Method of nuclear reprogramming

Abstract

A method of producing an induced pluripotent stem cell includes introducing into a somatic cell one or more non-viral expression vectors. The vectors include one or more of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and Nanog gene. The somatic cell is then cultured in a medium that supports pluripotent stem cells. At least a portion of the one or more introduced non-viral expression vectors is not substantially integrated in the chromosome.

1 . A method of producing a human induced pluripotent stem (iPS) cell population, the method comprising the steps of:
 a) obtaining a starting population of human somatic cells;   b) obtaining one or more iPS reprogramming plasmids comprising:
 (i) a DNA sequence comprising a replication origin in each of said one or more iPS reprogramming plasmids that allows autonomous episomal replication of the one or more iPS reprogramming plasmids in the somatic cell; and 
 (ii) one or more expression cassettes encoding iPS reprogramming factors, wherein the iPS reprogramming factors comprise Oct-4; 
   c) introducing in vitro the one or more iPS reprogramming plasmids into the cytoplasm of the human somatic cells, wherein the one or more iPS reprogramming plasmids are maintained episomally in the cytoplasm and are not integrated into a chromosome;   d) culturing the human somatic cells that comprise the iPS reprogramming plasmids to effect expression of said iPS reprogramming factors, such that at least a portion of the human somatic cells become induced pluripotent stem cells that have traits consistent with embryonic stem cells; and   e) further culturing cells having the embryonic stem cell traits for a sufficient number of generations in the absence of selection pressure for the presence of the iPS reprogramming plasmids to provide human iPS cells that are essentially free of iPS reprogramming plasmids.   
     
     
         2 . The method of  claim 1 , wherein cells of the starting cell population are fibroblasts, hematopoietic cells, mesenchymal cells, liver cells or stomach cell. 
     
     
         3 . The method of  claim 1 , wherein the iPS reprogramming factors comprise Sox-2. 
     
     
         4 . The method of  claim 3 , wherein the iPS reprogramming factors further comprise Nanog or Lin28. 
     
     
         5 . A method of producing somatic cells comprising differentiating a human iPS cell population provided by the method of  claim 1 . 
     
     
         6 . A method of producing a human reprogrammed cell population, the method comprising the steps of:
 a) obtaining a starting population of human somatic cells;   b) obtaining one or more nuclear reprogramming plasmids comprising:
 (i) a DNA sequence comprising a replication origin in each of said one or more nuclear reprogramming plasmids that allows autonomous episomal replication of the one or more nuclear reprogramming plasmids in the cytoplasm of the human somatic cells; and 
 (ii) one or more expression cassettes encoding nuclear reprogramming factors, wherein the nuclear reprogramming factors comprise Oct-4; 
   c) introducing in vitro the one or more nuclear reprogramming plasmids into the cytoplasm of the human somatic cells, wherein the one or more nuclear reprogramming plasmids are maintained episomally in the cytoplasm and are not integrated into a chromosome;   d) culturing the human somatic cells that comprise the nuclear reprogramming plasmids to effect expression of said nuclear reprogramming factors, such that at least a portion of the human somatic cells become reprogrammed cells that are in a more undifferentiated state than the somatic cells so as to form an ES-like colony; and   e) further culturing the reprogrammed cells for a sufficient number of generations in the absence of selection pressure for the presence of the nuclear reprogramming plasmids to provide human reprogrammed cells that are essentially free of nuclear reprogramming plasmids.   
     
     
         7 . The method of  claim 1 , wherein the starting cell population of human somatic cells are fibroblasts and wherein the one or more iPS reprogramming plasmids encode one or more of Oct4, Sox-2 and Klf4. 
     
     
         8 . The method of  claim 7 , wherein the one or more iPS reprogramming plasmids encode all of Oct-4, Sox-2 and Klf4. 
     
     
         9 . The method of  claim 1 , wherein the replication origin is SV40 replication origin.