Using fibroblast growth factor to establish a line of embryonic stem cells

Abstract

This disclosure provides an improved system for culturing human pluripotent stem cells. Traditionally, pluripotent stem cells are cultured on a layer of feeder cells (such as mouse embryonic fibroblasts) to prevent them from differentiating. In the system described here, the role of feeder cells is replaced by components added to the culture environment that support rapid proliferation without differentiation. Effective features are a suitable support structure for the cells, and an effective medium that can be added fresh to the culture without being preconditioned by another cell type. Culturing human embryonic stem cells in fresh medium according to this invention causes the cells to expand surprisingly rapidly, while retaining the ability to differentiate into cells representing all three embryonic germ layers. This new culture system allows for bulk proliferation of pPS cells for commercial production of important products for use in drug screening and human therapy.

1 . A method for establishing a line of human embryonic stem cells, comprising: 
 a) isolating cells from the inner cell mass of a human blastocyst;    b) forming colonies comprising undifferentiated cells from the isolated blastocyst cells;    c) passaging the colonies into a culture environment that contains an extracellular matrix instead of feeder cells; and    d) culturing the colonies in the culture environment in a nutrient medium containing fibroblast growth factor, thereby establishing a line of human embryonic stem cells;    wherein the embryonic stem cell line can proliferate in culture for at least 64 days without differentiation, while maintaining a normal karyotype and the potential to differentiate to cells of endoderm, mesoderm, and ectoderm tissues.    
     
     
         2 . The method of  claim 1 , wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts in said culture environment.  
     
     
         3 . The method of  claim 1 , wherein the extracellular matrix is made from one or more extracellular matrix components selected from laminin, fibronectin, proteoglycan, and entactin.  
     
     
         4 . The method of  claim 1 , wherein the nutrient medium is a serum-free medium.  
     
     
         5 . The method of  claim 1 , wherein the medium is a fresh medium not previously conditioned by culturing with feeder cells.  
     
     
         6 . The method of  claim 1 , wherein the fibroblast growth factor is recombinant basic human fibroblast growth factor.  
     
     
         7 . The method of  claim 1 , wherein the medium also contains added stem cell factor (SCF).  
     
     
         8 . The method of  claim 1 , wherein the medium also contains added Flt3 ligand.  
     
     
         9 . The method of  claim 1 , wherein the embryonic stem cell line expresses SSEA-4, telomerase reverse transcriptase (TERT), and OCT-4.  
     
     
         10 . The method of  claim 1 , further comprising culturing and passaging the embryonic stem cell line in the culture environment for at least 40 days so that the cells proliferate without differentiation and while maintaining a normal karyotype.  
     
     
         11 . The method of  claim 1 , wherein cells in the established stem cell line comprise a genetic alteration.  
     
     
         12 . The method of  claim 11 , wherein the cells have been genetically altered by transfecting with a DNA-lipid complex; selecting cells that have been genetically altered with the complex; and expanding the selected cells in a feeder-free culture environment that contains an extracellular matrix and fibroblast growth factor.  
     
     
         13 . A method for producing a population of differentiated cells, comprising: 
 a) providing a line of human embryonic stem cells that have been established in culture without using feeder cells;    b) growing undifferentiated embryonic stem cells from said line in a culture environment that contains an extracellular matrix instead of feeder cells in a nutrient medium containing fibroblast growth factor; and then    c) causing the cells grown in b) to differentiate into the population of differentiated cells.    
     
     
         14 . The method of  claim 11 , wherein the extracellular matrix is made from one or more extracellular matrix components selected from laminin, fibronectin, proteoglycan, and entactin.  
     
     
         15 . The method of  claim 11 , wherein the medium is a fresh medium not previously conditioned by culturing with feeder cells.  
     
     
         16 . The method of  claim 11 , wherein the fibroblast growth factor is recombinant basic human fibroblast growth factor.  
     
     
         17 . The method of  claim 11 , wherein the medium also contains added stem cell factor (SCF).  
     
     
         18 . The method of  claim 11 , wherein the medium also contains added Flt3 ligand.