Abstract
Abstract: :
Purpose: The existence of stem cells is not restricted to embryonic development. Somatic stem cells reside in postnatal and adult tissues and have been implicated in the regenerative potential of certain organs. We investigated the presence of somatic neural stem cells in the postnatal retina in vivo and in vitro. Methods: Dissociates from postnatal day 1 and 3 (P1 and 3) rat retina were cultured in serum-free medium containing bFGF2 (20ng/ml)/ EFG (20ng/ml) and BrdU for 4 days up to 8 weeks to obtain neural spheres. Spheres were harvested and plated on poly-L-ornithine coated glass coverslips. Differentiation conditions (addition of 1% FCS, no growth factors) were applied for additional 7 days before cells were fixed in 4% paraformaldehyde and processed for immunocytochemical analysis. Results: Postnatal retinal progenitors (P1 and P3) proliferate in culture for up to 8 weeks. They express early neuronal (beta III Tubulin) and glial (GFAP, Vimentin) markers as well as a marker for neuronal precursos (Nestin). They also express the retinal progenitor marker Flk-1 and RT-PCR studies further identified Flk-1 transcripts for both P1 and P3 progenitors. After introducing differentiation in P3 cultures, the late neuronal markers NF 200 and Map2c were expressed as well as the photoreceptor specific Ca2+-binding protein Recoverin. At P3, the progenitors tend to express more of the adult neuronal markers than at P1. About 15% of BrdU-positive cells express beta III Tubulin while some 17% express GFAP. This ratio is also described for other neural stem cells in vitro. Conclusion: Even after birth, the mammalian retina has the capacity to generate progenitor cells. Later, when the retina is fully developed, this capacity vanishes. Here we show that progenitor cells can be propagated in vitro and thus may be useful tools for further transplantation experiments to asses their ability to generate retinal cells types in models of retinal degeneration.
Keywords: 554 retina • 560 retinal culture • 564 retinal development