

(b) Generation of Isl1+ multipotent progenitor cells (IMP) by addition of Wnt3a by addition of BMP4 after 1-3 days.

Isl1+ mesodermal cells can be generated by treating hESCs grown in MEF-CM or media of known composition by adding BMP4 for 2-4 days after the initial addition of Wnt3a for 1-3 days.

(c) Generation of Isl1+ multipotent progenitor cells (IMP) by adding BMP4 and GSK inhibitors such as BIO to hESCs in MEF-CM.

BG02hESCs grown on Matrigel (Matrigel) in MEF-CM were passaged with trypsin at 1.5X 10 per 60mm plate⁶Cells were seeded back on Matrigel (Matrigel) in MEF-CM supplemented with BIO (2. mu.M) and BMP4(100 ng/ml). The medium was changed once a day. Within 240 hours (10 days), Q-PCR analysis was performed to assess differentiation. The treated cells were altered in morphological indicators of differentiation compared to hESCs. Transcript levels analyzed by Q-PCR showed that the hESC marker Nanog, Oct4, Lefty a decreased at 48 hours, while the mesendoderm marker (T, MixLl) peaked at 48 hours but decreased at 96 hours. As mesendoderm marker levels decreased, early mesoderm markers (FoxFl, GATA4, Isl1, Tbx20, PDGFR α, PDGFR β) began to increase from 24-48 hours. These markers are indicative of the formation of IMPs.

(d) The production of Isl1+ multipotent progenitor cells (IMP) by adding BMP4 and GSK inhibitors such as BIO to hESCs cultured in media of known composition.

hESCs can differentiate into Isl1+ progenitor cells by adding BMP4 and BIO to hESCs cultured in media of known composition. Treatment with BMP4 and BIO was performed for 6 days.

(d) 1-3 days later by addition of GSK inhibitors such as BIO, addition of BMP4, production of Isl1+ multipotent progenitor cells

Isl1+ mesodermal cells can be generated from hESCs grown in MEF-CM or media of known composition by adding BMP4 for 2-4 days after 1-3 days of maintenance with GSK inhibitors such as BIO.

(e) Isl1+ multipotential progenitor cells (IMP) were generated by adding Wnt3a and BMP4 and TGF signaling inhibitors (e.g., SB431542) to hESC cultures

Isl1+ mesodermal cells can be generated from hESCs grown in MEF-CM or media of known composition by removing TGF and continuing culture with Wnt3a and BMP4 for 2-4 days after 1-4 days maintenance by addition of Wnt3a, BMP4 and a TGF inhibitor (e.g., SB 431542).

(f) Generation of BMP4, Isl1+ multipotent progenitor cells (IMP) was added after 1-4 days maintenance by addition of Wnt3a and TGF β signalling inhibitors (e.g. SB 431542).

Isl1+ mesodermal cells can be generated from hESCs grown in MEF-CM or media of known composition by addition of Wnt3a and a TGF β inhibitor (e.g., SB431542) for 1-4 days followed by addition of BMP4 for 2-4 days.

(g) Generation of Isl1+ multipotent progenitor cells (IMP) by addition of BMP4 and SB431542 after 1-3 days of maintenance by addition of Wnt3 a.

Isl1+ mesodermal cells were generated from hESCs grown in MEF-CM or media of known composition by addition of Wnt3a and SB431542 for 1-3 days followed by addition of BMP4 for 2-4 days.

According to the method of the present invention, the cells obtained as described above were divided 1:6 into known-composition media containing BIO (2. mu.M) and BMP4(l00ng/ml) on days 3-6. The cells were maintained in the medium indefinitely and were sorted at a ratio of 1:4 to 1:6 every 4-6 days. The cells thus generated maintained the expression of Isl1+ and Nkx2.5 in subsequent passages (FIGS. 2A-B). These cells lost the hESC pluripotency marker Nanog and the epithelial cell marker E-cadherin, whereas β -catenin was found to have lost localization throughout the cell, including within the nucleus (fig. 2C-D).

2. Clonal expansion of self-renewing ISL1+ pluripotent progenitor cells (IMPs).

The invention also includes a method of clonal propagation of mesodermal (Isl 1+ multipotent progenitor cell, IMP) cells under self-renewal conditions comprising a GSK inhibitor and BMP, comprising: (a) mesodermal cells (Isl 1+ multipotent progenitor cells), (b) grown for 1-5 days in methylcellulose (final concentration of 0.9%, purchased from Stem cell technologies) and self-renewing medium, (c) further grown for 3-20 days in self-renewing medium to form single colonies. Individual colonies were collected and expanded and allowed to differentiate further (figure 3). Reference is made to example 3 of PCT/US2008/001222(WO2008/094597) for information on IMP cell production from hESCs.

The ability of IMPs to passage and expand at clonal density allows for a rigorous check of the potential of these cells, and also allows for the use of these cells in high-density/high-throughput screening assays. For example, since Isl1+ cells are in the adult heart, it is important to identify small molecules that affect IMP cell proliferation/expansion and differentiation into functional cell types. IMP cells represent a model to identify drugs/compounds that can be used to control the behavior of Isl1+ cells in the heart. As a result, this would stimulate Isl1+ cells to participate in myocardial repair/regeneration.

Method for generating clonable IMP (Isl 1+ multipotent progenitor cell) cell lines

This example describes the steps of generating a clonable IMP cell line (i.e., a cell line derived from a single cell) and plating it at low density, suitable for high throughput drug screening. IMP cells can be maintained (self-renewal) in media of known composition supplemented with GSK inhibitors (e.g., BIO at 2. mu.M) and BMP4 (e.g., 100ng/ml) and passed through Accutase^TMPassage (fig. 3). These cells are present in "low density" (10-500, preferably 20-200 cells/mm)²) Is spread on Matrigel^TMCoating quiltOf the biocompatible thickeners on the plate, a cellulose thickener is preferred, and methylcellulose is preferred (final concentration 0.9%) for 3 days. After 3 days, the medium was changed daily. After 14 days, individual colonies were isolated and sub-cultured into stable, clonable cell lines. Alternatively, clonally expanded IMP cells may be passaged in clumps (rounds) using enzymes such as collagenase.

3. Method for producing endothelial, smooth muscle and cardiomyocytes from self-renewing ISL1+ multipotent progenitor cells (IMPs)

a) Cardiomyocyte production from self-renewing IMP' s

Methods involving the production of cardiomyocytes from self-renewing IMP cells (see above and PCT/US2008/001222, WO 2008/094597). In principle, these cells can be derived from direct sources of hESCs from Isl1+ cells.

Several methods of producing cardiomyocytes are described below:

separating self-renewing IMP's by 25-250 × 10³Cells/cm²Inoculated and grown in media of known composition, which is depleted of activin A and +/-IGF, and present in either:

i)BMP(l0ng/ml)

ii)BMP(10ng/ml)+DKKl(150ng/ml)

iii) BMP (10ng/ml) + DKK (150ng/ml) + VEGF (10ng/ml) (FIG. 4)

iv)DKKl(150ng/ml)+VEGF(10ng/ml)

Cells were grown in these media for an additional 14 days. The resulting cultures contained 10-30% cardiomyocytes as determined by the expression of cTNT, SM-actin and sarcomeric actin (figure 4).

As an alternative strategy, Isl1+ IMP cells were generated by:

v) B27 supplement in RPMI medium (1 x; yingjun (Invitrogen)

Can be converted into cardiomyocytes.

In addition, the single condition (i-v) above may be supplemented with all-trans retinoic acid (0.1-5 μm) to enhance cardiomyocyte differentiation.

b) Production of endothelial cells from self-renewing IMPs

Relates to a method for producing endothelial cells from self-renewing IMP cells. In principle, these cells can be derived from direct sources of hESCs from Isl1+ cells.

