Adult Rodent Spinal Cord-Derived Neural Stem Cells: Isolation and Characterization

Self-renewing multipotent stem/progenitor cells have been isolated from various areas of the adult central nervous system, both neurogenic and non-neurogenic, in vitro by using a variety of mitogens and culture conditions. Mitogenic growth factors stimulate the proliferation of multipotent stem/progenitor cells from embryonic and adult rodent, primates, and human. Neural stem cells were isolated from the adult rat spinal cord as follows: Adult rat spinal cords were dissected, and tissue was mechanically and enzymatically dissociated. The cell suspension was filtered and further purified from debris and enriched for progenitors by using a Percoll density gradient. The dissociated cells were grown on polyornithine- and laminin-coated plates in N2 medium containing fibroblast growth factor-2. The cells grew as attached layers. Cultures were passaged by trypsinization, and rapidly proliferating cells that could be passaged, frozen, and thawed were isolated. Clonal cultures derived from single founder cells identified by marker genes generate neurons, astrocytes, and oligodendrocytes, confirming the multipotent nature of the parent cell. Subcloning analysis showed that after serial passaging, recloning, and expansion, these cells retained multipotentiality, indicating that they are self-renewing.