Lineage-Restricted Neuronal Precursors

Abstract

A self-renewing restricted stem cell population has been identified in developing (embryonic day 13.5) spinal cords that can differentiate into multiple neuronal phenotypes, but cannot differentiate into glial phenotypes. This neuronal-restricted precursor (NRP) 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. NRP cells self renew over multiple passages in the presence of fibroblast growth factor (FGF) and neurotrophin 3 (NT-3) and express a characteristic subset of neuronal epitopes. When cultured in the presence of RA and the absence of FGF, NRP cells differentiate into GABAergic, glutaminergic, and cholinergic immunoreactive neurons. NRP cells can also be generated from multipotent NEP cells cultured from embryonic day 10.5 neural tubes. Clonal analysis shows that E-NCAM immunoreactive NRP cells arise from an NEP progenitor cell that generates other restricted CNS precursors. The NEP-derived E-NCAM immunoreactive cells undergo self renewal in defined medium and differentiate into multiple neuronal phenotypes in mass and clonal culture. Thus, a direct lineal relationship exists between multipotential NEP cells and more restricted neuronal precursor cells present in vivo at embryonic day 13.5 in the spinal cord. Methods for treating neurological diseases are also disclosed.

1 . A method for screening for potentially neurologically therapeutic compounds comprising exposing mammalian neuronal restricted precursor cells or derivatives thereof or mixtures thereof cultured in vitro to the compound and monitoring a response of said cells. 
     
     
         2 . The method of  claim 1  wherein said mammalian neuronal restricted precursor cells are exposed to a compound at varying dosages. 
     
     
         3 . The method of  claim 1  wherein said mammalian neuronal restricted precursor cells are exposed to a compound for various time periods. 
     
     
         4 . The method of  claim 1  wherein said mammalian neuronal restricted precursor cells are exposed to a compound at varying dosages for various time periods. 
     
     
         5 . The method of  claim 1  wherein the monitored response of said cells is a change in level of expression of an enzyme, receptor, cell surface molecule, neurotransmitter, amino acid, neuropeptide, or biogenic amine upon exposure to the compound. 
     
     
         6 . The method of  claim 5  wherein the change in level of expression is an increase. 
     
     
         7 . The method of  claim 5  wherein the change in level of expression is a decrease. 
     
     
         8 . The method of  claim 1  wherein the monitored response of said cells is promoting division of said cells measured by an increase in cell number. 
     
     
         9 . The method of  claim 1  wherein the monitored response of said cells is promoting DNA synthesis in said cells. 
     
     
         10 . The method of  claim 1  further comprising applying an agent to said cells in conditions where said cells are expected to die and monitoring cell survival in the presence of the compound. 
     
     
         11 . A method for screening for a compound that inhibits binding to a selected receptor on a neuronal restricted precursor cell, 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. 
     
     
         12 . A method for screening for compounds which activate a selected receptor on a neuronal restricted precursor cell, said method comprising contacting neuronal restricted precursors with a compound and measuring a physiological alteration in said cells associated with activation of said receptor.