Study of progenitor cells from avian retina

J TAU; M REYNALDO; NG CARRI

Cordoba

Congreso; IRCN; 2009

Reunion Conjunta del TN y SAN

Retinal progenitor/stem cells from hot-blood vertebrate eyes have become quite significant for retinal regeneration research. These cells were found in the ciliary-circumferential marginal zone (CMZ) in embryos and postnatal birds. The layering of CMZ has been immunoanalyzed, but little is known about the proliferation and differentiation of retinal progenitor cells in culture. Thus, the purpose of this study was to analyze their morphological and immuno profile under basal conditions and trophic stimulation. Briefly, these cells were obtained as primary culture after mechanical dissociation from specific small pieces removed from CMZ of chick and quail embryos at E13 to E19. Isolated cells were then cultured in DMEM-F12, bFGF, EGF. Cells clumped together and formed typical neurospheres a few hours later. To characterize these we performed a tridimensional neurosphere bioassay inside a gelified collagen I. They were studied, during 4 days, under the action of NGF, NT-3, and NTN, factors known to be very active in retinal development. Progenitors could be identified by their clumping behavior forming neurospheres, a long preservation period in basal quiescent state for at least nine to ten months, by assaying for the expression of proliferate markers (PCNA, KI67), neural progenitor markers (A2B5, Foxn4, Pax6), and by the incorporation of BrdU. In the 3-D-bioassay, trophic stimulated cells generated outgrowths in which most cells were glial and a discrete number of neurites emitted by neurons. Several cells expressed calbindin and vimentin. Our bioassay of trophic stimulation allowed us to conclude that isolated progenitors were able to proliferate and differentiate, and clearly generate large glial cells and neurons.