Proliferation of Retina Neuroblasts in Cocultures of Muller Cells and Retinal Neurons: Effects of Docosahexaenoic Acid on Photoreceptor Differentiation

POLITI L.E.; SIMON M.V.; DE LOS SANTOS EB; ROTSTEIN N.P.

Fort Lauderdale, Florida, EEUU

Congreso; ARVO Annual Meeting; 2009

Association for Research in Vision and Ophthalmology (ARVO)

<!-- /* Font Definitions */ @font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-1610611985 1073750139 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin-top:0in; margin-right:0in; margin-bottom:10.0pt; margin-left:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-fareast-font-family:Calibri; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:ES-AR;} @page Section1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.Section1 {page:Section1;} --> Proliferation of Retina Neuroblasts in Cocultures of Muller Cells and Retinal Neurons: Effects of Docosahexaenoic Acid on Photoreceptor Differentiation Luis Politi, Victoria Simón, Beatriz de los Santos and Nora Rotstein. Instituto de Investigaciones Bioquímicas de Bahía Blanca, CONICET and Universidad Nacional del Sur. E-mail: inpoliti@criba.edu.ar Purpose:  Photoreceptor (PHR) loss is the main cause of blindness in neurodegenerative diseases of the retina. Muller stem cells might be used as a source of new PHRs as a promising treatment for these diseases. This requires establishing the conditions by which these cells promote proliferation of neuroblasts (NB) and their further differentiation into PHRs.   Methods:  to investigate these questions we used pure primary cultures of Muller cells or retinal neurons, and neuro-glia co-cultures grown in serum containing media or in a serum-free medium, supplemented or not with docosahexaenoic acid (DHA), a lipid molecule that promotes PHR survival and differentiation. At different developmental times, we harvested co-culture cells and reseeded them to obtain secondary subcultures. We then determined BrdU uptake, to determine proliferation, TUNEL labeling, to quantitate apoptosis and the presence of nestin, NeuN and Crx and opsin, as stem cell, neuronal and photoreceptor markers, respectively . Results: NB were absent in primary or secondary pure glial cultures. In pure neuronal cultures, a significant amount of NB was initially present, which rapidly differentiated into PHRs and disappeared from the cultures. In contrast, in neuro-glia co-cultures, NB could be maintained up to 15 days. In secondary or tertiary cultures obtained from the co-cultures, NB proliferated and expressed nestin. Moreover, some cells that took up BrdU, later went on to express NeuN, Crx and opsin, implying that under these conditions proliferating NB could give rise to new PHRs. Noteworthy, when we incubated secondary co-cultures with DHA, the percentage of PHR-like cells expressing opsin and Crx significantly increased with a parallel reduction in their apoptosis.     Conclusion: the interaction of retinal neurons with Muller cells is critical for the generation and preservation of NB. DHA promoted these NB to survive and advanced their differentiation into PHRs. Our findings might provide important tools for successfully using stem cells in the treatment of retinal diseases.   Grants  from ANPCYT, CONICET and UNS