EPIDERMAL GROWTH FACTOR IS A NEURONAL DIFFERENTIATION FACTOR FOR RETINAL STEM CELLS IN VITRO

¹ Unit of Gene Therapy and Stem Cell Biology, Jules Gonin Eye Hospital, Lausanne, Switzerland and Department of Ophthalmology, University of Lausanne, Lausaane, Switzerland
² Department of Ophthalmology, University of Lausanne, Lausaane, Switzerland; and IRO - Institute of Research in Ophthalmology, Sion, Switzerland

^* To whom correspondence should be addressed. E-mail: yvan.arsenijevic{at}ophtal.vd.ch.

	Abstract

Stem cells are a tool to elucidate in vitro the putative role of factors on cell fate. Here, we analyze the role of EGF on progeny derived from retinal stem cells (RSCs). We isolated cells from neuroretinas of neonate mice. All the proliferating cells harbored the radial glia marker RC2, expressed transcription factors usually found in radial glia (Mash1, Pax6), and met the criteria of stem cells: high capacity of expansion, maintenance of an undifferentiated state, and multipotency demonstrated by clonal analysis. We analyzed the differentiation seven days after transfer of the cells in different culture media. In absence of serum, EGF led to the expression of the neuronal marker -tubulin-III, and acquisition of neuronal morphology, in 15% of the cells. Analysis of cell proliferation by bromodeoxyuridine incorporation revealed that EGF mainly induced the formation of neurons without stimulating cell cycle progression. Moreover, a pulse of 2h EGF stimulation was sufficient to induce neuronal differentiation. Some neurons were committed to the retinal ganglion cell (RGC) phenotype, as revealed by the expression of retinal ganglion markers (Ath5, Brn3b and melanopsin), and in few cases to other retinal phenotypes (photoreceptors (PRs) and bipolar cells). We confirmed that the late RSCs were not restricted over-time and conserved multipotentcy characteristics by generating retinal phenotypes that usually appear at early (RGC) or late (PRs) developmental stages. Our results show that EGF is not only a factor controlling glial development, as previously shown, but also a potent differentiation factor for retinal neurons at least in vitro.

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