Embryonic stem cells and neural progenitor cells derived therefrom

Abstract

The present invention provides undifferentiated human embryonic stem cells, methods of cultivation and propagation and production of differentiated cells. In particular it relates to the production of human ES cells capable of yielding somatic differentiated cells in vitro, and committed progenitor cells such as neural progenitor cells capable of giving rise to mature somatic cells including neural cells and/or glial cells and uses thereof. The invention also provides methods that generate in vitro and in vivo models of controlled differentiation of ES cells towards the neural lineage. The model, and the cells that are generated along the pathway of neural differentiation may be used for the study of the cellular and molecular biology of human neural development, for the discovery of genes, growth factors, and differentiation factors that play a role in neural differentiation and regeneration, for drug discovery and for the development of screening assays for teratogenic, toxic and neuroprotective effects.

1 . A method of generating neural progenitor cells, the method comprising:
 (a) continuously culturing undifferentiated pluripotent stem cells on a fibroblast feeder layer to generate colonies;   (b) selecting from said colonies, small piled, tightly packed, differentiated cells; and   (c) culturing said differentiated cells with growth factors to produce neurospheres containing the neural progenitor cells, thereby generating neural progenitor cells.   
     
     
         2 . The method of  claim 1 , wherein said neurospheres do not express alphafetoprotein. 
     
     
         3 . The method of  claim 1 , wherein said continuous culturing is effected for 2-3 weeks. 
     
     
         4 . The method of  claim 1 , wherein said growth factors comprise epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). 
     
     
         5 . The method of  claim 1  wherein said pluripotent stem cells comprise human embryonic stem (ES) cells. 
     
     
         6 . The method of  claim 5 , wherein said ES cells are prepared according to a method comprising:
 obtaining an in vitro fertilised human embryo and growing the embryo to a blastocyst stage of development;   removing inner cells mass (ICM) cells from the embryo;   culturing ICM cells under conditions which do not induce extraembryonic differentiation and cell death, and promote proliferation of undifferentiated stem cells; and   recovering stem cells.   
     
     
         7 . The method of  claim 6 , wherein the method for preparing ES cells is further characterized by:
 culturing the ICM cells on a fibroblast feeder layer to promote proliferation of embryonic stem cells prior to recovering the stem cells from the feeder layer, wherein the fibroblast feeder cells are arrested in their growth;   replating the stem cells from the fibroblast feeder layer onto another fibroblast feeder layer; and   culturing the stem cells for a period sufficient to promote proliferation of morphologically undifferentiated stem cells.   
     
     
         8 . The method of  claim 1 , wherein said culturing undifferentiated pluripotent stem cells does not generate embryoid bodies. 
     
     
         9 . The method of  claim 1 , wherein said culturing said differentiated cells is effected as monolayers or spheres.