Generation of induced pluripotent stem cells from normal human mammary epithelial cells

Abstract

Described herein are reprogramming techniques allowing for production of mammary-derived iPSCs (“m-iPSCs”). The m-iPSCs described herein exhibit all the hallmarks of stem cell identity including round cluster, bright colony morphology, clonal expansion, and pluripotent marker expression (alkaline phosphatase expression, Oct-4, nanog, etc.) Further refined techniques allow for generation of m-iPSCs under essentially defined conditions.

1 - 22 . (canceled) 
     
     
         23 . A method of generating human epithelial cell-derived induced pluripotent stem cells (ep-iPSCs), comprising:
 providing a quantity of human epithelial cells;   transfecting the human epithelial cells with vectors encoding Oct4, Sox2, Nanog, Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T Antigen (SV40LT) and p53 shRNA;   plating the cells on a culture vessel coated with a substrate; and   culturing the cells in an induction media, wherein the transfection, plating and the culturing generates colonies of human epithelial-derived induced pluripotent stem cells (ep-iPSCs), wherein the induction media comprises HA-100, CHIR99021, PD0325901, and A83-01, wherein the plating of the transfected cells on the culture vessel coated with the substrate further comprises culturing in norm oxygen conditions, and wherein the plated mammary cells express the transfected Oct4, Sox2, Nanog, KLF4, L-Myc, Lin28, SV40LT and p53 shRNA.   
     
     
         24 . The method of  claim 23 , wherein the epithelial cells are from primary cells. 
     
     
         25 . The method of  claim 23 , wherein the epithelial cells are from a tumor. 
     
     
         26 . The method of  claim 23 , wherein the epithelial cells are from a cell line. 
     
     
         27 . The method of  claim 23 , wherein the at least one vector comprises an episomal vector. 
     
     
         28 . The method of  claim 27 , wherein the episomal vector comprises an oriP/EBNA1 vector. 
     
     
         29 . The method of  claim 23 , wherein transfecting epithelial cells comprises nucleofection or lipofection. 
     
     
         30 . The method of  claim 23 , wherein plating the cells on a culture vessel coated with a substrate further comprises culturing in norm oxygen conditions. 
     
     
         31 . The method of  claim 23 , wherein the substrate comprises Matrigel. 
     
     
         32 . The method of  claim 23 , wherein culturing the cells in an induction media is for a period of 10-31 days. 
     
     
         33 . A composition of ep-iPSCs produced by the method of  claim 23 . 
     
     
         34 . The composition of  claim 33 , wherein the ep-iPSCs are capable of serial passaging as a cell line. 
     
     
         35 . A multi-potent cell produced by culturing the composition of  claim 34  in the presence of a differentiation agent. 
     
     
         36 . A method of reprogramming a human epithelial cell, comprising:
 providing a quantity of human epithelial cells;   expanding the human mammary cells to about 90% confluence;   culturing the human epithelial cells with vectors encoding Oct4, Sox2, Nanog, Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T Antigen (SV40LT) and p53 shRNA;   plating the cells on a culture vessel coated with a substrate; and
 culturing the cells in an induction media, wherein the expanding, the transfecting, the plating and the culturing reprograms the human mammary cells to a less differentiated state, wherein the induction media comprises HA-100, CHIR99021, PD0325901, and A83-01, wherein the plating of the transfected cells on the culture vessel coated with the substrate further comprises culturing in norm oxygen conditions, and wherein the plated mammary cells express the transfected Oct4, Sox2, Nanog, KLF4, L-Myc, Lin28, SV40LT and p53 shRNA. 
   
     
     
         37 . The method of  claim 36 , wherein the epithelial cells are from primary cells. 
     
     
         38 . The method of  claim 36 , wherein the epithelial cells are from a tumor. 
     
     
         39 . The method of  claim 36 , wherein the epithelial cells are from a cell line.