Generation and maintenance of stem cells

Abstract

The present invention provides for the generation and maintenance of pluripotent cells by culturing the cells in the presence of an ALK5 inhibitor.

1 - 57 . (canceled) 
     
     
         58 . A method of manufacturing cells for therapeutic use comprising culturing pluripotent cells through at least one cell division in the presence of a sufficient amount of:
 (a) a compound selected from one or more of a MEK inhibitor, an Erk inhibitor, a p38 inhibitor, and an FGF receptor inhibitor;   (b) a GSK3β inhibitor; and   (c) nutrients;   to allow for at least one cell division while maintaining cell pluripotency, and thereby obtaining cells for therapeutic use.   
     
     
         59 . The method of  claim 58 , wherein the culturing step further comprises culturing the cells in the presence of a Leukemia inhibitory factor (LIF). 
     
     
         60 . The method of  claim 58 , wherein the GSK3β inhibitor is CHIR99021. 
     
     
         61 . The method of  claim 58 , wherein the compound is a MEK inhibitor. 
     
     
         62 . The method of  claim 61 , wherein the MEK inhibitor is PD0325901. 
     
     
         63 . The method of  claim 58 , wherein the pluripotent cells are cultured through at least five cell divisions while maintaining cell pluripotency. 
     
     
         64 . The method of  claim 58 , further comprising introducing a heterologous nucleic acid into the pluripotent cells and culturing the resulting cells to allow for at least one additional cell division while maintaining pluripotency. 
     
     
         65 . The method of  claim 58 , wherein the pluripotent cells are rat, human, non-human primate, ovine, bovine, feline, canine, or porcine cells. 
     
     
         66 . A therapeutic composition comprising pluripotent animal cells for therapeutic use, wherein the pluripotent mammalian cells are obtained by a method comprising culturing pluripotent cells through at least one cell passage in the presence of a sufficient amount of:
 (a) a compound selected from one or more of a MEK inhibitor and an FGF receptor inhibitor; and   (b) a GSK3β inhibitor;   to allow for at least one cell division while maintaining cell pluripotency.   
     
     
         67 . The therapeutic composition of  claim 66 , wherein the pluripotent cells are further in the presence of Leukemia inhibitory factor (LIF). 
     
     
         68 . The therapeutic composition of  claim 66 , wherein the GSK3β inhibitor is CHIR99021. 
     
     
         69 . The therapeutic composition of  claim 66 , wherein the compound is a MEK inhibitor. 
     
     
         70 . The therapeutic composition of  claim 69 , wherein the MEK inhibitor is PD0325901. 
     
     
         71 . The therapeutic composition of  claim 66 , wherein the pluripotent animal cells are human cells or rat cells. 
     
     
         72 . The therapeutic composition of  claim 66 , wherein the pluripotent animal cells are embryonic stem cells.