Interactions with Müller glial cells induce and preserve stem cell characteristics in retinal progenitors after several passages

SIMON, M.V.; DE GENARO, P.; DE LOS SANTOS, E.B.; ROTSTEIN, N.P.; POLITI, L.E.

Fort Lauderdale

Congreso; ARVO Annual Meeting; 2011

ARVO

Interactions With Müller Glial Cells Induce And Preserve Stem Cell Characteristics In Retinal Progenitors After Several Passages Simón MV, De Genaro P, de los Santos B, Rotstein N, Politi L. (Mail: mvsimon@criba.edu.ar) Instituto de Investigaciones Bioquímicas, UNS-CONICET, Bahía Blanca, Bs. As. Purpose: Müller glial cells (MGC) are now established as stem cells in the vertebrate eye, becoming excellent candidates to replace neurons lost during retina degenerative diseases. However, their regenerative potential in mammals is very limited, so uncovering ways to expand the number of stem cells in the retina would be of therapeuthical relevance. We have previously shown that when co-cultured with MGC, retinal progenitors remained mitotically active and could be re-seeded. Here we analyzed if the interaction with MGC induced stem cell characteristics in retinal progenitors and if they promoted the eventual differentiation of these progenitors as photoreceptors (PHRs). Methods: Retinal pure glial cultures and neuron-glia co-cultures were prepared from post-natal day 2 rats. Co-cultures were consecutively harvested and re-seeded until their fifth passage. Retinal progenitors were identified by Pax-6 and nestin-expression and by 5-bromo-2-deoxyuridine (BrdU) uptake, and quantified by flow cytometry. Their differentiation into PHRs was analyzed by Crx and opsin expression. Results: Secondary co-cultures showed MGC and small round cells, and 5% of the later exhibited characteristic stem cell markers (BrdU uptake, Nestin- and Pax-6 expression). Progenitors with these stem cell features were preserved in co-cultures even after four successive re-seedings. On the contrary, these progenitors were no longer found after re-seeding of pure glial cultures. Progenitors that took up BrdU also expressed the early photoreceptor marker Crx and opsin in secondary co-cultures, implying that they had exited the cell cycle to adopt a PHR fate. Noteworthy, Crx-positive cells were also found after four re-seedings. Conclusions: our results suggest that in addition to giving rise to new neurons, MGC cells have a complementary mechanism to contribute to retina regeneration: they might preserve and/or generate a pool of proliferative progenitors with stem cell characteristics even after several passages, which might later differentiate as PHRs.