Several methods of producing endothelial cells are described below:

separating self-renewing IMP's by 25-250 × 10³Cells/cm²Inoculated and grown in media of known composition, which is depleted of activin A and +/-IGF, and present in either:

v)BMP(10ng/ml)

vi)BMP(l0ng/ml)+DKKl(150ng/ml)

vii)BMP(10ng/ml)+DKKl(150ng/ml)+VEGF(10ng/ml)

viii)DKKl(150ng/ml)+VEGF(10ng/ml)

cells were grown in these media for an additional 14 days.

c) Production of smooth muscle cells from self-renewing IMPs

Self-renewing IMPs can be grown for 21 days in medium of known composition in the presence of Wnt3a (25ng/ml) and BMP4(l00 ng/ml).

4. Formation of endothelial, smooth muscle and cardiomyocytes from hESCs via IMP progenitor intermediates

(i) Production of smooth muscle cells from IMPs.

hESCs were grown for 6 days in the presence of Wnt3a (25ng/ml) and BMP4(l00ng/ml) in a medium of known composition. Cells were divided into 1:4-1:6 portions of the same medium and maintained for 4 days. Cells were fixed and stained for smooth muscle markers, smooth muscle actin, calponin (calponin), calponin (caldesmin) and SM-MHC. Most cells stain these smooth muscle markers.

(ii) Production of cardiomyocytes and endothelial cells from IMPs.

IMPs were prepared by three treatments. Processing one; hESCs were grown in the presence of activin a (100ng/ml) in the known composition medium for the first 24 hours, Wnt3a (25ng/ml) for days l-4, and BMP4(100ng/ml) for days 2-6. Processing II; hESCs (BG02) were grown for days 1-2 in medium of known composition with IGF-I, heterozygotes (Heregulin) and FGF2 removed and in the presence of Wnt3a (25ng/ml) and for days 2-6 in the presence of BMP4(100 ng/ml). Processing III; hESCs were grown in the presence of activin A (100ng/ml) in the known composition medium for the first 24 hours, Wnt3a (25ng/ml) for days 1-2, and BMP4(l0ng/ml) for days 2-6. At the end of day 6, cells were placed in medium of known composition for 14 additional days. Cells were harvested and Q-PCR analysis indicated that treatment 2 produced endothelial cell markers (CD 31/Picamel (Pecaml) and CDH 5/VE-cadherin) and treatment 3 produced cardiomyocyte markers (ACTCl/cardiac alpha actin and cTNT) (FIG. 21). These results indicate that IMP cells are capable of differentiating into cardiomyocytes and endothelial cells.

a) Production of endothelial cells from IMPs.

hESCs were grown for 4-6 days in the presence of Wnt3a (25ng/ml) and BMP4(100ng/ml) of known composition media. At 25-250X 10³Cells/cm²Cells were separated and grown in medium of known composition, which removed activin a and +/-IGF, and present either:

ix)BMP(10ng/ml)

x) BMP (10ng/ml) + DKK (150ng/ml) (FIG. 5)

xi)BMP(10ng/ml)+DKKl(150ng/ml)+VEGF(10ng/ml)

xii)DKKl(150ng/ml)+VEGF(10ng/ml)

Cells were grown in these media for an additional 14 days. 20-40% of the cells obtained were endothelial in origin (FIG. 5).

Alternatively, instead of splitting the initial IMP cultures, they are maintained and subjected to the treatment described by (ix-xii) without passage.

b) Production of smooth muscle cells from IMPs.

hESCs were grown for 21 days in the presence of Wnt3a (25ng/ml) and BMP4(l00ng/ml) in a medium of known composition. The cells were split between 1:4 and 1:8 and grown in the same medium for a further 24 hours. The resulting culture was > 90% smooth muscle (fig. 6A-C).

c) The production of cardiomyocytes from self-renewing IMP's.

Methods involving the generation of cardiomyocytes from IMP cells derived directly from hESCs (see also PCT/US2008/001222, WO 2008/094597).

Several methods of producing cardiomyocytes are described below:

separating IMP's by 25-250 × 10³Cells/cm²Inoculated and grown in media of known composition, which is depleted of activin A and +/-IGF, and present in either:

i)BMP(l0ng/ml)

ii)BMP(10ng/ml)+DKKl(150ng/ml)

iii)BMP(10ng/ml)+DKKl(150ng/ml)+VEGF(10ng/ml)

iv)DKKl(150ng/ml)+VEGF(10ng/ml)

cells were grown in these media for an additional 14 days. The resulting cultures contained 10-30% or more cardiomyocytes as determined by the expression of cTNT, SM-actin and sarcomeric actin.

As an alternative strategy, Isl1+ IMP cells were generated by:

v) B27 supplement in RPMI medium (1 x; yingjun (Invitrogen)

Treatment may be converted to cardiomyocytes.

In addition, the single condition (i-v) above may be supplemented with all-trans retinoic acid (0.1-5 μm) to enhance cardiomyocyte differentiation.

5. Further determination of IMP cells by cell surface marker analysis

Based on our observations in the laboratory, an increase in PDGFR α transcription factor was associated with IMP formation, which we now indicate is also associated with detection of PDGFR α on the cell surface by flow cytometry (fig. 7). When hESCs differentiate in the presence of Wnt3a and BMP4 towards IMPs cells, they down-regulate the hESCs marker SSEA3 and up-regulate PDGFR α.

Differentiation of MMCs into c-KIT + cardiovascular progenitors

MMCs are self-renewing, multipotent populations derived from hESCs, mesodermally derived progenitor cells with the potential to differentiate into a variety of cell types, including, inter alia, the cardiovascular system such as cardiomyocytes, smooth muscle and endothelial cells (PCT/US2008/001222, publication No. WO2008/094597, incorporated herein by reference). MMCs can be frozen, restored, and then grown for an extended period of time while retaining their multipotent differentiation potential. Here, the differentiation of MMCs into c-kit + CXCR4+ cell types is described (see FIG. 8). This cell type can be used, but is not limited to, repairing damaged cardiac muscle and cardiovascular tissue. The cells can be used as a cell therapy agent, by direct injection into the damaged tissue site or by systemic administration, wherein the cells can "home" to the damaged tissue site. Fig. 15. Due to the multipotent nature of these cells, the repair function of these cells is not limited to cardiovascular applications and can be used to control inflammatory diseases and repair other damaged tissues/organs.

Culture of human embryonic stem cells (as described in WO2008/094597)

Methods of growing hescs.

The method comprises the following steps: the watch reaches the standardThe hESCs of a marker such as POU domain transcription factor Oct4 are preferably grown in mouse embryo feeding conditioned medium MEF-CM or a medium of known composition and Matrigel (Matrigel) as a growth substrate (for example). The cells are usually arranged in a 1-1.5X 10 order⁶Plating was performed every 60mm plate. Cells were passaged separately at approximately 1:4 to 1:10 every 4-5 days.

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

hESCs can be grown on Matrigel (Matrigel) (BD Biosciences) or other matrices (McLean et al, Stem cells (Stem cells) 25:29) supporting hESC maintenance, preferably at a dilution of 1:20-1:200, in mouse embryo fibroblast conditioned medium (MEF-CM) in the presence of Fgf 2. The cells can be cultured by using enzymes (trypsin, Accutase)^TMCollagenase), artificial (mechanical) and non-enzymatic methods. The cells were plated at 1.5X 10 per 60mm dish⁶Is plated and divided at 1:4 to 1:10 passage every 4 to 5 days.

