Low oxygen culturing of central nervous system progenitor cells

Abstract

The present invention relates to the growth of cells in culture under conditions that promote cell survival, proliferation, and/or cellular differentiation. The present inventors have found that proliferation was promoted and apoptosis reduced when cells were grown in lowered oxygen as compared to environmental oxygen conditions traditionally employed in cell culture techniques. Further, the inventors found that differentiation of precursor cells to specific fates also was enhanced in lowered oxygen where a much greater number and fraction of dopaminergic neurons were obtained when mesencephalic precursors were expanded and differentiated in lowered oxygen conditions. Thus at more physiological oxygen levels the proliferation and differentiation of CNS precursors is enhanced, and lowered oxygen is a useful adjunct for ex vivo generation of specific neuron types. Methods and compositions exploiting these findings are described.

1 . A method of increasing cell differentiation comprising culturing undifferentiated central nervous system (CNS) cells in low ambient oxygen conditions, wherein said low ambient oxygen conditions promotes the cellular differentiation of said neuronal cells.  
     
     
         2 . The method of  claim 1 , wherein said low ambient oxygen conditions comprise an ambient oxygen condition of between about 0.25% to about 18% oxygen.  
     
     
         3 . The method of  claim 1 , wherein said low ambient oxygen conditions comprise an ambient oxygen condition of between about 0.5% to about 15% oxygen.  
     
     
         4 . The method of  claim 1 , wherein said low ambient oxygen conditions comprise an ambient oxygen condition of between about 1% to about 10% oxygen.  
     
     
         5 . The method of  claim 1 , wherein said low ambient oxygen conditions comprise an ambient oxygen condition of between about 1.5% to about 6% oxygen.  
     
     
         6 . The method of  claim 1 , wherein said low ambient oxygen conditions mimic physiological oxygen conditions for CNS cells.  
     
     
         7 . The method of  claim 1 , wherein said cells are primary tissue culture cells.  
     
     
         8 . The method of  claim 1 , wherein said cells are derived from a cell line.  
     
     
         9 . The method of  claim 1 , wherein said cells are selected from the group consisting of a central nervous system stem cells, spinal cord-derived progenitor cells, glial cells, astrocytes, neuronal stem cells, central nervous system neural crest-derived cells, neuronal precursor cells, neuronal cells, hepatocytes, and bone marrow derived cells.  
     
     
         10 . The method of  claim 9 , wherein said cells are fetal cells.  
     
     
         11 . The method of  claim 9 , wherein said cells are adult cells.  
     
     
         12 . The method of  claim 1 , wherein said cells are selected from the group consisting of mecencephalic progenitor cells, lateral ganglion precursor cells, cortical precursor cells, astrocytes and neuroblasts.  
     
     
         13 . The method of  claim 1 , wherein said differentiation is determined by monitoring a differentiation specific phenotype in said cells.  
     
     
         14 . The method of  claim 13 , wherein said differentiation specific phenotype determined by monitoring message level, protein level, subcellular localization, functional assays or morphological changes.  
     
     
         15 . The method of  claim 14 , wherein said message level is monitored using PCR™, in situ hybridization, RNAse protection assay, or single cell PCR™.  
     
     
         16 . The method of  claim 14 , wherein said protein level is monitored using antibody staining, HPLC, western blotting or immunoprecipitation.  
     
     
         17 . The method of  claim 14  wherein said message level monitored is the message for nestin, tyrosine hydroxylase, GAPDH; BDNF; GDNF; FGFR3; En1; FGF8; SHH; Ptx3; Nurr1; VEGF; EPO; HIF1α or VHL.  
     
     
         18 . The method of  claim 15  wherein said protein level monitored is the level of nestin, tyrosine hydroxylase, dopamine β-hydroxylase or dopamine transporter.  
     
     
         19 . The method of  claim 14 , wherein said functional assay monitors the rate of dopamine production.  
     
     
         20 . The method of  claim 1 , wherein said low oxygen conditions produce a cell population that is enriched in dopaminergic neurons as compared to a similar cell population that is grown in 20% oxygen incubator conditions.  
     
     
         21 . The method of  claim 1 , wherein said low oxygen conditions produce a cell population that is enriched in serotoninergic neurons as compared to a similar cell population that is grown in 20% oxygen incubator conditions.  
     
     
         22 . The method of  claim 1 , wherein said low oxygen conditions produce a cell population that is depleted in GABAnergic neurons as compared to a similar cell population that is grown in 20% oxygen incubator conditions.  
     
     
         23 . The method of  claim 1 , wherein said low oxygen conditions produce a cell population that is depleted in glutaminergic neurons as compared to a similar cell population that is grown in 20% oxygen incubator conditions.  
     
     
         24 . The method of  claim 1 , further comprising growing said cells in the presence of a neuronal growth stimulant, mitogen, cytokine, neuroprotective factor or an anti-apoptotic agent.  
     
     
         25 . The method of  claim 1 , wherein said differentiated phenotype is retained after transfer of said cells from said low ambient oxygen conditions to 20% oxygen culture conditions.  
     
     
         26 . The method of  claim 25 , wherein said cells are grown in low ambient oxygen conditions for multiple generations prior to transfer to 20% oxygen culture conditions.  
     
     
         27 . The method of  claim 1 , wherein said cells are continuously maintained in low ambient oxygen conditions.  
     
     
         28 . A method of inhibiting apoptosis of a CNS cell in culture comprising growing said cell in low ambient oxygen conditions.  
     
