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B.A. Angenieux, D.F. Schorderet, F.L. Munier, Y. Arsenijevic; EGF PROMOTES RETINAL GANGLION CELL DIFFERENTIATION FROM EXPANDED PROGENITOR CULTURES IN VITRO. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5352.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Until now, it has been admitted that early retinal progenitor cells (RPCs) can only give rise to early–born neurons and that late RPCs give rise to late–born neurons (Cepko, 2001). However, in a recent paper, Ahmad's group (James et al, 2003) has shown that late RPCs (isolated at embryonic day E18) can generate retinal ganglion cells (RGCs), which are the earliest–born neurons of the retina. Here, we analyze whether the RPCs can generate RGCs after cell expansion and determine which factor(s) can contribute to such induction. Methods: RPCs were isolated from postnatal DBA mice (n=4). The cells were grown in EGF and FGF–2 and were passed 3 to 21 times before inducing differentiation. For the induction of differentiation, 40'000 single cells were transferred onto a poly–ornithin substrate with laminin. After 7 DIV, the cells were either fixed or the RNA was extracted Results: Prior to differentiation, all RPCs express nestin filament and Pax6, two markers of precursor cells. They also show a high proliferative capacity, 5 million cells can generate more than 7*1028 cells. In a serum–free media without factors, no cells survive. Upon addition of EGF, 15% of the cells express ß–tubulinIII, an early neuronal marker, and acquire mature neuronal morphology with long and fine processes looking like ganglion cells. Moreover, after RT–PCR analysis, the cells express math5 (n=3), a transcription factor required during the first step of RGC differentiation and, sometimes, Brn3b, another transcription factor implied in the later stage. Interestingly, EGF does not highly stimulate the neuroblast proliferation. Actually, only 20% of the ß–tubulinIII–positive cells undergo proliferation, during EGF stimulation. To determine if the action of EGF is direct or indirect, low density cultures are needed. Preliminary data indicate that at low density, around 25% of the cells are immature ß–tubulinIII–positive cells whereas increasing cell density leads to a smaller number of cells positive for ß–tubulinIII, although morphologically more mature. Thus, it seems that EGF promotes directly neuronal formation and that cell–cell contacts are necessary to maintain the neuronal maturation, as well as EGF presence. Conclusions: It appears that EGF is not only a factor controlling glial fate, as it was previously shown, but also a potent inducer of RGC phenotype. Moreover, it appears that a new dogma is emerging: the RPC keeps its multipotent characteristic but it is the environment that changes the fate of RPCs over time.
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