Dopaminergic neurons and proliferation-competent precursor cells for treating Parkinson's disease

Abstract

This disclosure provides improved methods for obtaining populations of neural progenitor cells and differentiated neurons from pluripotent stem cells. The technology can be used to produce progenitors that proliferate through at least 40 doublings, while maintaining the ability to differentiate into a variety of different neural phenotypes. Cell populations have been obtained that contain a high proportion of cells staining for tyrosine hydroxylase, which is a feature of dopaminergic neurons. The neural progenitors and terminally differentiated neurons of this invention can be generated in large quantities for use in drug screening and the treatment of clinically important neurological disorders, such as Parkinson's disease.

1 . A system for generating human neuronal cells, comprising: 
 a first cell population comprising undifferentiated cells from a line of human embryonic stem (hES) cells; and    a differentiated cell population cultured in vitro, wherein at least ˜30% of MAP-2 positive cells have the characteristic that they are progeny of the hES cells, and express tyrosine hydroxylase.    
     
     
         2 . A system for generating human neuronal cells, comprising: 
 a first cell population comprising undifferentiated cells from a line of human embryonic stem (hES) cells; and    a differentiated cell population cultured in vitro, wherein at least ˜5% of all the cells in the population have the characteristic that they are progeny of the hES cells, and express tyrosine hydroxylase.    
     
     
         3 . A system for generating human neuronal cells, comprising: 
 a first cell population comprising undifferentiated cells from a line of human embryonic stem (hES) cells; and    a neuronal precursor cell population cultured in vitro, in which at least ˜60% of the cells are progeny of the hES cells; express A2B5, polysialylated NCAM, or Nestin; and which upon culturing for 7 days with added neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF), neurotrophin 4 (NT-4) and nerve growth factor (NGF), but no added mitogens, generates a cell population in which at least ˜30% of MAP-2 positive cells express tyrosine hydroxylase.    
     
     
         4 . A system for generating human neuronal cells, comprising: 
 a first cell population comprising undifferentiated cells from a line of human embryonic stem (hES) cells; and    a neuronal precursor cell population cultured in vitro, in which at least ˜60% of the cells are progeny of the hES cells; express A2B5, polysialylated NCAM, or Nestin; and which upon culturing for 7 days with added neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF), neurotrophin 4 (NT-4) and nerve growth factor (NGF), but no added mitogens, generates a cell population in which at least ˜5% of all the cells in the population express tyrosine hydroxylase    
     
     
         5 . The system of  claim 3 , wherein the neuronal precursor cell pupation is capable of at least 20 population doublings in culture, and which after 20 doublings maintains an ability to form differentiated cell populations according to  claim 1  or  2  upon culturing with NT-3, BDNF, NT-4 and NGF, but no added mitogens.  
     
     
         6 . The system of  claim 3 , wherein the neuronal precursor cell population provides clinical improvement in a nigrostriatal lesion animal model of Parkinson's disease.  
     
     
         7 . The system of  claim 3 , wherein at least ˜38% hES derived neural cells express β-tubulin III.  
     
     
         8 . The system of  claim 3 , produced by: 
 a) obtaining a line of hES cells;    b) culturing some of the hES cells in a medium containing one or more neurotrophins and one or more mitogens, and    c) harvesting from the culture a cell population in which at least ˜60% of the cells express A2B5, polysialylated NCAM, or Nestin; which is capable of at least 20 doublings in culture, and which after 20 doublings maintains an ability to be differentiated into a population comprising at least 20% MAP-2 positive cells.    
     
     
         9 . The system of  claim 8 , wherein the added mitogen(s) include a mitogen selected from epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), and erythropoietin (EPO).  
     
     
         10 . The system of  claim 8 , wherein the added neurotrophins include neurotrophin 3 (NT-3) or brain-derived neurotrophic factor (BDNF).  
     
     
         11 . The system of  claim 3 , produced by: 
 a) obtaining a line of hES cells;    a) culturing some of the hES cells in a medium containing one or more added TGF-β superfamily antagonists, and    b) harvesting from the culture a neural cell population in which at least 50% of the cells express either polysialylated NCAM or β-tubulin III.    
     
     
         12 . The system of  claim 11 , wherein the neural cell population was produced by culturing the hES cells in a medium containing both noggin and follistatin.  
     
     
         13 . The system of  claim 8 , wherein the neural cell population was produced by passaging the cells at least 6 times in a medium comprising an added neurotrophin and an added mitogen.  
     
     
         14 . The system of  claim 1 , produced by: 
 a) obtaining a line of hES cells;    b) differentiating some of the hES cells to produce a neuronal precursor cell population in which at least ˜60% of the cells express A2B5, polysialylated NCAM, or Nestin, and then    c) culturing the neuronal precursor cells in a medium containing one or more factors selected from neurotrophins, cAMP, and ascorbic acid in the absence of added mitogens, so as to generate a cell population in which at least ˜30% of MAP-2 positive cells express tyrosine hydroxylase