Neural Stem Cell Research
We study the properties of multipotential neural stem cells (NSC) as well as neuronal restricted precursors (NRP) and glial restricted precursors (GRP), and we test their therapeutic potential after grafting into injured adult spinal cords (Han and Fischer, 2001). NSCs have the potential to differentiate into neuronal and glial cells, and are therefore candidates for cell replacement after CNS injury. Their phenotypic fate in vivo is dependent on the engraftment site, suggesting that the environment exerts differential effects on neuronal and glial lineages.
We and others have shown that upon grafting into the adult spinal cord, NSCs remain undifferentiated or show a fate restricted to a glial lineage, indicating that the non-neurogenic adult spinal cord environment is not permissive for neuronal differentiation. We focused our experiments on the in vitro and in vivo characterization of lineage-restricted neural precursors because of the potential for controlling their fate in the adult non-neurogenic environment. These studies have been greatly facilitated by the use of transgenic rats that express the human alkaline phosphatase gene, allowing for effective labeling of the grafted cells and reliable identification of their phenotype (Mujtaba et. al., 2002).
Our transplantation experiments address fundamental issues of potential and fate with respect to neural stem cells and lineage-restricted progenitors, as well as development of therapeutic strategies for repair of spinal cord injury. Our work with neural stem cells is focused on strategies that allow the grafted cells to differentiate into neurons and contribute to regeneration and possibly cell replacement.
The failure to obtain neuronal phenotypes after grafting NSCs into the adult spinal cord is a serious obstavle to testing therapeutic strategies directed at neuronal cell replacement. Our hypothesis has been that the adult spinal cord environment will support differentiation of lineage-restricted precursors because their intrinsic program no longer needs to initiate the commitment signals.
Our studies demonstrate that lineage-restricted precursor cells are a promising alternative to multipotent stem cells. We hypothesize that multipotent neural stem cells do not differentiate into neurons following transplantation because the stem cells fail to progress into lineage-restricted prescursors.
Neurospheres grown in EGF and bFGF and stained for nestin (B)
Fig. 1. E14 spinal cord cells. (A) Typical neurosphere generated from a single cell in medium containing EGF and bFGF for 10 days, under Nomarski optics. (B) Fluorescent image of a neurosphere expanded for 5 days showing that cells were immunopositive for nestin, an intermediated filament characteristic of undifferentiated cells, throughout the neurosphere. Scale bars: 25μm.
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