(ii) Determination of Conditions (DC)

(a) A known component medium for routine hESCs culture is StemPurchased from Yingjun (Invitrogen) (Wang et al, Blood (Blood)110: 4111). Except Accutase^TMFor passaging the cells as single cell suspensions, media was used according to the manufacturer's recommendations. The following formulation was able to maintain hESCs in a pluripotent state. Serum-free medium conditions of known composition, but not limited to this particular formulation, were found to work well and included feeder-free culture: DMEM F12(Gibco), 2% BSA (serum grade, #82-047-3), 1 XPcillin/streptomycin (Pen/Strep) (Gibco), 1 XP nonessential amino acids (Gibco), 1 XP trace elements A, B and C ((Sigma, # A4034), l 0. mu.g/ml transferrin (Gibco, #11107-018), 0. lnM. beta. -mercaptoethanol, 8ng/ml Fgf2(Sigma, # F0291), 200ng/ml LR-IGF (JRH Biosciences), #85580), l0ng/ml activin A (development System (R RH Biosciences) (# 85580)&D Systems), #338-AC), 10ng/ml Heterozygium (Heregulin) beta (Peprote technology)ch)；#100-03)。

(b) hESCs can also be cultured in commercially available media formulations of known composition, as suggested by the manufacturer, such as mTeSRl (BD/Stem Cell Technologies; Ludwig et al, nature biotechnology (Nat Biotechnol.)24: 185). Accutase^TMPassages can also be used in combination with this medium.

Generation of pluripotent migratory cells (MMCs)

Example 8 based on PCT/US2008/001222, WO2008/094597

Growth at Stem, as described aboveBG02hESCs in medium of known composition using Accutase^TMPassage and plating onto Matrigel (Matrigel) coated dishes (1.0X 10)⁶Cells per 60mm dish), in addition the medium was supplemented with BIO (2 μ M) and SB431542(20 μ M; sigma). Medium was changed daily and cells were incubated with Accutase^TMPassages were performed every 5-6 days, separated by 1:5-1:10 at each passage. When cultured under these conditions, the pluripotency marker Nanog decreased in the first passage (P0) and T transcript levels increased, while Sox17, FoxF1, CXCR4 and PDGFR α remained low. Four days after treatment with BIO and SB431542, 90% of the cells stained positive for T, indicating that they were transformed by the mesendoderm state at a certain time. During this time, Nanog, Oct4, and E-cad were significantly down-regulated as shown by immunostaining. The disappearance of E-cadherin indicates that the cells undergo a transition from epithelial to mesenchymal, consistent with the process of differentiation into mesendoderm. Upon continued passage, T expression was reduced (as determined by Q-PCR) and the pluripotent marker Nanog was not re-appeared at P1-P10. This was confirmed by immunostaining, in which P7 cells did not express Nanog, Oct4 or E-cadherin, as opposed to hESCs. Mesodermal and endodermal markers were not increased during this time. Cells were serially passaged under the same conditions and they maintained robust proliferative activity for more than 20 passages and maintained morphology (using the same medium containing BIO and SB431542 as described above). The MMCs produced are standardThe method is carried out at low temperature>A plating efficiency of 10% was recovered. The growth and morphological characteristics of cryorecovered (cryorecovered) MMCs were indistinguishable from previously cryopreserved (precryupered) MMCs.

The generation of additional self-renewing progenitors of mesodermal origin using a combination of an inhibitor of GSK3, an activin/Nodal (Nodal) signaling inhibitor and a BMP signaling inhibitor (GABi cells).

As an extension of the principles already established in this application and in the previously filed PCT applications (PCT/US2008/001222, WO2008/094597), which are incorporated herein by reference in their entirety, it is possible to generate mesoderm-derived self-renewing progenitor cells from hESCs that can remain in culture for a long time (>10 passages) and that exhibit the potential for pluripotent differentiation. These progenitor cells can be developed from hESCs grown under the conditions described herein (examples above and PCT/US2008/001222, WO 2008/094597).

These progenitor cells can be generated by treating hESCs with a combination of GSK3 inhibitor and activin/Nodal (Nodal) signaling (e.g., SB431542) and/or BMP signaling inhibitor (e.g., noggin or compound C). Due to the action of GSK3 inhibitors, hESCs were differentiated to mesendoderm by EMT in the presence of the above specific chemical inhibitors, and subsequent cultures switched to a progenitor phenotype. (FIG. 11).

Additional examples of progenitor cell production from hESCs include:

(i) GSK3 inhibitors, such as BIO (2. mu.M) plus activin/Nodal (Nodal) signal inhibitors (e.g., SB431542) -these are referred to as MMCs (as described in PCT/US2008/001222, incorporated herein by reference in its entirety)

(ii) GSK3 inhibitors, such as BIO plus BMP signal inhibitors (e.g., noggin, Compound C) -prophetic examples

hESCs cells were cultured at 2.0X 10⁶Density of/60 mm plates was plated on Matrigel (Matrigel) plates. Differentiation medium contained DMEM/F12(50/50), approximately 2% prealbumin (probumin), antibiotics (1 XPicillin/streptomycin (Pen/St)rep)1 × non-essential amino acids (NEAA)), trace elements A, B, C (e.g., 1 ×, from media technology (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), heregulin (e.g., about l0ng/ml), BIO (e.g., about 2 μ M), and Compound C (e.g., about l μ M). Noggin may also be substituted for compound C.

Cells were grown continuously with Accutase^TM(Innovative Cell Technologies) were passaged at 1:5 division every 5-7 days. These cells can be frozen, thawed with high degrees of recovery, and differentiated into multiple lineages. Cells can also be passaged as single cell suspensions or clumps with other dispersing agents (enzymatic and non-enzymatic).

(iii) GSK3 inhibits such as BIO plus BMP signal inhibitor plus activin/Nodal (Nodal) signal inhibitor (prophetic examples)

hESCs cells at 2.0X 10⁶Density of/60 mm plates was plated on Matrigel plates. Differentiation medium contained DMEM/F12(50/50), about 2% probumin (albumin), antibiotics (1 x penicillin/streptomycin (Pen/Strep)1 x nonessential amino acids (NEAA)), trace elements a, B, C (e.g., from media technology (Mediatech)), ascorbic acid (about 50 μ g/ml), transferrin (e.g., about l0 μ g/ml), β -mercaptoethanol (about 0.lmM), bFGF (e.g., about 88ng/ml), LR-IGF (e.g., about 200ng/ml), hybridin (e.g., about 10ng/ml), BIO (e.g., about 2 μ M), compound C (e.g., about l μ M) and SB 432 (e.g., mg/ml).

Cells were grown continuously with Accutase^TM(Innovative Cell Technologies) were passaged at 1:5 division every 5-7 days. These cells can be frozen, thawed with high degrees of recovery, and differentiated into multiple lineages. Cells can also be passaged as single cell suspensions or clumps with other dispersing agents (enzymatic and non-enzymatic).

Example (i) has been broadly described herein, and the pluripotent lineages thus generated are referred to as pluripotent migratory cells (MMCs).

Progenitor cells described in example (ii) can in principle be generated from several hESC lines, including BG02, WA09, WA07, and maintain self-renewing colonies for more than 10 passages.

Progenitor cells described in example (iii) can in principle be generated from several hESC lines, including BG02, WA09, WA07, and can maintain a self-renewing population for more than 10 passages.

