Defined media for expansion and maintenance of pluripotent stem cells

Abstract

The present invention provides methods to promote the proliferation of undifferentiated pluripotent stem cells in defined media. Specifically, the invention provides a defined cell culture formulation for the culture, maintenance, and expansion of pluripotent stem cells, wherein culturing stem cells in the defined cell culture formulation maintains the pluripotency and karyotypic stability of the cells for at least 10 passages. Further disclosed is a cell population, grown under defined media conditions, that expresses OCT4, SOX2, NANOG, and FOXA2.

What is claimed is: 
     
         1 . A defined cell culture formulation for the culture, maintenance, and expansion of pluripotent stem cells, wherein the defined cell culture formulation comprises basal medium, insulin, transferrin, selenium, fatty-acid free albumin, a TGF-β ligand, bFGF, and ascorbic acid; and wherein culturing stem cells in the defined cell culture formulation maintains the pluripotency and karyotypic stability of the cells for at least 10 passages. 
     
     
         2 . The defined cell culture formulation of  claim 1 , wherein the cell culture formulation comprises DMEM-F12. 
     
     
         3 . The defined cell culture formulation of  claim 1 , wherein the cell culture formulation further comprises insulin growth factor 1 (IGF-1). 
     
     
         4 . The defined culture formulation of  claim 3 , wherein the cell culture formulation comprises DMEM-F12. 
     
     
         5 . The defined cell culture formulation of  claim 1 , wherein the cell culture formulation further comprises Trace Elements C, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, lithium chloride, glucose, Defined Lipids, and L-alanyl-L-glutamine dipeptide. 
     
     
         6 . The defined cell culture formulation of  claim 1 , wherein the cell culture formulation comprises MCDB-131. 
     
     
         7 . The defined cell culture formulation of  claim 1 , wherein ITS-X provides the insulin, transferrin, and selenium. 
     
     
         8 . The defined cell culture formulation of  claim 1 , wherein the fatty acid free albumin is reagent grade. 
     
     
         9 . The defined cell culture formulation of  claim 1 , wherein the TGF-β ligand is TGF-β1. 
     
     
         10 . A defined cell culture formulation consisting essentially of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, a TGF-β ligand, bFGF, and IGF-1. 
     
     
         11 . The defined cell culture formulation of  claim 10 , wherein the formulation consists of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, a TGF-β ligand, bFGF, and IGF-1. 
     
     
         12 . The defined culture formulation of  claim 11 , wherein the formulation is further supplemented with ascorbic acid. 
     
     
         13 . A defined cell culture formulation consisting essentially of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, a TGF-β ligand, bFGF, IGF-1, and ascorbic acid. 
     
     
         14 . The defined cell culture formulation of  claim 12 , wherein the formulation consists of DMEM-F12 basal medium, insulin, transferrin, selenium, fatty-acid free albumin, a TGF-β ligand, bFGF, IGF-1, and ascorbic acid. 
     
     
         15 . A defined cell culture formulation consisting essentially of MCDB-131, Trace Elements C, ascorbic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, lithium chloride, glucose, defined lipids, insulin, transferrin, selenium, fatty acid free albumin, a TGF-β ligand, bFGF, and L-alanyl-L-glutamine dipeptide. 
     
     
         16 . The defined cell culture formulation of  claim 15 , wherein the formulation consists of MCDB-131, Trace Elements C, ascorbic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, lithium chloride, glucose, defined lipids, insulin, transferrin, selenium, fatty acid free albumin, a TGF-β ligand, bFGF, and L-alanyl-L-glutamine dipeptide.