COMPOSITIONS FOR MESODERM DERIVED ISL1+ MULTIPOTENT CELLS (IMPs), EPICARDIAL PROGENITOR CELLS (EPCs) AND MULTIPOTENT CD56C CELLS (C56Cs) AND METHODS OF PRODUCING AND USING SAME

Abstract

The present invention relates to inter alia, methods for the generation and maintenance of Mesoderm-derived ISL1+ Multipotent Progenitors (IMPs), the production of a number of pluripotent cells including and epicardial pluripotent cells (EPCs) and using these cells to produce endothelial cells, cardiomyocytes, smooth muscle cells, vascular cells and other cells and related methods as otherwise disclosed herein. The invention also relates to compositions comprising a population of cells.

1 . A method for maintaining or self-renewing a population of Islet 1+ multipotent progenitor cells (IMPs) comprising providing a population of IMPs and exposing said population to a cell culture media in combination with an effective amount of a GSK inhibitor and a bone morphogenic protein on a substrate protein optionally, in the presence of a thickener and isolating said cells after said exposing step. 
     
     
         2 . The method according to  claim 1  wherein said GSK inhibitor is BIO or Wnt3a. 
     
     
         3 . The method according to  claim 1  or  2  wherein said bone morphogenic protein is BMP4. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . A method of expanding a population of Islet 1+ multipotent progenitor cells (IMPs) comprising providing a population of IMPs and exposing said population to a cell culture media in combination with an effective amount of a GSK inhibitor and a bone morphogenic protein on a substrate protein at low density for a period of time effective to increase said population optionally, in the presence of a thickener, and isolating said cells after said exposing step. 
     
     
         10 . The method according to  claim 9  wherein said GSK inhibitor is BIO (2′Z,3′E)-6-Bromoindirubin-3′-oxime (inhibitor IX). 
     
     
         11 . The method according to  claim 9  or  10  wherein said bone morphogenic protein is BMP4. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . A method of producing cardiomyocytes and/or endothelial cells from IMPs comprising: providing a population of IMP cells; growing said population of IMP cells in a differentiation media which does not contain Activin A and optionally contains insulin growth factor (IIGF) in the presence of an effective amount of at least one differentiation agent selected from the group consisting of BMP, DKK1, VEGF and mixtures thereof; and optionally, isolating said cells. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . A method of producing smooth muscle cells from IMPs comprising providing a population of IMP cells; growing said population of IMP cells in a cell culture media which does not contain Activin A and optionally contains insulin growth factor (IGF) in the presence of an effective amount of a wingless protein and a bone morphogenic protein; and optionally, isolating said cells. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . A population of ISL1+ multipotent progenitor cells (IMPs) having the following characteristics:
 i) These cells express Isl1, Nkx2.5, Fgf10, Gata4, FoxF1, PDGFRα;   ii) These cells optionally express Tbx3 and/or Hand1;   iii) These cells are karyotypically normal;   iv) These cells do not express the Oct4, Nanog, T or eomesodermin;   v) These cells may express PDGFRβ and/or cadherin 11 on the cell surface; and   vi) These cells can be differentiated into cardiomyocytes, smooth muscle cells and/or endothelial cells.   
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . A method of preparing a population of multipotent CXCR4+CD56+ cells (C56Cs) from a population of multipotent migratory cells (MMCs) comprising exposing said MMCs in a differentiation medium in the absence of an Activin A inhibitor and a GSK3 inhibitor to an effective amount of a wingless protein, a bone morphogenic protein and a butyrate salt. 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . (canceled) 
     
     
         48 . (canceled) 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . (canceled) 
     
     
         52 . (canceled) 
     
     
         53 . (canceled) 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . (canceled) 
     
     
         58 . A population of epicardial pluripotent cells (“EPCs) having the following characteristics:
 i) These cells express Wilm's tumor suppressor protein (Wt1), Tcf21 (epicardin) and Raldh2 (Aldh1a2); 
 ii) These cells also express one or more of Tbx18, COL3A1, GATA6, Tbx3 and Tbx5. 
 
