Dopaminergic neurons differentiated from pluripotent stem cells and uses of thereof

Abstract

The present invention provides populations of neural cells derived from pluripotent cells, and methods for making and using the same. Disclosed herein are methods for generating dopaminergic neurons in vitro using a combination of agents that cause differentiation of the pluripotent cells into dopaminergic neurons. Also disclosed are methods for treating a neurodegenerative disease in a patient by generating dopaminergic neurons in vitro, and transplanting them into the brain of the patient, such that the dopaminergic neurons are sufficient to reduce or eliminate the symptoms of the neurodegenerative disease.

1 . A population of cells derived from pluripotent stem cells contacted with an effective amount of retinoic acid, human sonic hedgehog (SHH) protein, and FGF8A protein. 
     
     
         2 . The population of cells of  claim 1 , wherein the pluripotent stem cells are further contacted with an effective amount of WNT1 protein. 
     
     
         3 . The population of cells of  claim 1 , wherein the cells have a neuronal morphology in culture. 
     
     
         4 . The population of cells of  claim 1 , wherein the pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells. 
     
     
         5 . The population of cells of  claim 1 , wherein the cells express FOXA2 + . 
     
     
         6 . The population of cells of  claim 1 , wherein the cells express β-tubulin + . 
     
     
         7 . The population of cells of  claim 1 , wherein the cells express tyrosine hydroxylase. 
     
     
         8 . The population of cells of  claim 1  wherein the cells are FOXA2 + /β-tubulin + /TH + . 
     
     
         9 . The population of cells of  claim 1 , wherein said population is enriched using flow cytometry. 
     
     
         10 . The population of  claim 9 , wherein said populations comprises at least 50% FOXA2 + /β-tubulin + /TH +  cells. 
     
     
         11 . The population of  claim 1 , wherein said pluripotent stem cells are not co-cultured with feeder cells. 
     
     
         12 . A therapeutic composition comprising the population of cells of  claim 1 , wherein the cell population is in an amount sufficient to treat a disease or condition. 
     
     
         13 . The therapeutic composition of  claim 12 , wherein the population of cells is suspended in a physiologically acceptable carrier. 
     
     
         14 . The therapeutic composition of  claim 13 , wherein the physiologically acceptable carrier is artificial cerebrospinal fluid. 
     
     
         15 . The therapeutic composition of  claim 12 , wherein the population of cells is encapsulated. 
     
     
         16 . The therapeutic composition of  claim 15 , wherein the population of cells is contained within an inert biomatrix. 
     
     
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         21 . A method for generating SN-A9 dopaminergic neurons, the method comprising:
 (a) contacting pluripotent stem cells with an effective amount of retinoic acid;   (b) contacting the pluripotent stem cells with an effective amount of human sonic hedgehog (SHH) protein, and FGF8A protein, and   (c) culturing said pluripotent stem cells under conditions sufficient to cause differentiation of said pluripotent stem cells into SN-A9 dopaminergic neurons.   
     
     
         22 . The method of  claim 21 , wherein said pluripotent stem cells are further contacted with a WNT1 protein. 
     
     
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         41 . An isolated population of cells, wherein at least 1% of the cells of said population expresses the FOXA2 + /β-tubulin + /TH +  phenotype. 
     
     
         42 . The population of cells of  claim 41 , where said population comprises cells derived from pluripotent stem cells.