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B.A. Angenieux, D.F. Schorderet, F.L. Munier, Y. Arsenijevic; EGF Induces Neuron Formation in Retinal Progenitor Cultures . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1672.
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Purpose: The generation of neurons from retinal progenitors (RPC) is a promising tool for the treatment of retinal degeneration. Here, we explore the influence of epigenetic factors on retinal progenitor differentiation and the generation of neurons. Previously, EGF has been used to induce proliferation of neuronal progenitors in vitro or to drive progenitors towards the astrocyte phenotype. The present study aims to reveal the RPC fate under EGF stimulation Methods: We have isolated retinal progenitors from postnatal DBA mice (P0 to P2, n=3). Progenitors were grown in EGF and FGF-2 and were passed 3 to 11 times before inducing differentiation. For the induction of differentiation, 50'000 single cells were transferred to a poly-ornithin substrate with or without laminin. Results: Prior to differentiation most RPCs (60 to 90%) were nestin positive. In a serum-free media no cells survived. Upon addition of EGF (20ng/ml), up to 7% of cells presenting neuronal morphology and expressing beta-tubulin, an early neuronal marker, were observed. Then short-term (2-4 hours) or long-term (7-11 day in vitro (DIV)) exposures to EGF were undertaken in order to identify the function of EGF: differentiation versus proliferation. Cells were analyzed 7 DIV or 11 DIV after stimulation. Short-term stimulations are sufficient to induce neuronal differentiation: 11.5% of the cells were b-tubulin positive; whereas with long-term stimulations few b-tubulin positive cells were observed (0.7%, (p=0.024)) but with a complete mature morphology. Laminin also appears to be essential for neuronal differentiation. On the other hand, FBS alone or in addition to EGF completely abolishes neuronal formation. Conclusions: It appears that EGF is not only a factor controlling glial fate as it was previously shown, but is also a potent inducer of retinal neuron formation. EGF short pulse is sufficient for neuronal formation but its sustained action is necessary to obtain mature morphological neurons.
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