     
     
         59 . The population of cells according to  claim 58  wherein the cells also express at least two of Tbx18, COL3A1, GATA6, Tbx3 and Tbx5. 
     
     
         60 . The population of cells according to  claim 58  wherein the cells also express at least three of Tbx18, COL3A1, GATA6, Tbx3 and Tbx5. 
     
     
         61 . The population of cells according to  claim 58  wherein the cells also express at least four of Tbx18, COL3A1, GATA6, Tbx3 and Tbx5. 
     
     
         62 . The population of cells according to  claim 58  wherein the cells also express all five of Tbx18, COL3A1, GATA6, Tbx3 and Tbx5. 
     
     
         63 . (canceled) 
     
     
         64 . (canceled) 
     
     
         65 . A method of producing a population of epicardial pluripotent cells (EPCs) from ISL1+ multipotent progenitor cells (IMPs) comprising providing a population of IMPs; exposing said population of IMPs in a differentiation media to an effective amount of a GSK inhibitor, a bond morphogenic protein and retinoic acid to produce a population of EPCs; and optionally, isolating said EPCs. 
     
     
         66 . (canceled) 
     
     
         67 . (canceled) 
     
     
         68 . (canceled) 
     
     
         69 . (canceled) 
     
     
         70 . (canceled) 
     
     
         71 . (canceled) 
     
     
         72 . (canceled) 
     
     
         73 . A method of producing a population of epicardial pluripotent cells (EPCs) from human pluripotent stem cells (hPSCs) comprising providing a population of hPSCs; exposing said hPSCs on a support protein in differentiation media to an effective amount of a GSK inhibitor, a bone morphogenic protein and optionally, an Activin A inhibitor to produce ISL1+ multipotent progenitor cells (IMPs) and thereafter, exposing said population of IMPs in a differentiation media to an effective amount of a GSK inhibitor, a bond morphogenic protein and retinoic acid to produce a population of EPCs; and optionally, isolating said EPCs. 
     
     
         74 . (canceled) 
     
     
         75 . (canceled) 
     
     
         76 . (canceled) 
     
     
         77 . (canceled) 
     
     
         78 . (canceled) 
     
     
         79 . (canceled) 
     
     
         80 . A method of producing a population of endothelial cells from epicardial pluripotent cells (EPCs) comprising: providing a population of EPCs; exposing said EPCs in a differentiation media to an effective amount of VEGF and optionally, an Activin A inhibitor. 
     
     
         81 . The method according to  claim 80  wherein said VEGF is VEGF 165 . 
     
     
         82 . The method according to  claim 80  or  81  wherein said Activin A inhibitor is SB431542. 
     
     
         83 . (canceled) 
     
     
         84 . (canceled) 
     
     
         85 . (canceled) 
     
     
         86 . A method of producing a population of smooth muscle cells and/or cardiac fibroblasts from epicardial pluripotent cells (EPCs) comprising: providing a population of EPCs; exposing said EPCs in a differentiation media to an effective amount of fetal bovine serum (FBS) and a pyruvate salt; and optionally, isolating said smooth muscle cells or said cardiac fibroblasts. 
     
     
         87 . (canceled) 
     
     
         88 . (canceled) 
     
     
         89 . A method of producing a population of smooth muscle cells from epicardial pluripotent cells (EPCs) comprising: providing a population of EPCs; exposing said EPCs in a differentiation media to effective amounts of
 i) VEGF;   ii) VEGF and PDGFβ;   iii) VEGF and HDKK1; or   iv) Fetal bovine serum (FBS) or a mixture of FBS and i, ii or iii; and optionally, isolating said smooth muscle cells.   
     
     
         90 . (canceled) 
     
     
         91 . (canceled) 
     
     
         92 . A method of treating or repairing damaged cardiac and/or vascular tissue in a patient in need thereof comprising administering to said patient an effective amount of a population of ISL1+ multipotent progenitor cells (IMPs) or epicardial progenitor cells (EPCs). 
     
     
         93 . (canceled) 
     
     
         94 . (canceled) 
     
     
         95 . (canceled) 
     
     
         96 . (canceled) 
     
     
         97 . (canceled) 
     
     
         98 . A pharmaceutical composition comprising a population of cells according to any of  claims 26 ,  33  or  58  in combination with a pharmaceutically acceptable carrier, additive or excipient. 
     
