System for expanding and differentiating human 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 obtaining a population of differentiated 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 cells from the colonies onto an extracellular matrix in a culture environment that is essentially free of feeder cells;    d) culturing the passaged cells on the extracellular matrix in a nutrient medium containing fibroblast growth factor; and    e) differentiating the cultured cells into a population comprising lineage restricted cells or terminally differentiated cells.    
     
     
         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 , further comprising genetically altering the cells before they are differentiated.  
     
     
         10 . The method of  claim 9 , wherein the cells are genetically altered by transfecting cells in the population with a DNA-lipid complex; and selecting cells that have been genetically altered with the complex.  
     
     
         11 . The method of  claim 1 , wherein the cells are differentiated by culturing the cells in the absence of serum or serum replacement.  
     
     
         12 . The method of  claim 1 , wherein the cells are differentiated by adding retinoic acid, butyrate, DMSO, or a growth factor to the growth environment.  
     
     
         13 . The method of  claim 1 , wherein the cells are differentiated by culturing on a solid surface that promotes differentiation.  
     
     
         14 . The method of  claim 1 , wherein the cells are differentiated by a process that comprises forming embryoid bodies.  
     
     
         15 . The method of  claim 1 , wherein the cells are differentiated into neural precursor cells, neuronal cells or glial cells.  
     
     
         16 . The method of  claim 1 , wherein the cells are differentiated into cardiomyocytes.  
     
     
         17 . The method of  claim 1 , wherein the cells are differentiated into hepatocytes.  
     
     
         18 . The method of  claim 1 , wherein the population obtained is at least about 80% lineage restricted cells or terminally differentiated cells.  
     
     
         19 . The method of  claim 1 , wherein the population obtained is at least about 50% neuronal cells.  
     
     
         20 . The method of  claim 10 , wherein the population obtained is at least about 50% cardiomyocyte lineage cells.