Serum free cultivation of primate embryonic stem cells

Abstract

Disclosed herein are methods for culturing primate embryonic stem cells. These cells are cultured on a prolonged and stable basis in the presence of exogenously supplied fibroblast growth factor and in the absence of animal serum. Preferably there is also a fibroblast feeder layer. Also disclosed is a culture media containing fibroblast feeder layer and the fibroblast growth factor.

1 - 16 . (canceled)  
   
   
       17 . A method of culturing primate embryonic stem cells in defined media without serum, the method comprising: 
 culturing the primate embryonic stem cells in a culture medium which can support stem cells, the culture medium being essentially free of mammalian serum and containing exogenously supplied human fibroblast growth factor in a concentration between 0.4 and 500 ng/ml, so that the stem cells proliferate in culture and remain undifferentiated in the absence of serum in the medium.    
   
   
       18 . The method of  claim 17 , wherein the fibroblast growth factor is basic fibroblast growth factor.  
   
   
       19 . The method of  claim 17 , wherein the fibroblast growth factor is human basic fibroblast growth factor which has been produced from a recombinant gene.  
   
   
       20 . The method of  claim 17 , wherein the primate embryonic stem cells are human embryonic stem cells.  
   
   
       21 . A method of culturing primate embryonic stem cells in defined media without serum, the method comprising: 
 culturing the primate embryonic stem cells in a culture medium which can support stem cells, the culture medium being essentially free of mammalian fetal serum and containing exogenously supplied mammalian fibroblast growth factor in a concentration of between 0.4 and 500 ng/ml, said culturing step being conducted for over one month with the embryonic stem cells proliferating in culture while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.    
   
   
       22 . A method of culturing primate embryonic stem cells in defined media without serum, the method comprising: 
 culturing the primate embryonic stem cells in a culture medium which can support stem cells, the culture medium being essentially free of mammalian fetal serum and in the presence of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is exogenously supplied to the culture at a concentration of between 0.4 and 500 ng/ml, said culturing step being conducted for over one month with the embryonic stem cells proliferating in culture while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.    
   
   
       23 . In a method of culturing primate embryonic stem cells without serum, the improvement comprising: 
 culturing the primate embryonic stem cells in a culture free of added mammalian fetal serum and which can support stem cells, and in the presence of a fibroblast growth factor that is exogenously supplied to the culture at a concentration of between 0.4 and 500 ng/ml, so that the stem cells proliferate in culture and remain undifferentiated in the absence of serum in the medium.