Lineage-Restricted Neuronal Precursors

Abstract

A cell population has been identified and isolated that can differentiate into multiple neuronal phenotypes, but cannot differentiate into glial phenotypes. This mammalian CNS neuron-restricted cell expresses highly polysialated or embryonic neural cell adhesion molecule (E-NCAM) and is morphologically distinct from neuroepithelial stem cells (NEP cells) and spinal glial progenitors derived from embryonic day 10.5 spinal cord. Methods for isolating these cells and uses thereof are also disclosed.

1 . An isolated cellular composition comprising mammalian CNS neuron-restricted cells or derivatives or mixtures thereof. 
     
     
         2 . A pharmaceutical composition comprising a therapeutically effective amount of the composition of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         3 . A method for treating a neuronal disorder in a mammal comprising administering to said mammal a therapeutically effective amount of the composition of  claim 1 . 
     
     
         4 . A method for treating a neuronal disorder in a mammal comprising administering to said mammal a therapeutically effective amount of the pharmaceutical composition of  claim 2 . 
     
     
         5 . A method for treating neurodegenerative symptoms in a mammal comprising the steps of:
 (a) genetically transforming mammalian CNS neuron-restricted cells of  claim 1  with a gene encoding a growth factor, neurotransmitter, neurotransmitter synthesizing enzyme, neuropeptide, neuropeptide synthesizing enzyme, or substance that provides protection against free-radical mediated damage thereby resulting in a transformed population of neuronal restricted precursor cells that express said growth factor, neurotransmitter, neurotransmitter synthesizing enzyme, neuropeptide, neuropeptide synthesizing enzyme, or substance that provides protection against free-radical mediated damage; and   (b) administering an effective amount of said genetically transformed cells to said mammal.   
     
     
         6 . A method of isolating the mammalian CNS neuron-restricted cells of  claim 1  comprising the steps of:
 (a) plating mammalian embryonic stem cells in neural differentiation conditions so that the ES cells alter their morphology and express neuronal and glial markers nestin, NCAM, MAP2 kinase, GFAP and cyclophilin/DM20/PLA; 
 (b) removing A2B5+ cells from the differentiated cells of step (a) via specific antibody capture with an antibody that specifically recognizes A2B5; 
 (c) purifying from supernatant from step (b) a subpopulation expressing embryonic neural cell adhesion molecule via a procedure selected from the group consisting of specific antibody capture, fluorescence activated cell sorting, and magnetic bead capture, using an embryonic cell adhesion molecule antibody that specifically recognizes polysialated neural cell adhesion molecule; 
 (d) plating the purified subpopulation of cells in feeder-cell-independent culture on a substratum and in a FGF-containing medium; and 
 (e) incubating the plated cells in the FGF-containing medium to obtain an isolated, pure population of mammalian CNS neuron-restricted precursor cells. 
 
     
     
         7 . A method of obtaining postmitotic neurons comprising:
 (a) culturing mammalian CNS neuron-restricted cells of  claim 1  in proliferating conditions; and   (b) changing the culture conditions of the neuron-restricted precursor cells from proliferating conditions to differentiating conditions, thereby causing the neuron-restricted precursor cells to differentiate into postmitotic neurons.   
     
     
         8 . A method for screening for potentially neurologically therapeutic compounds comprising exposing mammalian neuronal restricted precursor cells of  claim 1  or derivatives thereof or mixtures thereof cultured in vitro to the compound and monitoring a response of said cells. 
     
     
         9 . A method for screening for a compound that inhibits binding to a selected receptor on a neuronal restricted precursor cell of  claim 1 , said method comprising contacting neuronal restricted precursor cells with a compound and determining the ability of the compound to block a response elicited by binding of an agonist to the selected receptor on the neuronal restricted precursor cells. 
     
     
         10 . A method for screening for compounds which activate a selected receptor on a neuronal restricted precursor cell of  claim 1 , said method comprising contacting neuronal restricted precursors with a compound and measuring a physiological alteration in said cells associated with activation of said receptor.