Differentiation of MMCs into a C-kit + CXCR4+ progenitor cell population (C56Cs)

To further differentiate MMCs, MMC cells obtained as described above were plated at 2.5X 10⁶The/60 mm plates were density coated on Matrigel (Matrigel) plates. Differentiation involves removal of GSK3 inhibitors (i.e., BIO) and SB431542, which are used to maintain MMCs. Differentiation medium contained DMEM/F12(50/50), about 2% prealbumin (probumin) (albumin), antibiotics (1 x penicillin/streptomycin (Pen/Strep)1 x nonessential amino acids (NEAA)), trace elements a, B, C (e.g., 1 x from media technology (Mediatech)), ascorbic acid (e.g., about 50 μ g/ml), transferrin (e.g., about l0 μ g/ml), β -mercaptoethanol (about 0.lmM), bFGF (e.g., about 8ng/ml), LR-IGF (e.g., about 200ng/ml), activin a (e.g., about 10ng/ml), hybridin (heregulin) (e.g., about 10ng/ml), BMP4 (e.g., about 100ng/ml), Wnt3a (e.g., 25/ml) and sodium butyrate (e.g., about 0.5 mM). Importantly, the GSK3 (i.e., BIO) inhibitor and SB431542 were removed from the differentiation step and BMP4 (or other BMP, e.g., BMP2 with similar activity) and Wnt3a (or other Wnt with similar activity) were added (along with sodium butyrate) for a period of about 1 to 8 days or more, 2 to 7 days, 2 to 6 days. Cells were tested by quantitative RT-PCR (figure 9) and flow cytometry (figures 10A-C) on days 2,4 and 6.

Over a differentiation period of 4-6 days, increased CXCR4, c-Kit, CD56(N-CAM) was found as judged by real-time quantitative PCR analysis and flow cytometry analysis of transcript levels (fig. 9, 10). Flow cytometry analysis showed no detection of CD31, kdr (flkl) and SSEA3, but a slight increase in PDGFR α during 4-6 days of differentiation (fig. 10). The Isl1 transcript levels were also increased in these experiments (FIG. 9). Brightfield pictures of c-kit + CXCR4+ cells generated by MMCs by BMP4, Wnt3a, and sodium butyrate treatment for 2-6 days are shown in fig. 10D.

Alternatively, C56Cs can be obtained from pluripotent stem cells by first exposing the pluripotent stem cells, particularly hESCs, to conditions that produce MMC's, and once the MMCs are obtained, exposing them to differentiating conditions.

CXCR4+ CD56+ cell (C56Cs)

Method for producing C56Cs

The pathway to produce C56Cs is shown in fig. 14. Production of MMCs from hESCs has been described previously. The method of providing MMCs is applicable to any human pluripotent stem cell such as an induced pluripotent stem cell (iPS cell) or the like. Description of hESCs general methods for the production of MMCs are applicable to pluripotent stem cells, as described elsewhere herein. To produce C56Cs, MMCs were treated with BMP4(l00ng/ml), Wnt3a (25ng/ml), sodium butyrate (0.5mM) for about 1 to 8 days (preferably 3-6 days) in a basal medium containing DMEM/F12(50/50), about prealbumin (probumin) (albumin), antibiotic (1 XPicillin/streptomycin (Pen/Strep)1 × nonessential amino acids (NEAA)), trace elements A, B, C (e.g., 1X, from Medium technology (Mediatech)), ascorbic acid (50 μ g/ml), transferrin (l0 μ g/ml), β -mercaptoethanol (about 0.lmM), bs (e.g., about 8ng/ml), LR-IGF (e.g., about 200ng/ml), activin A (e.g., about 10ng/ml), hybrid antibiotics (e.g., Heregulin), about 10 ng/ml). After which C56Cs was passaged. The C56Cs thus produced was of high purity and was available for treatment without further purification.

It is envisioned that MMCs may also be used for the therapeutic applications described herein, but experiments were performed in this cell type due to the generally higher levels of CXCR4 in C56 Cs.

CXCR4+ CD56+ cell (C56Cs) biomarkers

A more detailed study of the cell surface markers associated with C56Cs is presented below. These cells exhibit higher levels of CXCR4 and CD56 on the cell surface. Thus, these cells have been designated C56Cs, i.e. CXCR4+ CD56+ cells. The c-kit, CD56, CD166, CD105, CD44, CD133 and CD90 biomarkers may also be present in this cell population. Representative flow cytometry plots are shown in fig. 17, 18, and these results are summarized in fig. 19. Briefly, although MMCs are also CXCR4+, the expression of CXCR4 was increased in C56Cs as judged by flow cytometry. MMCs and C56C were positive for both CD56 and CD 133. As with CD105, when MMCs were converted to C56C, C-kit levels increased-however, the entire population was not completely positive for these 2 markers at all times. As the MMCs shift to C56Cs, CD105, CD166, and CD104 also have an increasing trend. Although some transcripts were detected (data not shown), Flkl/KDR positivity was not shown in MMCs or C56C cells. MMCs and C56Cs also exhibited low levels of PDGFR α and were negative for CD 31. Based on their properties, C56Cs is similar to but not identical to mesenchymal stem cells and is considered to represent a pre-mesenchymal stem cell-like state.

Homing of CXCR4+ CD56+ (C56Cs) cells to ischemic tissues and bone

Since C56Cs expresses CXCR4, we propose that they can home to sites of inflammation and tissue damage through the SDF-1/CXCR4 signaling axis (as reviewed in Dalton, 2008; regenerative medicine (Regen Med.), 3: 181-. This is similar to the migration of bone marrow-derived mesenchymal Stem cells into peripheral blood as described previously (Kucia et al 2005, Stem cell 23: 879-. A schematic of how MMCs and C56Cs can be administered as systemic cellular therapeutic agents is shown in figure 20. The cells may also be administered systemically, together with other compounds or cell types (i.e., Isl1+ multipotent cardiovascular progenitor cells, for example), or directly to the site of tissue damage. FIG. 21 and FIGS. 23 to 26 show images, wherein [ alpha ]¹¹¹In]Oxime radiolabeled cells (Caveliers et al, 2007 Nuclear medicine and molecular imaging journal (Q J Nucl Med Mol)51:61-66) passed through a previous history of receiving coronary artery ligation (Dow II: (C.O.)Sprague Dawley) rats were administered systemically via the tail vein. Femoral intravenous injection can also be used to achieve the effect. Within 3 days, whole animal "live" images were captured by gamma camera. During this time the cells appeared to localize to organs such as liver and lung, bone and importantly ischemic heart (fig. 21, 23-16). Within the required 2 hours, the injected cells immediately remained in the lungs and then partially migrated to the liver. Initially, background accumulation of the lungs makes the labeling of the heart region less apparent, which becomes clear after 10-24 hours, revealing a significant accumulation of cells in the heart. Thereafter, heart tissue from rats that had been perfused with labeled cells was fixed and sectioned axially, and autoradiography confirmed that "homing" of C56Cs had occurred (fig. 22).

Functional recovery from myocardial ischemia using C56Cs in rodent model to determine whether C56Cs promotes functional recovery in rodent models of myocardial ischemia, C56Cs was injected into the tail vein of nude rats after myocardial ischemia was induced by surgical methods. In male athymic Stent-Dow (Sprague Dawley) rats (rh, rnu-rnu, 240-Dawley, Harlon), the anterior descending left coronary artery was closed (occlusion) by thoracotomy (thoracotomy) and suture for 30-60 minutes and then perfused for 24 hours (Laflamm et al, 2007, Nature Biotechnol.) (nat. Biotechnol.)) 25:1015-1024) to generate acute myocardial infarction.

