Neurogenic potential in Vsx2 (Chx10)+ human prenatal retinal progenitor cultures

Purpose To examine a proliferating Vsx2+ progenitor population in primary human prenatal retinal cultures and to determine the neurogenic potential of these cells. Methods Retinal progenitor cells from human prenatal eyes from 60 to 125 days were cultured as neurospheres in serum-free medium with mitogens. Cells were prepared for ICC immunostained for markers of neural and retinal proliferation and differentiation, and compared to cryosections from aged-matched whole eyes. Results Vsx2+ proliferating cells were present in primary neurosphere cultures derived from human prenatal retina. The 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+ which co-localized with retinal progenitor markers. Treatment with the Notch inhibitor, DAPT, increased Ascl1 expression and PKCα (+) cells, but did not alter the % of recoverin (+) cells. After 1 month in culture, very few cells expressed either Vsx2 or Ascl1, and DAPT treatment no longer demonstrated an effect on neuronal differentiation. Conclusion 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. 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.