Neurotrophin channeling of neural progenitor cell differentiation

The act of defining neuropoietic progenitor/stem cells is still in its earl y phases. Epidermal growth factor (EGF) stimulates extended proliferation o f aggregates of subventricular striatal cells, taken from E15 mouse striatu m, termed neurospheres in liquid culture. We have shown here and in previou s work, using either immunohistochemistry or RT-PCR, that neurosphere cells express 13 cytokines (32 tested) and 20 cytokine receptors (28 tested), wi th 11 potential paracrine and nine potential autocrine loops. The neurotrop hin receptors, Trk A, B, and C, were all expressed. Using a newly developed FAGS single cell deposition technique, we evaluated the rapacity of single EGF stimulated neurosphere cells to respond to the ligands for Trk A and B , nerve growth factor (NGF), and brain-derived neurotrophin factor (BDNF), Addition of NGF or BDNF to EGF for 14 days had no effect, but removal of EG F at day 14 with subsequent addition of BDNF or NGF resulted in an increase in neuronal and astroglial, but not oligodendrocyte, colony cells at 21 an d 28 days of culture for BDNF, and of both cell types at 28 days for NGF, T ri-lineage colonies increased at day 21 with BDNF and at day 28 for both NG F and BDNF. Gross colony morphology also showed changes with neurotrophin a ddition, forming multiple individual cell balls or filamentous spreads. Whe n EGF was withdrawn, a threshold effect was observed, with small, hut not l arge, colonies ceasing growth. BDNF and NGF showed no effects on cell proli feration when compared to EGF controls, as determined by 5'-bromo-2-deoxyur idine (BrdU) incorporation and thus, they appear to affect differentiation of progenitor cells. These data indicate a sequential action of cytokines w ith EGF maintaining viability and proliferation and blocking differentiatio n. Removal of EGF is then permissive for the differentiating effects of BDN F and NGF, These data further indicate that the majority of EGF neurosphere clones have neurotrophin dependent tri-lineage potential. (C) 2001 John Wi ley & Sons. Inc.