Cells (usually 1-3X 10) are plated daily for 3-4 days, starting 24 hours after infarction⁶At 0.1ml) was injected into the tail vein. The animals received cyclosporin a (0.75 mg/day) as an immunosuppressant 24 hours before and during the first 7 days after cell infusion. Animals were then imaged at different times post injection using transthoracic echocardiography (fig. 16, 17; Zhu et al, 2008, nuclear medicine rapid news (nuclear. med. commun.)29: 764-. Left Ventricular Ejection Fraction (LVEF) (Laflamme et al, 2007, Nature Biotechnology) was calculated by the published methodSurgery (nat. Biotechnol.)25: 1015-1024).

Summary of the results of C56C administration:

the fraction of injections (EFs) in rats with acute myocardial infarction was calculated as described by LaFlamme et al (2007, Nat. Biotechnol.)25: 1015-1024). The mean injection score after infarction (n-3) on saline cells alone was 56.33+/-7.4 and 59.7+/-16.4 at 2 and 4 weeks post-injection, respectively. EFs of infarcted rats (n-4) receiving C56Cs were 80.8+/-5.9 and 82.8+/-4.4 at 2 and 4 weeks post injection, respectively.

Echocardiography (fig. 27, 28) and MRI analysis (fig. 29, 30) showed significant and repeatable functional recovery in all animals receiving C56Cs infusions (n-4). MRI analysis demonstrated that muscle regeneration of ischemic myocardial tissue occurred after C56Css2 perfusion weeks (fig. 29, 30). Thickening of the heart wall in the infarct zone and recovery of the beating myocardial muscle are readily observed by echocardiography and MRI imaging.

Of all the measures used, C56Cs administration had a major therapeutic effect on cardiac regeneration in acute myocardial infarction.

Homing of C56Cs to stroke lesions in rodent models

In addition to ischemic heart models, another application for MMCs and C56Cs is in the recovery/repair of stroke. To investigate the ability of GFP + C56Cs to home to stroke, rodent models were used. C57Bl mice received craniotomy and stroke with light thrombosis. Each animal received 3-4 xl0 by tail vein injection after-24 hours of stroke with light thrombus formation⁶A cell. Cells were resuspended in texas red solution and injected. GFP + cells were observed in the circulation immediately after injection, not 48 hours later. GFP + cells in the ischemic border zone (penumbra) and choroid plexus (FIGS. 31, 32) were identified using 2-photon microscopy.

Further embodiments

Production of IMPs from hipSCs

Production of IMPs cells from hiPSCs is similar to the method of production of IMPs cells from hESCs, as listed above and described herein.

(a) Methods for growing hESCs and hipSCs

hESCs and hipSCs expressing markers such as Oct4 and Nanog are preferably grown in mouse embryo feeding conditioned medium (MEF-CM) or medium of known composition (DM) using Matrigel (Matrigel) as the growth medium. The cells are usually in the range of 1-1.5X 10⁶Plates were plated per 60mm plate. Cells were passaged separately at 1:4-1:10 every 4-5 days.

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

hESCs and hipSCs (e.g., hFib2-iPS4) can be grown in mouse embryo fibroblast conditioned media (MEF-CM) in the presence of Fgf2 on Matrigel (Matrigel) (BD Biosciences); preferably 1:20-1:200 dilution) or other substrates (McLean et al, 2007; Stem Cells (Stem Cells)25, 29-38; Park et al, 2008; Nature (Nature)451, 141-. Cells can be passaged by a variety of methods using enzymes (trypsin, akuyasa (Accutase), collagenase), artificial passage (mechanical), and non-enzymatic methods. The cells were plated at 1.5X 10 per 60mm dish⁶And passaging at 1:4-1:10 divided every 4-5 days.

(ii) Medium conditions of known composition

(a) Known composition media for routine culture of hESCs and hipSCs as StemPurchased from Yingjun (Invitrogen) (Wang et al, Blood (Blood)110: 4111-4119). Except that Acuitase (Accutase) (Chemicon) was used for cell passaging as a single cell suspension, the medium was used according to the manufacturer's recommendations. The following formulation was able to maintain hESCs and hiPSCs in a pluripotent state. The following exemplary serum-free media conditions, known to be composed, work well, but are not limited to this particular formulation and include feeder-free culture: DMEM F12 (Gibc)o), 2% BSA (serum grade, #82-047-3), 1 xpicillin/streptomycin (Pen/Strep) (Gibco), 1 xperient amino acids (Gibco), 1 xperient trace elements a, B and C (Cellgro; #99-182-Cl, #99-176-Cl, #99-175-Cl), 50. mu.g/ml ascorbic acid ((Sigma, # A4034), l 0. mu.g/ml transferrin (Gibco, #11107-&D Systems), #338-AC), 10ng/ml heterozygote (heregulin) β ((Peprotech; # 100-03).

(b) According to the manufacturer's recommendations, hESCs and hipSCs can also be cultured in other commercially available media formulations of known composition, such as mTeSRl (BD/Stem cell technologies; Ludwig et al, Nature Biotechnology (Nat Biotechnology.). 24: 185). Acuitase (Accutase) passaging can also be used in combination with this medium.

(a) Production of Isl1+ pluripotent progenitor cells (IMP) by addition of Wnt3a and BMP4 to hiPSC cultures

As described above, hFib2-iPS4hipSCs grown in the above StemPro medium of known composition were passaged using Acuitase (Accutase) and plated on Matrigel (Matrigel) coated plates (1.0X 10)⁶Cells per 60mm dish), except that the medium was supplemented with BMP4(l00ng/ml, development System (R)&D Systems)) plus human Wnt3a (25ng/ml, developed a system (R)&D Systems)). The medium was changed daily. Immunostaining was performed after 4 days (96 hours). As judged by immunostaining, two markers of pluripotent stem cells, Oct4 and Nanog, were positive in hiPSCs (fig. 37). Immunostaining showed a large downregulation of these markers 4 days after treatment with BMP4 and Wnt3a (figure 37). Furthermore, E-cadherin expression disappeared and snell (Snail) expression began to rise (fig. 37). This indicates that after BMP-Wnt treatment, hiPSCs have lost their epithelial structure and undergo an epithelial to mesenchymal transition. When Nanog and Oct4 disappeared, Isl1 was differentiated for 4 days with nearly 400-fold increase in transcript levels, and Hande (Hand)2, GAT A4, mRNAs were increased by 7500 and 175-fold during this period, respectively.

In general, as previously described, this expression profile is characteristic of IMPs cells derived from hESCs (see above, also PCT/US2008/001222, publication No. WO 2008/094597). In summary, hiPSCs were treated with effective amounts of BMP4 in combination with Wnt3a to generate cell types distinguishable from Isl1+ multipotent progenitor cells (IMPs).

(b) Generation of Isl1+ multipotent progenitor cells (IMP) by addition of BNP4 for 1-3 days after 1-3 days of maintenance by addition of Wnt3 a. (prophetic example)

hiPSCs grown in MEF-CM or media of known composition can be treated by adding BMP4 after 1-3 days of initial Wnt3a maintenance for 1-5 more days, preferably 2-4 more days, to produce Isl1+ mesodermal cells.

(c) Production of Isl1+ multipotent progenitor cells (IMP) by addition of BMP4 and GSK3 inhibitors such as BIO to hipSCs in MEF-CM. (prophetic example)

The same as in (a) and (b), except that an inhibitor of GSK3 may be used in place of, or in combination with, Wnt3 a.

(d) The production of Isl1+ multipotent progenitor cells (IMP) by adding BMP4 and GSK3 inhibitors such as BIO to hiPSCs cultured in media of known composition.

hiPSCs could differentiate into Isl1+ progenitor cells by adding BMP4 and BIO to hiPSCs cultured in media of known composition, treated with BMP4 and BIO for 6 days.