     
         29 . The method of  claim 28 , wherein said low ambient oxygen conditions comprise an ambient oxygen condition of between about 0.25% to about 18% oxygen.  
     
     
         30 . A method of increasing the expansion of a CNS cell in culture comprising growing said cell in low ambient oxygen, wherein said cell exhibit increased expansion in said low ambient oxygen as compared to growing said cell in 20% oxygen incubator conditions.  
     
     
         31 . The method of  claim 30 , wherein said low ambient oxygen conditions comprise an ambient oxygen condition of between about 0.25% to about 18% oxygen.  
     
     
         32 . The method of  claim 30 , wherein said low ambient oxygen conditions mimic physiological oxygen conditions for CNS cells.  
     
     
         33 . The method of  claim 30 , wherein said cell is a primary tissue culture cell.  
     
     
         34 . The method of  claim 30 , wherein said cell is derived from a cell line.  
     
     
         35 . The method of  claim 30 , wherein said cell is a fetal cell.  
     
     
         36 . The method of  claim 30 , wherein said cell is an adult cell.  
     
     
         37 . A method of increasing cell proliferation in culture comprising growing CNS cells in low ambient oxygen, wherein said growth in low ambient oxygen increases cell proliferation compared to growing said cells in 20% oxygen incubator conditions.  
     
     
         38 . A method of preparing a cell for use against a neurodegenerative disorder comprising 
 a) obtaining a population of CNS cells and    b) growing said cells in low ambient oxygen conditions    wherein said low ambient oxygen conditions increases the expression of a gene involved in said neurodegenerative disease.    
     
     
         39 . The method of  claim 38 , wherein said neurodegenerative disease is Parkinson's Disease.  
     
     
         40 . The method of  claim 38  wherein said gene is tyrosine hydroxylase (TH).  
     
     
         41 . The method of  claim 38 , wherein said cell is a primary cell.  
     
     
         42 . The method of  claim 38 , wherein said cell is derived from a cell line.  
     
     
         43 . The method of  claim 38 , further comprising contacting said cell with a first polynucleotide encoding a dopamine biosynthetic protein under conditions suitable for the expression of said protein wherein said polynucleotide is under the transcriptional control of a promoter active in said cells.  
     
     
         44 . The method of  claim 38 , further comprising contacting said cell with a first polynucleotide encoding a dopamine releasing protein under conditions suitable for the expression of said protein wherein said polynucleotide is under the transcriptional control of a promoter active in said cells.  
     
     
         45 . The method of  claim 43 , further comprising contacting said cell with a second polynucleotide encoding a dopamine releasing protein under conditions suitable for the expression of said protein wherein said polynucleotide is under the transcriptional control of a promoter active in said cells.  
     
     
         46 . The method of  claim 44 , further comprising contacting said cell with a second polynucleotide encoding a dopamine biosynthetic protein under conditions suitable for the expression of said protein wherein said polynucleotide is under the transcriptional control of a promoter active in said cells.  
     
     
         47 . The method of  claim 43 , wherein said dopamine biosynthesis protein is selected from the group consisting of TH; L-amino acid decarboxylase (AADC) and erythropoietin.  
     
     
         48 . The method of  claim 44 , wherein said dopamine releasing protein is a vesicular monoamine transporter (VMAT).  
     
     
         49 . The method of  claim 45 , wherein said first and second polynucleotides are under control of different promoters.  
     
     
         50 . The method of  claim 46 , wherein said first and second polynucleotides are under control of different promoters.  
     
     
         51 . The method of  claim 43 , wherein the promoter is selected from the group consisting of CMV IE, SV40 E, β-actin, TH promoter, AADC promoter, and nestin promoter.  
     
     
         52 . The method of  claim 45 , wherein said first and second polynucleotides each are covalently linked to a polyadenylation signal.  
     
     
         53 . The method of  claim 46 , wherein said first and second polynucleotides each are covalently linked to a polyadenylation signal.  
     
     
         54 . A cell produced according to the method comprising obtaining a starting CNS cell and growing said cell in low ambient oxygen conditions wherein said conditions produce a differentiated neuronal cell.  
     
     
         55 . The cell of  claim 54 , wherein said starting cell is a nestin-positive cell.  
     
     
         56 . The cell of  claim 54 , wherein said low ambient conditions produce a nestin-negative cell.  
     
     
         57 . The cell of  claim 54 , wherein said low ambient conditions produce a TH positive cell.  
     
     
         58 . The cell of  claim 54 , wherein said cell further comprises an expression vector comprising a polynucleotide encoding an exogenous gene wherein said polynucleotide is operatively linked to a promoter.  
     
     
         59 . A method of treating Parkinson's disease in a subject comprising: 
 a) obtaining cells suitable for transplanting to said subject;    b) growing said cells in low ambient oxygen conditions; and    c) implanting said cells grown from step (b) into said subject    wherein said implanted cells have an increased capacity to produce dopamine in said subject as compared to similar cells grown in 20% oxygen incubator conditions.    
     
     
         60 . The method of  claim 59 , wherein said cells are from said subject and have been transduced with a polynucleotide that expresses a protein that increases dopamine production.  
     
     
         61 . The method of  claim 59 , wherein said cells are CNS cells from a source other than said subject.  
     
     
         62 . The method of  claim 59 , wherein said cells are transduced with a polynucleotide that expresses a protein that increases dopamine production.