in serum-free liquid culture of epidermal growth factor (EGF) responsive. nestin-positive stem cells from adult mice has opened possibilities for fully exploring the nature of the neural stem cell regulatory system and the elucidation of the neuropoietic system. We have reproduced the original work of Reynolds, Weiss and colleagues and in extensive studies established the in vitro clonal growth of EGF responsive progenitor cells in semi-solid soft ager culture. WE have established an initial matrix of cytokine receptor expression by neurosphere cells (determined by RT-PCR) showing the expression of 22 separate cytokine receptors along with expression of a number their ligands. We've also shown that one of these receptors (c-fms) is expressed and that the neurosphere cells respond both with proliferation and with differentiation to CSF-1. We've characterized several of the neurotrophins and c-kit by antibody staining and used the monoclonal antibody to c-kit to physically separate neural cells for further analysis. We have also characterized neurosphere cells with regard to light scatter and Hoechst staining by fluorescence-activated cell sorting showing a differential expression of c-kit in different phases of cell cycle. We plan to continue to develop the cytokine receptor matrix of neurosphere cells, using RT-PCR, antibody staining and functional effects of a variety of cytokines and analyzing cytokine receptor induction by exposure to specific cytokines. We will utilize the knowledge of cytokine receptor expression, cell cycle status and other physical and metabolic parameters to separate sub-populations of neurosphere cells by fluorescent activating cell sorting. This data should allow us to elucidate the heterogeneity of the neuropoietic progenitor/stem cell system and determine the regulatory features of this system, including both microenvironmental and cytokine requirements.
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