(d) Isl1+ multipotent progenitor cells are generated by adding GSK3 inhibitors, such as BIO1-3 days followed by BMP 4.

hiPSCs grown in MEF-CM or media of known composition can produce Isl1+ mesodermal cells by 1-3 days with the addition of GSK3 inhibitor such as BIO followed by 2-4 days with the addition of BMP 4.

(e) Isl1+ multipotent progenitor cells (IMP) were generated by adding Wnt3a and BMP4 and TGF β signalling inhibitors (e.g. SB431542) to hiPSC cultures.

hiPSCs grown in MEF-CM or media of known composition, Isl1+ mesodermal cells can be generated by adding Wnt3a, BMP4 and a TGF β inhibitor (e.g., SB431542) for 1-4 days, then removing the TGF β inhibitor and proceeding with Wnt3a, BMP4 for an additional 2-4 days.

(f) After 1-4 days of maintenance by addition of Wnt3a and a TGF signaling inhibitor (e.g., SB431542), BMP4, Isl1+ pluripotent progenitor cell (IMP) production was added.

Isl1+ mesodermal cells can be generated by growing hipSCs in MEF-CM or medium of known composition by adding Wnt3a and a TGF inhibitor (e.g., SB431542) for 1-4 days followed by 2-4 days of additional BMP 4.

(g) Generation of Isl1+ multipotent progenitor cells (IMP) by addition of BMP4 and SB431542 after 1-3 days of maintenance by addition of Wnt3 a.

Isl1+ mesodermal cells can be generated from hipSCs grown in MEF-CM or media of known composition by addition of Wnt3a and SB431542 for 1-3 days followed by addition of BMP4 for 2-4 days.

Generation of EPCs from (ISL 1+) IMPs

The following describes the differentiation of IMP cells produced by hESCs or hipSCs into multipotent protoepicardial/Epicardial Progenitor Cells (EPCs). This cell type plays an important role because it produces lineages that make up the coronary vasculature. Fig. 34, 35, 43.

(a) Generation of protoepicardial/Epicardial (EPCs) from Isl1+ multipotent progenitor cells (IMPs) by the addition of effective amounts of Wnt3a, BMP4, and all-trans retinoic acid

hESC/hFib2-iPS4hiPSCs grown in StemPro known composition medium/known composition medium can be differentiated into IMPs (as described above and elsewhere). At day 4, IMP cell stage, media known to be composed was supplemented with BMP4(50ng/ml, range about 2-l00ng/ml, R & DSystems)), Wnt3a (25ng/ml, range about 1-100+ ng/ml, R & D Systems), and all-trans retinoic acid (4. mu.M, range about 0.25-25. mu.M Sigma), with media changed every two days (1-4 days) for about 10-16 days (about 7-25 days) (FIG. 39). The expression of Wt-1, Tbx18, Raldh2 and Tcf21 (epicatechin) was confirmed by Q-PCR (FIGS. 40, 42), and the expression of Wt-1 was confirmed by immunofluorescence (FIG. 41). This approach typically produced cultures that were > 80% WT1 positive.

Protoepicardial/epicardial production from IMPs by the addition of effective amounts of Wnt mimetibodies, such as GSK3 α/β inhibitors (i.e., BIO), BMP 4/other BMPs and all-trans retinoic acid.

Protoepicardium/epicardium can be produced from IMPs by adding BIO (GSK3 α/β inhibitor), BMP4 and all-trans retinoic acid to a medium of known composition for-16 days or longer.

Production of endothelial cells, smooth muscle and cardiac fibroblasts from EpCs

a) Production of endothelial cells from EpCs

IMPs were grown for 16 days in the presence of Wnt3a (25ng/ml), BMP4(50ng/ml) and all-trans retinoic acid (Sigma; 4. mu.M) in a medium of known composition. Cells were passaged and treated at 125000 cells/cm²Inoculated and grown in medium of known composition +/-activin A (development System (R)&D Systems)), any one of the following substances is present in the medium:

v)VEGF₁₆₅(development System (R)&D Systems))#293-VE；l0ng/ml)

vi)VEGF₁₆₅(10ng/ml) + SB431542 (Torkshires biosciences; 20. mu.M)

Cells were grown in these media for an additional 10-14 days. 20-30% of the resulting cultures were of endothelial cell origin as judged by immunostaining for CD31 and VE-cadherin. (FIG. 44 a).

(b) Smooth muscle and cardiac fibroblasts are produced epicardially.

IMPs were grown in medium of known composition in the presence of Wnt3a (25ng/ml) and BMP4(100ng/ml)It is 16 days long. Cells were passaged and treated at 125000 cells/cm²Inoculated and grown in 10% FBS, DMEM, 1 XPcillin/streptomycin (Pen/Strep) (Gibco), 1 XPbutyrate (Medium technology (Mediatech)) and L-alanyl-L-glutamine (Medium technology (Mediatech)). Cultures generated therefrom as determined by immunostaining for smooth muscle actin>90% are smooth muscle (fig. 45). Myocardial fibroblasts were detected by staining with antigen collagen antibodies (fig. 44b, 45). These cells constitute 5-10% of the culture.

Smooth muscle was also prepared using a medium of known composition supplemented with:

i)VEGF₁₆₅(l0ng/ml)

ii)VEGF₁₆₅(10ng/ml) + PDGFB (development System (R)&D Systems)；5ng/ml)

Ui)VEGF₁₆₅(10ng/ml) + hDKK (development System (R)&D Systems)；150ng/ml)

iv) 10% fetal bovine serum FBS

Compositions of matter for IMPS derived from human IPSCS and hecs

Microarray analysis of IMP cells produced by hiPSCs indicated;

IMP cells derived from hipSCs always express Isl1

IMP cells derived from hipSCs express Pdgfr alpha, FoxF1, Nkx2.5, Gata4

IMP cells derived from hipSCs also optionally express Tbx3 and Hande (Hand)1

A table summarizing some of the most upregulated genes is shown in figure 46, table 1.

Composition of matter for EPCS derived from human pluripotent cells

Microarray analysis of EPCs generated from three hESC lines and the human iPSC line indicated EPC cell expression;

nephroma inhibitory protein 1(Wt1), Tcf21 (epicatechin), Raldh2(Aldhla2)

These transcripts are the primary identifiers of EPCs, the primary epicardial/epicardial cell type produced by cultured pluripotent stem cells.

EPCs can also be expressed;

one or more (two, three, four or five) of Tbxl8, COL3Al, GATA6, Tbx3, Tbx5

A table summarizing some of the most upregulated genes is shown in figure 47, table 2.

Use of EPCs:

EPCs can be used for the identification of secreted factors produced by the epicardium that affect cardiomyocyte proliferation, survival, function and differentiation.

EPCs can be used as a source of cells for drug screening for cardiovascular applications.

EPCs can be used as a source of cells for therapeutic purposes in the repair of ischemic heart, regenerating coronary vessels.

EPCs can be used for tissue engineering purposes, which obtain cardiac or coronary vessel components.

EPCs can be used as research tools for researching cardiovascular development and diseases.

Method for producing vascular tubes from EPCs

We attached a strategy to generate blood vessels containing endothelial cells and smooth muscle cells, as shown in fig. 48. This involves the production of IMP cells (Isl 1+) from hESCs. IMP cells are then converted to EPCs (Wt1+) and then to vascular structures containing smooth muscle and endothelial cells.

