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D.M. Gamm, J.N. Melvan, C.N. Svendsen; Effects of RPE Conditioned Media on Human Retinal Progenitor Cell Growth and Differentiation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3236.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop a culture method to expand populations of human retinal progenitor cells as neurospheres (retinal neurospheres) for use in basic developmental and transplantation studies. Methods: Human embryonic retinas were cultured in media optimized for cortical neurosphere growth, with or without the addition of conditioned media (CM) from human retinal pigment epithelium (RPE) cultures. Growth assays measured changes in individual sphere volume over time, and immunocytochemical studies examined BrdU incorporation and expression of nestin, beta–tubulin III, GFAP and recoverin. Results: In the absence of RPE CM, all retinal neurosphere populations senesced within 4 weeks of initial culture. However, parallel cultures treated with RPE CM demonstrated continuous growth and expansion (greater than 15 passages) as well as preservation of early sphere morphology. Likewise, BrdU incorporation decreased dramatically in retinal neurospheres after one week in culture unless RPE CM was added. Immunocytochemical analysis of undifferentiated cells contained within retinal neurospheres exposed to RPE CM demonstrated nearly 100 percent nestin expression with minimal staining for GFAP and beta–tubulin III. Under differentiating conditions, these cells adopted a predominantly glial morphology and expressed GFAP, although an occasional population of recoverin–positive cells was observed. Conclusions: By modifying a standard neurosphere culture protocol to include RPE CM, populations of undifferentiated, nestin–positive human retinal progenitor cells could be maintained and expanded in vitro for a number of months. This system may offer an avenue to study late human retinal development and provide a source of cells for ex vivo growth factor delivery strategies.
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