Methods for Generation for Pluripotent and Multipotent Cells

Abstract

This disclosure relates to methods of producing induced pluripotent (iPS), multipotent, and/or lineage-committed stem cells from differentiated cells, maintaining iPS, multipotent, and/or lineage-committed cells in culture, and re-differentiating the iPS and multipotent stem cells into any desired lineage-committed cell type.

1 . A method for inducing pluripotent or multipotent stem cells, comprising:
 obtaining primary cells from an animal;   culturing the primary cells; and   contacting the cultured primary cells with an agent that blocks CD47 signaling, thereby inducing pluripotent or multipotent stem cells.   
     
     
         2 . The method of  claim 1 , further comprising identifying and isolating a subset of pluripotent or multipotent stem cells that express stem cell marker genes. 
     
     
         3 . The method of  claim 2 , wherein the stem cell marker genes comprise at least one of c-Myc, Sox2, Klf4, or Oct4. 
     
     
         4 . The method of  claim 1 , further comprising culturing the primary cells in serum free media. 
     
     
         5 . The method of  claim 1 , wherein the induced pluripotent or multipotent stem cells form embryoid bodies. 
     
     
         6 . The method of  claim 1 , wherein the agent that blocks CD47 signaling comprises an anti-CD47 antibody or fragment thereof, a CD47-binding peptide, a CD47 antisense oligonucleotide, a CD47 morpholino, an anti-TSP1 antibody or fragment thereof, a TSP1-binding peptide, a TSP1 antisense oligonucleotide, or a TSP1 morpholino. 
     
     
         7 . The method of  claim 1 , wherein the agent comprises a small molecule capable of binding to CD47 or a small molecule capable of binding to TSP1. 
     
     
         8 . The method of  claim 1 , wherein the primary cells comprise endothelial cells, fibroblasts, hematopoietic cells, adipose cells, mucosal tissue cells, umbilical cord cells, or placenta cells. 
     
     
         9 . The method of  claim 8 , wherein the primary cells comprise human umbilical vein endothelial cells. 
     
     
         10 . A method for generating differentiated cells, comprising:
 obtaining primary cells from an animal;   culturing the primary cells;   contacting the cultured primary cells with an agent that blocks CD47 signaling to produce contacted cells;   isolating from among the contacted cells, cells that express stem cell marker genes;   culturing the isolated cells that express stem cell marker genes in serum-free media to produce induced pluripotent or multipotent stem cells; and   culturing the induced pluripotent or multipotent stem cells in cell differentiation medium to produce differentiated cells.   
     
     
         11 . The method of  claim 10 , wherein the induced pluripotent or multipotent stem cells form embryoid bodies. 
     
     
         12 . The method of  claim 10 , wherein culturing the induced pluripotent or multipotent stem cells in cell differentiation medium comprises culturing the induced pluripotent or multipotent stem cells in neural cell differentiation medium, smooth muscle cell differentiation medium, hepatocyte cell differentiation medium, or mesenchymal cell differentiation medium. 
     
     
         13 . The method of  claim 1 , wherein the differentiated cells comprise ectoderm-derived lineage cells. 
     
     
         14 . The method of  claim 13 , wherein the ectoderm-derived lineage cells comprise neuronal cells or astrocytes. 
     
     
         15 . The method of  claim 1 , wherein the differentiated cells comprise mesoderm-derived lineage cells. 
     
     
         16 . The method of  claim 15 , wherein the mesoderm-derived lineage cells comprise smooth muscle cells, endothelial cells, hematopoietic cells, or myeloid cells. 
     
     
         17 . The method of  claim 1 , wherein the differentiated cells comprise endoderm-derived lineage cells. 
     
     
         18 . The method of  claim 17 , wherein the endoderm-derived lineage cells comprise hepatocytes or adipocytes. 
     
     
         19 . The method of  claim 1 , wherein the agent that blocks CD47 signaling comprises an anti-CD47 antibody or fragment thereof, a CD47-binding peptide, a CD47 antisense oligonucleotide, a CD47 morpholino, an anti-TSP1 antibody or fragment thereof, a TSP1-binding peptide, a TSP1 antisense oligonucleotide, or a TSP1 morpholino. 
     
     
         20 . The method of  claim 1 , wherein the agent comprises a small molecule capable of binding to CD47 or a small molecule capable of binding to TSP1. 
     
     
         21 . The method of  claim 1 , wherein the primary cells comprise endothelial cells, fibroblasts, hematopoietic cells, adipose cells, mucosal tissue cells, umbilical cord cells, or placenta cells. 
     
     
         22 . The method of  claim 21 , wherein the primary cells comprise human umbilical vein endothelial cells. 
     
     
         23 . A method for expanding differentiated cells, comprising:
 generating differentiated cells by the method of  claim 10 ; and   contacting the differentiated cells with an agent that blocks CD47 signaling, thereby expanding the differentiated cells.   
     
     
         24 . A method for expanding differentiated cells, comprising contacting the differentiated cells with an agent that blocks CD47 signaling. 
     
     
         25 . The method of  claim 1 , where cells are cultured continuously with an agent that blocks CD47 signaling. 
     
     
         26 . The method of  claim 1 , where cells are cultured transiently with an agent that blocks CD47 signaling. 
     
     
         27 . Cells produced by the method of  claim 1 . 
     
     
         28 . A method to increase stem cell numbers in an animal comprising administering to the animal an agent that blocks CD47 signaling. 
     
     
         29 . The method of  claim 28 , wherein the administering to the animal of an agent that blocks CD47 signaling comprises local administration of the agent or systemic administration of the agent. 
     
     
         30 . The method of  claim 28 , wherein the administering to the animal of an agent that blocks CD47 signaling comprises topical administration of the agent to the skin of the animal. 
     
     
         31 . The method of  claim 28 , wherein the agent that blocks CD47 signaling comprises an anti-CD47 antibody or fragment thereof, a CD47-binding peptide, a CD47 antisense oligonucleotide, a CD47 morpholino, an anti-TSP1 antibody or fragment thereof, a TSP1-binding peptide, a TSP1 antisense oligonucleotide, or a TSP1 morpholino. 
     
     
         32 . The method of  claim 31 , wherein the agent that blocks CD47 signaling comprises a small molecule capable of binding to CD47. 
     
     
         33 . The method of  claim 28 , wherein administering to the animal an agent that blocks CD47 signaling comprises parenteral administration. 
     
     
         34 . A method of treating a disorder in a subject comprising administering cells produced by the method of  claim 1  to the subject. 
     
     
         35 . The method of  claim 34 , wherein the cells are administered to the subject parenterally. 
     
     
         36 . The method of  claim 35 , wherein the cells are administered to the subject by transplantation. 
     
     
         37 . A kit, comprising one or more of:
 a container containing an agent that blocks CD47 signaling;   a container containing a cell culture medium; and   one or more cell culture dishes.   
     
     
         38 . The kit of  claim 37 , wherein the agent that blocks CD47 signaling comprises an anti-CD47 antibody or fragment thereof, a CD47-binding peptide, a CD47 antisense oligonucleotide, a CD47 morpholino, an anti-TSP1 antibody or fragment thereof, a TSP1-binding peptide, a TSP1 antisense oligonucleotide, or a TSP1 morpholino. 
     
     
         39 - 41 . (canceled)