As described previously, WA09 cells differentiated into Wt1+ Epicardial Progenitor Cells (EPCs) within-20 days. The cells were then harvested with 0.25% trypsin-EDTA to form a single cell suspension. Then the thin films are put intoCell size 1.25X 10⁵Cells/cm²(ii) was plated at a density of 8ng/mL FGF2 (Invitrogen), 200ng/mL LR-IGF (Sigma), 10ng/mL Heterozygosin (hetrgulin) beta (Peprotech) and 10ng/mL VEGF (development System (R) (Protecth))&D Systems)) in a medium conditioned with known components. At 37 ℃ 5% CO₂In the middle, cells were grown for 10-14 days, and the medium was changed every 2 days. VEGF was removed from the medium and the culture was allowed to stand at 37 ℃ with 5% CO₂Medium was maintained for 5-7 days without medium change to form tubes (fig. 49). The tubes were then fixed with 4% paraformaldehyde and treated with CD31 and CDH5 (development System (R)&D Systems)). The resulting immunofluorescence image shows the formation of tubes based on evidence of the presence of visible lumens (lumen) and three-dimensional structures composed of Z stacks (fig. 50). Images were obtained on a Zeiss (Zeiss) confocal microscope.

Characterization of migration characteristics of EPCs in vitro and in vivo

The epicardium/epicardium has the ability to form an outer layer over the surface of the myocardium and to migrate into the myocardium by invasive means (Olivey et al, 2004, dynamics of cardiovascular medicine (trends Med.)14, 247;. 251). The standard test to assess the protoepicardial/epicardial migration properties is to plate the cells on collagen I matrix.

(i) In vitro migration of EPCs: primary epicardium/epicardium isolated from myocardial tissue explants has the ability to become interstitial and migrate away from the attachment site (Gaudix et al, 2006 developmental dynamics (Dev Dyn.)235, 1014-. This is a characteristic feature of true epicardial/epicardial origin and includes epithelial to mesenchymal transition.

The standard test for assessing pro-epicardial/epicardial migration properties is plating cells onto a collagen matrix. To evaluate the migration ability of EPCs on collagen gel, the following procedure was performed. IMP cells were treated with retinoic acid, BMP4 and Wnt3a for 6 days to produce Wt1+ EPCs. Single cell suspension (1X 10)⁶Individual cell) A 60mm tissue culture dish coated with PHEMA (polyhydroxyethylmethacrylate) was plated and left for 24 hours to produce spheres. Plates were then plated on Geltrex or collagen I (10. mu.g/ml) coated plates (HAIF) in media of known composition and imaged at various time points (see FIG. 51). For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton Xl00 (permeabilized). hESCs (fig. 52-54) or EPC spheres (fig. 53, 54) were then fixed and cytokeratin or vimentin was probed with antibodies to establish a comparison of the epithelial and mesenchymal status of the cells. This analysis shows that EPC spheres undergo epithelial to mesenchymal transition after plating on a collagen-based matrix, such as Geltrex or collagen 1. This is very similar to the behavior of the original epicardium/epicardium isolated from tissue explants (Gaudix et al, 2006 developmental dynamics (Dev Dyn.)235, 1014-1026; Olivey et al, 2006 developmental dynamics (Dev Dyn.).235, 50-59; Dettman et al, 1998 developmental biology (Dev Biol.). 193, 169-181).

(ii) Migration of EPCs in vivo

Protoepicardial/epicardial tissue explants transplanted onto developing chicken myotubes show very unique properties. Transplanted protoepicardium/epicardium underwent epithelial to mesenchymal transition and invaded the myocardium (Guadix, et al, Developmental Dynamics, 235, 1014-.

To assess the developmental potential of HES-derived epicardial cells, PE aggregates were immediately transplanted into chicken embryos in HH stages 14-16 near the endogenous epicardium adjacent to the heart. Embryos were incubated for 3 to 6 days and hESC/EPC-derived transplanted cells were visualized by immunodetection with GFP antibody (fig. 55-57). This analysis indicated that the transplanted EPC spheres implanted in the tissues of the chickens and invaded the myocardium of the chickens. EPC cells thus exhibit in vivo, behavior consistent with truly epicardial/epicardial.

3. Demonstration that (Isl 1+) IMPs can integrate and differentiate into myosin heavy chain + (MHC +) cardiomyocytes when co-cultured with rodent cardiac tissue

Several reports have documented the ability of epicardial cells to differentiate into cardiomyocytes (see Zhou et al, 2008 Nature 454, 109-113). To determine the ability of EPCs to differentiate into cardiomyocytes, co-culture experiments were performed in which heart tissue explants were incubated with EPC spheres.

The right and left ventricles of the heart of 8 month old CD1 male mice were cut into small pieces (-2 mm square x lmm thick) and cultured in gelatin-coated 96-well plates in DMEM/M199/FBS/PSF for 24 hours. EPC spheres were then added and incubated for different times. Tissues were fixed with paraformaldehyde, embedded in paraffin, sectioned and probed with antibodies against the human β -myosin heavy chain to detect the presence of human cardiomyocytes. Large β -MHC + cells were detected in sections of tissue receiving Isl1+ cells but not in sections not receiving Isl1+ IMP cells (fig. 58).

Demonstration that IMP cells can integrate into tissues containing mesoderm and vascular structures after transplantation into chick embryos

To study GFP⁺Development potential of IMP, single cells were transplanted into the mesoderm of primitive gut embryonic chick embryos. This was done by uncovering the endoderm of Hamburg (Hamburger) and Hamilton (Hamilton) (HH) stage 4-5 embryos and overlaying a single cell suspension of 50-100 IMP cells on the mesoderm. The endoderm was then placed back and the embryos incubated for an additional 20-28 hours. IMP cells were identified by GFP immunodetection.

Analysis of the intact embryos and embryo sections showed that HES cells were extensively incorporated into the embryonic structure, obtaining the morphology of endogenous chicken cells (fig. 59). IMP cells were derived into several mesoderm developmental products, including the epithelial and visceral mesoderm of the body, the vascular endothelium, the perivascular mesoderm surrounding newly formed endothelial vessels (fig. 59A-F), and occasionally in the somites (not shown). IMP cells also enter the mesoderm in large numbers. IMP cells were observed throughout both lateral and medial mesoderm and in both foregut and liver primordia (fig. 59A-D, F). These data indicate that IMP has vascular potential when transplanted into the body.

Further determination of IMP cellular material composition

On the IMP cell surface, there were no previously defined cell surface markers. While KDR (Flk1) can be expressed on the surface of IMP cells, it is not a well-defined cell surface marker because it is widely expressed in stem and progenitor cell types. We now provide additional features. Transcriptional microarray analysis indicated significant upregulation of platelet-derived growth factor beta receptor and cadherin 11 in IMP cells derived from several hESC lines and hiPSCs (data not shown). To identify these as cell surface markers for IMP cells, we performed flow cytometry, indicating that IMPs can express PDGFR β and cadherin 11 on their cell surface (fig. 60). In contrast, hESCs (WA09) were not positive for these markers.

Determination of the migration mechanism operating in CS6C cells

To investigate the mechanism of migration of C56Cs to ischemic/damaged tissue, we tested these cells in the easy den (Boyden) chamber assay. 300,000C56C cells were seeded into the upper chamber of a Yidon (Boyden) chamber. In the lower chamber these data demonstrate that C56C cells were confirmed to respond to and migrate towards SDFI cytokines (fig. 61). Migration can be blocked with the antagonist AMD3100, suggesting that migration is mediated through CXCR4 receptor transduction.

The entire disclosures of all patents, patent applications, and publications, and available electronic materials, including, for example, nucleotide sequences entered into, e.g., gene banks (GenBank) and reference sequences (RefSeq), and amino acid sequences entered into, e.g., SwissProt, PIR, PRF, PDB, and translations of the coding regions annotated by gene banks (GenBank) and reference sequences (RefSeq), are incorporated herein by reference. Any inconsistency between incorporated reference material and that listed in the originally filed specification shall be resolved in favor of the originally filed specification. The foregoing detailed description and examples have been given for clarity of understanding only. It is to be understood that no unnecessary limitations are to be implied therefrom. The invention is not limited to the specific details shown and described, but rather, variations apparent to those skilled in the art are intended to be included within the scope of the invention as defined by the claims.

