Muller Glial Cells Promote The Differentiation Of Retinal Progenitors As Photoreceptors And Sphingosine-1-Phosphate And Docosahexaenoic Acid Prevent Their Apoptosis

POLITI, LE; SIMON, MV; DE GENARO, P; DE LOS SANTOS, B; ROTSTEIN, NP

Fort Lauderdale

Congreso; ARVO 2011 Annual Meeting Visionary Genomics; 2011

ARVO

Purpose: Müller glial cells (MGC) are retina stem cells (SC), shown to transdifferentiate as photoreceptors (PHRs). However, their therapeutical use requires stimulating this differentiation and preventing 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 could reduce their apoptosis. Methods: neuron-glia co-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. Moreover, 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. cGMP levels were decreased in the co-cultures, but not in pure neuronal cultures after light exposure. DHA or S1P reduced by half apoptosis 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 reduce the apoptosis of newly generated PHRs. As a whole, these findings might provide clues to develop new strategies for treating retinal degenerative diseases.