Abstract
Abstract: :
Purpose: In vitro expansion of retinal progenitor cells has been used as a tool to increase material for transplantation into experimental models of retinal degeneration. However, it is not yet possible to reliably bias retinal progenitors to adopt a desired cell fate. The experiments in this study have begun to investigate a mechanism to bias expanded retinal progenitors to adopt a photoreceptor phenotype. Methods: Retinal progenitor cells from E17 Sprague–Dawley rats and expanded in vitro for 7 days with EFG (epidermal growth factor) and bFGF (basic fibroblast growth factor) (20ng/ml). After 7 days, neurospheres that were free floating in the media were collected, dissociated and plated on glass coverslips coated with polyethyleneimine. To induce differentiation, EGF and bFGF were removed from the culture media (differentiation media) and in some cases IGF–1 (insulin like growth factor–1) was added (10ng/ml). Cultures were fed every 2–3 days by replacement of half the culture media. After 1–3 weeks cultures were fixed and processed with standard immunohistochemical techniques. Results: In media without IGF–1 we observed phenotypic indicators of some photoreceptor differentiation. A few small clumps of cells with a rounded morphology were observed. These clumps of cells were strongly immunoreactive for SNAP–25 and a subset of these cells were recoverin–immunoreactive, though no rhodopsin–immunoreacitivty was observed. However, even after three weeks, most cells displayed a flattened morphology and were immunoreactive for nestin, an intermediate filament protein present in undifferentiated neural progenitor cells. In contrast, when IGF–1 was added to the media, much greater differentiation of progenitors to a photoreceptor phenotype was observed. After two weeks there were many more clusters of cells immunoreactive for SNAP–25 and recoverin. After three weeks, many of these clusters of cells were also immunoreactive for rhodopsin. In addition, nestin–immunoreactivity was greatly reduced by the addition of IGF–1. Conclusions: Even after three weeks in differentiation conditions expanded E17 rat retinal progenitors showed very limited differentiation. Addition of IGF–1 to the differentiation media greatly increased the indicators of photoreceptor differentiation. These results demonstrate it is possible to bias differentiation of expanded retinal progenitors, and we have a model to investigate this process.
Keywords: photoreceptors • retinal development • microscopy: light/fluorescence/immunohistochemistry