Production of tyrosine hydroxylase positive neurons

Abstract

The present invention relates to a method of producing neurons that express the enzyme tyrosine hydroxylase (TH) by subjecting neural stem cells to FGF-1, a protein kinase A activator, a protein kinase C activator, and dopamine/L-DOPA. Surprisingly, when forskolin is used as a protein kinase A activator, it requires only low levels of FGF-1 and forskolin to efficiently produce TH positive neurons from fetal or adult neural stem cells. Also provided are compositions used to produce TH positive neurons and the resulting neural cell culture, as well as a method of treating disease or conditions which are associated with dopamine neuron loss or dysfunction.

1 . A method for producing tyrosine hydroxylase (TH) positive neurons from neural stem cells, comprising: 
 (a) providing at least one mammalian non-embryonic neural stem cell;    (b) contacting the neural stem cell with an effective amount of fibroblast growth factor 1 (FGF-1), a protein kinase A activator, a protein kinase C activator, and dopamine/L-DOPA; and    (c) allowing the neural stem cell to differentiate into TH positive neurons.    
     
     
         2 . The method of  claim 1  wherein the protein kinase A activator is selected from the group consisting of isobutylmethylxanthine (IBMX), pituitary adenylate cyclase activating polypeptide (PACAP), forskolin, and any combination thereof.  
     
     
         3 . The method of  claim 1  wherein the protein kinase C activator is a phorbol ester.  
     
     
         4 . The method of  claim 3  wherein the phorbol ester is 4-β-12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12-myristate 13-acetate (PMA).  
     
     
         5 . The method of  claim 1  wherein the neural stem cell is provided as a culture derived from the subventrical zone of an adult brain, and step (b) comprises contacting the culture with a TH culture medium comprising FGF-1, a protein kinase A activator, a protein kinase C activator, and dopamine/L-DOPA.  
     
     
         6 . The method of  claim 1  wherein the neural stem cell is located in the brain of a mammal.  
     
     
         7 . The method of  claim 1  further comprising contacting the neural stem cell with epidermal growth factor (EGF) and/or brain-derived neurotrophic factor (BDNF).  
     
     
         8 . A method for producing tyrosine hydroxylase (TH) positive neurons from neural stem cells in vitro, comprising: 
 (a) providing a culture of neural stem cells;    (b) incubating the neural stem cells in a TH culture medium comprising an effective concentration of fibroblast growth factor 1 (FGF-1), a protein kinase A activator, a protein kinase C activator and dopamine/L-DOPA, with the proviso that if the neural stem cells are embryonic neural stem cells and the protein kinase A activator is a combination of isobutylmethylxanthine (IBMX) and forskolin, the concentration of forskolin is less than about 3 μM; and    (c) allowing the neural stem cells to produce TH positive neurons.    
     
     
         9 . The method of  claim 8  wherein the protein kinase A activator is a combination of IBMX and pituitary adenylate cyclase activating polypeptide (PACAP).  
     
     
         10 . The method of  claim 8  wherein the protein kinase A activator is a combination of IBMX and forskolin, wherein the concentration of forskolin is about 1 μM.  
     
     
         11 . The method of  claim 10  wherein the concentration of IBMX is about 40 μM.  
     
     
         12 . The method of  claim 8  wherein the protein kinase C activator is a phorbol ester.  
     
     
         13 . The method of  claim 8  wherein the concentration of FGF-1 is less than about 1 ng/ml.  
     
     
         14 . The method of  claim 8  wherein the concentration of FGF-1 is about 0.2 ng/ml.  
     
     
         15 . The method of  claim 8  wherein the TH culture medium further comprises epidermal growth factor (EGF) and/or brain derived neurotrophic factor (BDNF).  
     
     
         16 . The method of  claim 8  wherein at least half of the TH culture medium in the culture of step (b) is replaced with an equal volume of fresh TH culture medium daily.  
     
     
         17 - 20 . (canceled)  
     
     
         21 . A culture of neural cells prepared by the method according to  claim 8 .  
     
     
         22 . A culture of neural cells prepared by the method according to  claim 5 .  
     
     
         23 . The culture of  claim 22  wherein the culture comprises neurons and at least about 25% of the neurons are TH positive.  
     
     
         24 - 39 . (canceled)