Methods and compositions for expanding, identifying, characterizing and enhancing potency of mammalian-derived glial restricted progenitor cells

Abstract

Methods for producing a population of human-derived glial restricted progenitor cells (GRPs) with decreased potentially unintended or undesired cellular phenotypes and/or decreased standard deviation in the cells of the population are provided. Also provided are antibody panels and gene expression profiles to characterize GRPs and a method for its use in characterizing GRP cells. In addition methods for use of these GRP cells to generate astrocytes and/or oligodendrocytes, to re-myelinate neurons and to treat glial cell related and other neurodegenerative diseases or disorders or injuries or damage to the nervous system are provided. A method to manufacture neural cells depleted of A2B5 positive cells is also provided.

What is claimed is: 
     
         1 . A method for manufacturing mammalian glial restricted progenitor (GRP) cells, said method comprising:
 (a) in vitro sorting A2B5 reactive cells from a mammalian tissue source capable of generating A2B5 positive cells;   (b) culturing the A2B5 positive cells for at least 10 days in vitro (DIV) on a substrate; and   (c) harvesting the cultured cells.   
     
     
         2 . The method of  claim 1  wherein A2B5 reactive cells are isolated using magnetic activated cell sorting or using fluorescence activated cell sorting or immunopanning. 
     
     
         3 . The method of  claim 2  wherein cells are passed two or more times over a column comprising magnetic beads to isolate A2B5 antibody-reactive cells or a fluorescence activated cell sorting or immunopanning dish. 
     
     
         4 . The method of  claim 1  wherein the cells are cultured for 10-20-DIV. 
     
     
         5 . The method of  claim 1  wherein the cells are cultured for 15-20 DIV. 
     
     
         6 . The method of  claim 1  wherein the cells are cultured for at least 20 DIV. 
     
     
         7 . The method of  claim 1  wherein the cells are cultured for 100 DIV or greater. 
     
     
         8 . A method for manufacturing mammalian glial restricted progenitor (GRP) cells according to  claim 1 , said method comprising:
 (a) dissociating mammalian neural tissue into a cell suspension;   (b) in vitro sorting A2B5 reactive cells from the cell suspension;   (c) culturing the A2B5-positive cells for at least 10 days in vitro (DIV) on a substrate; and   (d) harvesting the cultured cells.   
     
     
         9 . The method of  claim 8  wherein the mammalian neural tissue was obtained from a mammal after neural tube closure. 
     
     
         10 . The method of  claim 9  wherein the mammalian neural tissue is fetal cadaver forebrain tissue, fetal cadaver spinal cord tissue or mammalian biopsy brain or spinal cord tissue. 
     
     
         11 . The method of  claim 8  wherein dissociating neural tissue is performed enzymatically, mechanically or enzymatically and mechanically. 
     
     
         12 . A method for decreasing potentially unintended cellular phenotypes selected from neuronal progenitors, neurons, microglia and endothelial cells in a GRP cell population and/or decreasing standard deviations in cells of the GRP cell population at greater than 6 days in vitro (DIV) as compared to cells harvested at 6 days in vitro (DIV), said method comprising:
 (a) in vitro sorting A2B5 reactive cells from a mammalian tissue source capable of generating A2B5 positive cells; and   (b) culturing the A2B5 positive cells for at least 10 10 days in vitro (DIV) on a substrate.   
     
     
         13 . The method according to  claim 12 , said method comprising:
 (a) dissociating mammalian neural tissue into a cell suspension;   (b) in vitro sorting A2B5 reactive cells from the cell suspension;   (c) culturing the A2B5 positive cells for at least 10 days in vitro (DIV) on a substrate; and   (d) harvesting the cultured cells.   
     
     
         14 . A method for generating astrocyte precursor cells, astrocytes, oligodendrocyte precursor cells and/or oligodendrocytes comprising manufacturing mammalian glial restricted progenitor (GRP) cells, comprising the method steps:
 (a) in vitro sorting A2B5 reactive cells from a mammalian tissue source capable of generating A2B5 positive cells;   (b) culturing the A2B5 positive cells for at least 10 days in vitro (DIV) on a substrate; and   (c) harvesting the cultured cells, and   subsequently culturing the GRP cells under conditions which promote differentiation to astrocyte precursor cells, astrocytes, oligodendrocyte precursor cells and/or oligodendroctes.