NEURAL STEM CELLS : Development of a progenitor bioassay from mammalian retina.

MIRANDA, G.; REYNALDO M.; CARRI N.

Córdoba, Córdoba, Argentina

Workshop; curso sobre Investigación en Células Madre del programa PABSELA 2010; 2010

Fundación Crimson - UNC - IBRO

Retinal progenitor and stem cells have been found in adult humans. This finding has become quite significant for retinal regeneration research. The use of these cells to accomplish retinal regeneration requires an exhaustive knowledge on the molecular mechanisms that control neural progenitors proliferation and their subsequent differentiation into the corresponding phenotype. This subpopulation, found in the anterior part of the eye, has been well characterized by immunoanalysis but little is known about their cell culture. Human retinal stem cells constitute a great finding, but they cannot be used to analyze the molecular mechanisms that control them. Thus, the development of models to allow cell and molecular biology studies on this neural subpopulation results absolutely essential. These progenitor/stem cells have been found in avian retina and specific areas of the mammalian retina, mainly in the ciliary body, but there is still no evidence on these cells in the mammalian ciliary marginal zone (CMZ). We have started to develop cell culture systems (bioassays) that will allow us to isolate, culture, identify and characterize progenitor/Stem cells from the CMZ of rodent retinas. Our main objective is to obtain cells that maintain their multipotenciality in order to analyze their morphological and immuno-profile, study the regulation of their proliferation and differentiation, and then analyze the trophic stimulation effect on these cells. The neural retinal 3D bioassay developed in our lab consists of cultures prepared with entire embryonic rat retina E14-E16, or CMZ from neonatal (P1), adult (4-8 months) or senile rats (3 years); after mechanical or chemical+mechanical dissociation, cells are cultured in a chemically defined culture medium supplemented with trophic factors. After seeding, cells are grown in flotation and several of them clump together forming typical neurospheres that remain floating for a few days. Some cells in neurospheres surface incorporate BrdU indicating that they maintain their proliferative capacity. We are also analyzing the expression of different markers to trace the commitment state of the cells –Glial or Neuronal-, and culture them under different conditions to analyze the molecular mechanism of proliferation and differentiation.