Müller glial cells promote the differentiation of retinal progenitors as photoreceptors and sphingosine-1-phosphate and docosahexaenoic acid prevents their apoptosis

DE GENARO, P.; SIMÓN, V.; DE LOS SANTOS, E.B.; ABRAHAN,C.E.; ROTSTEIN, N.P.; POLITI, L.E.

Rio de Janeiro

Congreso; 1a Reunión del Latinoamerican Institute of Glia; 2011

iGlia

Müller Glial Cells Promote The Differentiation Of Retinal Progenitors As Photoreceptors And Sphingosine-1-Phosphate And Docosahexaenoic Acid Prevent Their Apoptosis Pablo De Genaro, Maria V. Simón, Beatriz De los Santos, Carolina Abrahan, Nora P. Rotstein, Luis E. Politi. Instituto de Investigaciones Bioquímicas, Bahía Blanca, Argentina. Purpose: Müller glial cells (MGC) are considered retina stem cells (SC) that have been shown to transdifferentiate as photoreceptors (PHRs). However, their potential therapeutical use would require the stimulation of this differentiation and prevention of their death, as shown to occur with most newly generated cells. We have established that MGC instructed retinal progenitors to acquire SC properties. We also demonstrated that docosahexaenoic acid (DHA) and sphinghosine-1-phosphate (S1P) prevent PHR apoptosis. We now investigated whether MGC induced retinal progenitors to differentiate into PHRs and if DHA and S1P might reduce their apoptosis. Methods: neuron-glia mixed cultures were incubated in serum containing media and re-seeded several times. PHR differentiation was evaluated analyzing Tuj1, Crx, opsin, and peripherin expression by immunochemistry. PHR functionality was investigated by evaluating [3H]-Glutamate uptake and by measuring cGMP levels in the dark and after light-exposure. Apoptosis was determined by TUNEL labeling, after incubating secondary co-cultures with serum-free medium without or with 6.7 µM DHA or 1 µM S1P. Results: Secondary co-cultures presented 80% and 10% of round cells expressing Crx and opsin, respectively, by day 7. Surprisingly, a significant amount of Crx-positive cells were found even after four consecutive passages. In secondary co-cultures these cells developed long processes, labeled with Tuj1, an early neuronal marker, showed apical processes that concentrated opsin and peripherin expression and took up [3H]-Glutamate. Light exposure diminished cGMP levels in co-cultures, but not in pure neuronal cultures. DHA or S1P reduced apoptosis of newly generated PHRs by half in secondary co-cultures. Conclusions: Our results suggest that in addition to preserving progenitor cells in co-culture, MGC might instruct them to acquire a PHR fate and then differentiate to become functional PHRs. They also imply that DHA and S1P significantly promote the survival of newly generated PHRs. As a whole, these findings might provide clues to develop new strategies for treating retinal degenerative diseases. Section: 04. Progenitor potential of glial cells in health and disease.