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V Enzmann, RM Howard, SR Whittemore, HJ Kaplan; Rat Neural Stem Cells (rNSC) Differentiate in Vitro into Cytokeratin-positive Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3691.
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Purpose: To investigate the differentiation of rNSC under the influence of vasoactive intestinal peptide (VIP) and co-culture with retinal pigment epithelium (RPE) cells or neurosensory retina (NSR). Methods: rNSC prepared from the cortex of embryonic rats (E14) were maintained in DMEM/F-12 medium containing FGF-2 and N2 supplement. The cells were cultured with different concentration of VIP in medium with 1% FBS, co-cultured with rat RPE (1:1; 1:5 or 1:10) or with rat NSR for 5 days. Cell morphology, proliferation and cytokeratin expression were studied. Other antibodies used to characterize the cells were: nestin - undifferentiated NSC, beta III tubulin - neuronal marker, GFAP - astrocyte marker and an anti-oligodendrocyte antibody. Results: The cell shape of rNSC changed under the influence of VIP from round to glial-like with processes. These cells stained positive for both glial and neuronal markers. Additionally, clusters of flattened, polygonal cells with an epithelial-like shape were observed which were cytokeratin positive. Treatment with VIP and 1% FBS enhanced the proliferation of rNSC in a dose-dependent manner compared to the control (no FBS). The highest concentration of VIP augmented proliferation over that observed with 1% FBS alone. Co-culture of rNSC with RPE cells also led to polygonal-shaped cytokeratin-positive cells. The ratio between stem cells and RPE cells did not influence differentiation. Co-culture of rNSC with NSR only resulted in cells with neural markers. Conclusion: Under the influence of VIP and in co-culture with RPE cells, rNSC differentiate in vitro into RPE-like, cytokeratin-positive cells.
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