Human feeder cells that support proliferation of undifferentiated pluripotent stem cells

Abstract

This invention provides media that support the growth of primate pluripotent stem cells in feeder-free culture, and cell lines useful for producing such media and for other purposes, such as use as a primary feeder layer. Conventionally, it has been necessary to grow pluripotent embryonic cells on feeder layers of primary embryonic fibroblasts, in order to prevent them from differentiating. It has now been discovered that standard culture media conditioned by special cell lines can be used to support proliferation of pluripotent stem cells while inhibiting differentiation in an environment free of feeder cells. This invention includes mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods for deriving such cell lines, processing media, and growing stem cells using the feeder cells or conditioned media are described and illustrated in this disclosure.

What is claimed as the invention is:  
     
         1 . A composition of cells comprising: 
 substantially undifferentiated human embryonic stem (hES) cells; and    cells from a human cell line that are capable of maintaining human embryonic stem cells in a substantially undifferentiated state through at least 4 passages when the cells are cultured together.    
     
     
         2 . The composition of  claim 1 , wherein the human cell line is a line of mesenchymal cells or fibroblasts from a non-malignant source.  
     
     
         3 . The composition of  claim 1 , wherein the human cell line has been obtained by differentiating human pluripotent stem cells.  
     
     
         4 . The composition of  claim 3 , wherein the pluripotent stem cells from which the human cell line has been obtained are embryonic stem cells.  
     
     
         5 . The composition of  claim 1 , wherein the human cell line is an immortalized cell line.  
     
     
         6 . The composition of  claim 1 , wherein the human cell line has been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.  
     
     
         7 . The composition of  claim 3 , wherein the human cell line has the same genome as that of the hES cells in the composition.  
     
     
         8 . The composition of  claim 3 , wherein the human cell line has a different genome from that of the hES cells in the composition.  
     
     
         9 . The composition of  claim 1 , wherein the cells of the human cell line that are present in the composition have been inactivated to prevent them from proliferating.  
     
     
         10 . The composition of  claim 9 , wherein the inactivating comprises irradiation or treatment with mitomycin c.  
     
     
         11 . The composition of  claim 1 , wherein the human cell line has the property that culturing it in a medium conditions the medium such that hES cells can be maintained in a substantially undifferentiated state in a growth environment essentially free of feeder cells but containing the conditioned medium.  
     
     
         12 . The composition of  claim 1 , wherein at least ˜60% of the hES cells in the composition are undifferentiated.  
     
     
         13 . A method of maintaining hES cells in a substantially undifferentiated state, comprising culturing the composition of  claim 1 .  
     
     
         14 . A method of maintaining hES cells in a substantially undifferentiated state, comprising coculturing the hES cells through at least 4 passages with an immortalized human cell line differentiated from human embryonic stem cells.  
     
     
         15 . A method of obtaining a differentiated cell population, comprising culturing the composition of  claim 1 , and subsequently causing progeny of the hES cells to differentiate.  
     
     
         16 . The method of  claim 15 , wherein the differentiated cell population is a population of hepatocytes, neuronal cells, glial cells, cardiomyocytes, or osteoblasts.