Neural progenitor cell populations

Abstract

This invention provides populations of neural progenitor cells, differentiated neurons, glial cells, and astrocytes. The populations are obtained by culturing stem cell populations (such as embryonic stem cells) in a cocktail of growth conditions that initiates differentiation, and establishes the neural progenitor population. The progenitors can be further differentiated in culture into a variety of different neural phenotypes, including dopaminergic neurons. The differentiated cell populations or the neural progenitors can be generated in large quantities for use in drug screening and the treatment of neurological disorders.

What is claimed as the invention is:  
     
         1 . A cell population that proliferates in an in vitro culture, obtained by differentiating primate pluripotent stem (pPS) cells, wherein at least ˜30% of the cells in the population are committed to form neuronal cells, glial cells, or both.  
     
     
         2 . A cell population that proliferates in an in vitro culture, obtained by differentiating primate pluripotent stem (pPS) cells, comprising at least ˜60% neural progenitor cells, wherein at least 10% of the cells can differentiate into neuronal cells, and at least 10% of the cells can differentiate into glial cells.  
     
     
         3 . A cell population that proliferates in an in vitro culture, obtained by differentiating primate pluripotent stem (pPS) cells, comprising at least ˜60% neural progenitor cells, wherein at least 10% of the cells express A2B5, and at least 10% of the cells express NCAM.  
     
     
         4 . The cell population of  claim 1 , wherein the pPS cells are human embryonic stem (hES) cells.  
     
     
         5 . The cell population of  claim 1 , obtained by differentiating pPS cells in a medium containing at least two ligands that bind growth factor receptors, selected from the group consisting of EGF, bFGF, PDGF, IGF-1, and antibodies to receptors for these ligands.  
     
     
         6 . The cell population of  claim 1 , obtained by differentiating pPS cells in a medium containing growth factors, sorting the differentiated cells for expression of NCAM or A2B5, and then collecting the sorted cells.  
     
     
         7 . The cell population of  claim 1 , which can be induced to produce a population of cells of which at least 30% of the cells have morphological features of mature neurons and are NCAM positive.  
     
     
         8 . The cell population of  claim 7 , wherein the cells having morphological features of mature neurons have at least three of the following characteristics: 
 a) at least 60% of the cells show calcium flux when administered acetylcholine;    b) at least 60% of the cells show calcium flux when administered GABA;    c) at least 10% of the cells show calcium flux when administered norepinephrine;    d) at least 60% of the cells show calcium flux when subjected to an external potassium concentration of 50 mM; or    e) at least 25% of the cells demonstrate action potentials when subject to stimulation in a whole-cell patch clamp apparatus.    
     
     
         9 . The cell population of  claim 1 , which can be induced to produce a population of cells in which at least 1% of the cells stain positively for tyrosine hydroxylase.  
     
     
         10 . The cell population of  claim 1 , comprising cells genetically altered to express telomerase reverse transcriptase.  
     
     
         11 . A cell population comprising mature neurons, astrocytes, oligodendrocytes, or any combination thereof, obtained by further differentiating the cell population according to  claim 1 .  
     
     
         12 . The cell population of  claim 11 , comprising a subpopulation of at least 30% of the cells that have the morphological characteristics of neurons and are NCAM positive, wherein the subpopulation has the following properties: 
 a) at least 60% show calcium flux when administered acetylcholine;    b) at least 60% show calcium flux when administered GABA;    c) at least 10% show calcium flux when administered norepinephrine;    d) at least 60% show calcium flux when subjected to an external potassium concentration of 50 mM; or    e) at least 25% demonstrate action potentials when subject to stimulation in a whole-cell patch clamp apparatus.    
     
     
         13 . The cell population of  claim 11 , in which at least 1% of the cells stain positively for tyrosine hydroxylase.  
     
     
         14 . The cell population of  claim 11 , obtained by culturing the cell population of  claim 1  in a medium containing an activator of cAMP, a neurotrophic factor, or a combination thereof.  
     
     
         15 . An isolated neural precursor cell, obtained by providing the cell population of  claim 1 , and selecting therefrom a cell having characteristics of a neural precursor cell.  
     
     
         16 . An isolated mature neuron, astrocyte, or oligodendrocyte, obtained by providing the cell population of  claim 11 , and selecting therefrom a cell having characteristics of a neuron, astrocyte, or oligodendrocyte, respectively.  
     
     
         17 . The isolated mature neuron of  claim 16 , which is a dopaminergic neuron.  
     
     
         18 . The isolated mature neuron of  claim 16 , obtained by culturing the cell of  claim 1  in a medium containing an activator of cAMP, a neurotrophic factor (such as nerve growth factor, neurotrophin 3, or brain-derived neurotrophic factor), or a combination thereof.  
     
     
         19 . A cell population comprising at least ˜60% neural progenitor cells and/or mature neurons that have the same genome as an established human embryonic stem (hES) cell line.  
     
     
         20 . The cell population of  claim 18 , wherein the neural progenitor cells and/or mature neurons express NCAM, A2B5, MAP-2, or Nestin.  
     
     
         21 . A method for obtaining neural precursor cells capable of producing a cell population comprising at least 1% tyrosine hydroxylase positive cells, comprising differentiating human embryonic stem cells.  
     
     
         22 . The method of  claim 21 , wherein the differentiating comprises culturing in a medium containing at least two ligands that bind growth factor receptors, selected from the group consisting of EGF, bFGF, PDGF, IGF-1, and antibodies to receptors for these ligands.  
     
     
         23 . A method for obtaining a cell population comprising at least 1% tyrosine hydroxylase positive cells, comprising differentiating human embryonic stem cells.  
     
     
         24 . The method of  claim 21 , comprising obtaining neural precursor cells according to  claim 19 , and then culturing the cells obtained thereby in a medium containing an activator of cAMP, a neurotrophic factor (such as nerve growth factor, neurotrophin 3, or brain-derived neurotrophic factor), or a combination thereof.  
     
     
         25 . The method of  claim 21 , further comprising genetically altering the cells to express telomerase reverse transcriptase before or after differentiating.  
     
     
         26 . A method of screening a compound for neural cell toxicity or modulation, comprising combining the compound with a cell population according to claim  1 ; determining any phenotypic or metabolic changes in the cell that result from contact with the compound; and correlating the change with neural cell toxicity or modulation.  
     
     
         27 . A method of screening a compound for neural cell toxicity or modulation, comprising combining the compound with a cell population according to claim  11 ; determining any phenotypic or metabolic changes in the cell that result from contact with the compound; and correlating the change with neural cell toxicity or modulation.  
     
     
         28 . A method for obtaining a polynucleotide comprising a nucleotide sequence contained in an mRNA more highly expressed in neural progenitor cells, the method comprising: 
 a) determining the level of expression of a plurality of mRNAs in one or more cells in the cell population of  claim 1 , in comparison to the level of expression of the same mRNAs in more mature neural cells;    b) identifying an mRNA expressed at a higher level in the cell(s) from the cell population, relative to that in the more mature cells; and    c) preparing a polynucleotide comprising a nucleotide sequence of at least 30 consecutive nucleotides contained in the mRNA selected in step b).    
     
     
         29 . A method of reconstituting or supplementing central nervous system (CNS) function in an individual, comprising administering to the individual a cell population according to  claim 1 .  
     
     
         30 . A method of reconstituting or supplementing CNS function in an individual, comprising administering to the individual a cell population according to claim  11 .