Abstract
Abstract: :
Purpose: To compare the behavior of human embryonic retinal and cortical progenitor cell–containing neurospheres in vitro using a novel culture method that avoids the use of proteases. Such an approach theoretically preserves surface receptors and native intercellular contacts and may impact the growth and differentiation potential of these cells. Methods: Thirty human embryonic retinas (aged 54 to 120 days) were mechanically chopped into 200 micron sections and cultured separately in media supplemented with EGF and FGF. An identical approach was utilized to propagate neurospheres derived from human embryonic frontal cortex. Growth assays were performed by measuring volumes of individual retinal and cortical spheres over time. Results were correlated with immunocytochemical studies examining BrdU incorporation and nestin, ß–tubulin III, and GFAP expression. Results: Spheres readily formed from both retinal and cortical tissue sections. Sphere populations were chopped and split every 10–14 days; however, most retinal neurospheres failed to demonstrate growth after 4–6 weeks in culture. Cultures of cortical neurospheres, on the other hand, undergo continuous expansion for numerous months before becoming senescent. Growth assays confirmed these observations, revealing limited, linear expansion of most retinal neurospheres with a minimum doubling time of 15 days. Likewise, cellular BrdU incorporation decreased in retinal neurospheres between 1 week and 1 month in culture. In contrast, cortical neurospheres expanded exponentially, doubling every 3–4 days and exhibiting steady cellular BrdU incorporation. Interestingly, rare retinal neurospheres demonstrated exponential growth after 6 weeks in culture (5–7 day doubling time) and could be expanded for at least 4 additional weeks. Immunocytochemical analysis of the major sphere populations revealed higher percentages of nestin expression in cortical neurospheres (90%) compared to retinal neurospheres (50%). Finally, ß–III tubulin expression was prominent in all retinal cultures, although it varied depending on the age of the donor tissue. Conclusions: 1.) Unlike cortical neurospheres, human embryonic retinal neurospheres can only be expanded for short periods in media supplemented with mitogens. However, a small subpopulation of retinal spheres did exhibit enhanced growth potential. 2.) The differentiation profile of cultured retinal neurospheres was strongly influenced by the age of the donor tissue and appeared to follow the course of normal retinal development.
Keywords: retinal culture • retinal development • retina