Enhanced Efficiency of Induced Pluripotent Stem Cell Generation

Abstract

Human somatic cells are reprogrammed to become induced pluripotent stem cells (iPS cells) by the introduction of a minicircle DNA vector. Cells of interest include adipose stem cells.

1 - 12 . (canceled) 
     
     
         13 . A population of induced human pluripotent stem cells comprising one or more minicircle DNA vectors comprising coding sequences encoding a plurality of reprogramming factors, wherein:
 (i) the coding sequence of each reprogramming factor is operably linked to a promoters   (ii) the plurality of reprogramming factors comprises Oct4, Sox2, Lin28, and Nanog; or Oct4, Sox2, c-Myc, and Klf4; and   (iii) each of the one or more minicircle DNA vectors is 0.3-10 Kb in length and lacks expression-silencing bacterial sequences.   
     
     
         14 - 16 . (canceled) 
     
     
         17 . The population of induced pluripotent stem cells of  claim 13 , wherein the plurality of reprogramming factors comprises Oct4, Sox2, Lin28, and Nanog. 
     
     
         18 . The population of induced pluripotent stem cells of  claim 13 , wherein the plurality of reprogramming factors comprises Oct4, Sox2, c-Myc, and Klf4. 
     
     
         19 . The population of induced pluripotent stem cells of  claim 28 , wherein the coding sequences of said reprogramming factors are operably linked to the same single promoter. 
     
     
         20 . The population of induced pluripotent stem cells of  claim 19 , wherein said coding sequences are separated from each other by sequences encoding self-cleaving peptides. 
     
     
         21 . (canceled) 
     
     
         22 . A minicircle DNA vector comprising coding sequences encoding a cocktail plurality of reprogramming factors sufficient to induce pluripotency of a somatic cell population of human adipose stem cells, wherein:
 (i) the coding sequence of each reprogramming factor is operably linked to a promoter;   (ii) the plurality of reprogramming factors comprises Oct4, Sox2, Lin28, and Nanog; or Oct4, Sox2, c-Myc, and Klf4; and   (iii) the minicircle DNA vector is 0.3-10 Kb in length and lacks expression-silencing bacterial sequences.   
     
     
         23 . The minicircle DNA vector of  claim 22 , wherein the cocktail plurality of reprogramming factors comprises Oct4, Sox2, Lin28, and Nanog. 
     
     
         24 . The minicircle DNA vector of  claim 22 , wherein the plurality of reprogramming factors comprises Oct4, Sox2, c-Myc, and Klf4. 
     
     
         25 . The minicircle DNA vector of  claim 22 , wherein the coding sequences of said reprogramming factors are operably linked to the same single promoter. 
     
     
         26 . The minicircle DNA vector of  claim 25 , wherein said coding sequences are separated from each other by sequences encoding self-cleaving peptides. 
     
     
         27 . (canceled) 
     
     
         28 . The population of induced pluripotent stem cells of  claim 13 , wherein a single minicircle DNA vector encodes said plurality of reprogramming factors. 
     
     
         29 . The population of induced pluripotent stem cells of  claim 19 , wherein said coding sequences are separated from each other by a sequence encoding a self-cleaving 2A peptide sequence. 
     
     
         30 . The population of induced pluripotent stem cells of  claim 13 , wherein the induced pluripotent stem cells are in feeder-layer free culture. 
     
     
         31 . The minicircle DNA vector of  claim 25 , wherein said coding sequences are separated from each other by a sequence encoding a self-cleaving 2A peptide sequence.