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L. S. Wright, I. Pinilla, J. M. Clermont, J. S. Meyer, K. A. Wallace, E. E. Capowski, D. M. Gamm; Vsx2 (Chx10) Expression Identifies a Retinal Progenitor With Neurogenic Potential in Human Prenatal Eye Cultures. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2651.
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© ARVO (1962-2015); The Authors (2016-present)
To examine a proliferating Vsx2+ progenitor population in primary human prenatal retinal cultures and to determine the neurogenic potential of these cells.
Retinal progenitor cells were prepared from postmortem human prenatal eyes at approximately 60 to125 days of gestation and cultured as neurospheres in serum-free medium supplemented with mitogens. Cells were prepared for immunocytochemical analysis, immunostained for markers of neural and retinal proliferation and differentiation, and compared to cryosections from aged-matched whole eyes.
A population of Vsx2+ proliferating cells was present in primary neurosphere cultures derived from human prenatal retina. These cells retained multiple characteristics of retinal progenitor cells found in situ, including Ki67, Sox2, nestin and Notch expression. Upon withdrawal of mitogens, there was an increase in Ascl1+ expression which co-localized with retinal progenitor markers. Treatment with the Notch inhibitor, DAPT, increased Ascl1 expression and PKCα immuno-positive cells, but did not alter the percentage of cells expressing recoverin. After 1 month in culture, very few cells expressed either Vsx2 or Ascl1, and DAPT treatment no longer demonstrated an effect on neuronal differentiation.
Under serum-free conditions, a population of Vsx2+ late retinal progenitor cells can be identified and propagated for a limited time in vitro. The presence of Vsx2 in retinal progenitor cells correlated with maintenance of neurogenic potential regardless of the culture method employed. Furthermore, DAPT-induced enhancement of neural differentiation did not occur following loss of Vsx2 expression in long-term cultures. Thus, Vsx2 is useful for identifying proliferative progenitor cells with neurogenic potential in human prenatal retina cultures.
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