     
         99 . (canceled) 
     
     
         100 . (canceled) 
     
     
         101 . (canceled) 
     
     
         102 . (canceled) 
     
     
         103 . (canceled) 
     
     
         104 . (canceled) 
     
     
         105 . (canceled) 
     
     
         106 . (canceled) 
     
     
         107 . (canceled) 
     
     
         108 . A method of producing a population of ISL+ multipotent progenitor cells/splanchnic mesoderm cells (IMP/Spl-m) from pluripotent stem cells (PSCs) comprising exposing said pluripotent stem cells to a combination of an effective amount of a wingless (Wnt) protein and a bone morphogenic protein (BMP) in a cell differentiation medium and optionally, isolating aid IMP/Spl-m cells. 
     
     
         109 . (canceled) 
     
     
         110 . (canceled) 
     
     
         111 . The method according to  claim 108  wherein said wingless protein is Wnt3a and said bone morphogenic protein is BMP4. 
     
     
         112 . (canceled) 
     
     
         113 . (canceled) 
     
     
         114 . (canceled) 
     
     
         115 . A method of producing a population of Wt+ epicardium-like cells (PE progenitor cells) comprising exposing a population of IMP/Spl-m cells to an effective amount of a wingless (Wnt) protein in combination with an effective amount of a bone morphogenic protein (BMP) in a cell differentiation medium in combination with at least one additional agent selected from the group consisting of a fibroblast growth factor, retinoic acid and mixtures thereof, and optionally, isolating said PE progenitor cells. 
     
     
         116 . (canceled) 
     
     
         117 . (canceled) 
     
     
         118 . (canceled) 
     
     
         119 . (canceled) 
     
     
         120 . (canceled) 
     
     
         121 . A method of producing smooth muscle cells and/or endothelial cells from PE progenitor cells comprising exposing a population of PE progenitor cells to a cell differentiation medium without Activin A comprising an effective amount of VEGF. 
     
     
         122 . (canceled) 
     
     
         123 . (canceled) 
     
     
         124 . (canceled) 
     
     
         125 . A method of producing a population of Wt+ epicardium-like cells (PE progenitor cells) from pluripotent stem cells comprising
 i. Exposing said pluripotent stem cells to a combination of an effective amount of a wingless (Wnt) protein and a bone morphogenic protein (BMP) in a cell differentiation medium and optionally, isolating aid IMP/Spl-m cells; and   ii. Exposing said IMP/Spl-m cells obtained in step i to to an effective amount of a wingless (Wnt) protein in combination with an effective amount of a bone morphogenic protein (BMP) in a cell differentiation medium in combination with at least one additional agent selected from the group consisting of a fibroblast growth factor, retinoic acid and mixtures thereof, and optionally, isolating said PE progenitor cells.   
     
     
         126 . (canceled) 
     
     
         127 . (canceled) 
     
     
         128 . (canceled) 
     
     
         129 . (canceled) 
     
     
         130 . (canceled) 
     
     
         131 . (canceled) 
     
     
         132 . (canceled) 
     
     
         133 . The method according to  claim 125  wherein said Wnt protein is Wnt3a included in said cell differentiation medium of step ii at a concentration of about 25 ng/ml, said bone morphogenic protein is BMP4 included in said differentiation medium of step ii at a concentration of about 50 ng/ml to about 100 ng/ml, said fibroblast growth factor is fibroblast growth factor 2, if included in said cell differentiation medium, is included at a concentration of about 100 ng/ml and said retinoic acid is all trans-retinoic acid, and if included in said cell differentiation medium, is included at a concentration of about 4 μM. 
     
     
         134 . (canceled) 
     
     
         135 . A method of inducing vascular formation in vivo comprising implanting into the epicardium of a patient or subject an effective amount of PE progenitor cells. 
     
     
         136 . A pharmaceutical composition comprising an effective number of PE cells in combination with a pharmaceutically acceptable carrier, additive or excipient and optionally, an additional bioactive agent. 
     
     
         137 . (canceled)