All headings are for the convenience of the reader and should not be used to limit the meaning of the headings below unless so specified. The embodiments described in the present or future tense are generally the foreseen embodiments.

Claims (43)

1. A method of maintaining or self-renewing a population of Islet 1+ multipotent progenitor cells (IMPs), the method comprising: providing a group of IMPs; and contacting the known composition medium with an effective amount of a combination of BIO and BMP4, on a substrate protein, with the population; isolating the cell after the contacting step.

2. The method of claim 1 wherein the combination of known composition media and an effective amount of BIO and BMP4 are contacted with the population on a substrate protein in the presence of a thickening agent.

3. The method of claim 1, wherein the substrate protein is selected from the group consisting of laminin, tenascin, thrombospondin, collagen, fibronectin, vitronectin, polylysine, polyornithine, or a mixture thereof.

4. The method of claim 3, wherein the substrate protein is laminin.

5. The method of claim 2, wherein the thickener is a cellulosic thickener.

6. The method of claim 5, wherein the thickener is methylcellulose.

7. The method of claim 1, wherein the step of contacting is performed for a period of two weeks.

8. The method of claim 1, wherein the step of contacting is performed for a two week period, wherein the step of contacting is performed in two parts: during the first part, cells were grown for three days without medium change; while during the second part the medium was changed daily.

9. A method of expanding a population of Islet 1+ multipotent progenitor cells (IMPs), the method comprising: providing a group of IMPs; contacting a medium of known composition with an effective amount of a combination of BIO and BMP4, at a low density on a base protein, with said population for a time effective to increase said population; and isolating the cell after the contacting step.

10. The method of claim 9, wherein the combination of media of known composition and an effective amount of BIO and BMP4 are contacted with the population at low density on a substrate protein in the presence of a thickening agent.

11. The method of claim 9, wherein the substrate protein is selected from the group consisting of laminin, tenascin, thrombospondin, collagen, fibronectin, vitronectin, polylysine, polyornithine, or a mixture thereof.

12. The method of claim 11, wherein the substrate protein is laminin.

13. The method of claim 10, wherein the thickener is a cellulosic thickener.

14. The method of claim 13, wherein the thickener is methylcellulose.

15. A method of producing cardiomyocytes and/or endothelial cells from IMPs, the method comprising: providing a group of IMPs; growing said population of IMPs in a differentiation medium in the presence of an effective amount of at least one differentiating agent selected from the group consisting of BMP, DKK1, VEGF, and mixtures thereof, said differentiation medium being devoid of activin a;

wherein the IMPs are isolated human IMPs having the following characteristics:

i) these cells express Isl1, nkx2.5, Fgf10, Gata4, FoxFl, PDGFR α;

ii) the cells are karyotypically normal;

iii) these cells do not express Oct4, Nanog, T or degermed proteins;

iv) these cells can express PDGFR β and/or cadherin 11 on the cell surface; and

v) these cells are capable of differentiating into cardiomyocytes, smooth muscle cells and/or endothelial cells.

16. The method of claim 15, wherein the differentiation medium contains insulin growth factor.

17. The method of claim 15 wherein said IMPs population is grown at 2.5 x 10 prior to said step of growing⁴Cells/cm²-2.5×10⁵Cells/cm²The concentration of (4) is inoculated.

18. The method of claim 15 wherein said IMPs are self-renewing.

19. The method according to claim 15 wherein said IMPs are derived from human induced pluripotent cells (hiPSCs).

20. A method of producing smooth muscle cells from IMPs, the method comprising: providing a group of IMPs; the IMPs populations were grown in the presence of effective amounts of wingless protein and BMP4 in a medium of known composition, which did not contain activin a.

21. The method of claim 20, wherein the medium of known composition contains insulin growth factor.

22. The method of claim 20, wherein said wingless protein is Wnt3 a.

23. The method according to claim 20 wherein said IMPs are prepared from human induced pluripotent stem cells (hiPSCs).

24. An isolated population of human ISL1+ multipotent progenitor cells (IMPs) having the following characteristics:

i) these cells express Isl1, nkx2.5, Fgf10, Gata4, FoxFl, PDGFR α;

ii) the cells are karyotypically normal;

iii) these cells do not express Oct4, Nanog, T or degermed proteins;

iv) these cells can express PDGFR β and/or cadherin 11 on the cell surface; and

v) these cells are capable of differentiating into cardiomyocytes, smooth muscle cells and/or endothelial cells.

25. The cell population according to claim 24, wherein the cells express Tbx3 and/or hande (Hand) 1.

26. The cell population of claim 24, which is produced by human induced pluripotent stem cells (hiPSCs).

27. The cell population of claim 24, which is produced by contacting human induced pluripotent stem cells in differentiation medium with an effective amount of BIO and BMP.

28. The cell population of claim 27, wherein the cell population is produced by contacting human induced pluripotent stem cells in differentiation medium with effective doses of a GSK inhibitor and a bone morphogenic protein and an activin a inhibitor.

29. The cell population of claim 28, wherein the activin a inhibitor is SB 431542.

30. Use of an effective amount of ISL1+ multipotent progenitor cells (IMPs) according to claim 24 in the manufacture of a medicament for treating or repairing damaged myocardial and/or vascular tissue in a patient in need thereof, comprising the step of administering the medicament to the patient.

31. The use of claim 30, wherein the cells are used to repair cardiac tissue of the patient.

32. The use of claim 30, wherein the population of cells is used to repair vascular tissue in the patient.

33. The use of any one of claims 30-32, wherein the cells are administered to damaged tissue.

34. A pharmaceutical composition comprising the cell population of any one of claims 24-28, and a pharmaceutically acceptable carrier, additive or excipient.

35. The composition of claim 34, wherein the carrier is a salt solution.

36. The composition of claim 34, wherein the composition further comprises a bioactive agent.

37. Use of an effective amount of a population of ISL1+ multipotent progenitor cells (IMPs) according to claim 24 in the manufacture of a medicament for repairing an endodermal organ in need of repair in a patient, the use comprising the step of administering the medicament to the patient.

38. The use of claim 37, wherein the population of cells is administered to an organ to be repaired.

39. The use of claim 37, wherein the organ is the liver, pancreas, or gastrointestinal tract of the patient.

40. A method of producing cardiomyocytes, the method comprising: culturing ISL1+ multipotent progenitor cells (IMPs) according to claim 24 in cell culture medium supplemented with an effective amount of cardiac tissue to produce a population of cardiomyocytes.

41. A method of isolating ISL1+ multipotent progenitor cells (IMPs) from a population of cells, the method comprising: contacting a population of said cells with an anti-PDGFR β antibody, allowing said antibody to bind to IMP cells in said population to form antibody-IMP complexes, and isolating said antibody-IMP complexes from said cell population.

42. The method of claim 41 wherein the antibody-IMP complex is treated to separate the antibodies in the complex from the IMP to provide a purified population of IMPs.

43.A method of identifying the presence or absence of ISL1+ multipotent progenitor cells (IMPs) in a population of cells, the method comprising: contacting a population of said cells with an anti-PDGFR β antibody linked to a reporter molecule, allowing said antibody to bind to IMP cells in said population to form an antibody-IMP-reporter complex, and measuring said complex to determine the presence and/or number of IMP cells in said population.