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R-T Yan, W-X Ma, S-Z Wang; Generation of Neural Spheres from RPE Cell Culture Induced by Neurogenin2 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):709.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate whether it is possible to derive retinal progenitors with stem cell properties from RPE cell cultures under neurogenin2 (ngn2) induction. Methods: RPE cells were cultured in serum-free medium, and infected with a retrovirus RCAS expressing ngn2. Neural spheres formed in the culture were dislodged and subcultured. To promote cell differentiation, cells in the neural spheres were dissociated, seeded onto polyornithine treated cover slips, and cultured in the presence of 10% fetal calf serum. Cells in the culture were fixed and stained with antibodies that identify various retinal cell types. Results: In cell culture, RPE cells grow as a monolayer attached to the bottom of the plastic culture dish. However, with ectopic expression of ngn2, numerous neural spheres were formed. The neural spheres were visible 4 days after the administration of RCAS-ngn2. Subsequently, their sizes increased dramatically. Cells in the neural spheres could be sub-cultured for at least several passages, one of the characteristics of neural stem cells. Upon differentiation, the cells expressed various markers that identify retinal neurons, including photoreceptor cells, ganglion cells, and horizontal cells. Inducing the formation of neural spheres from RPE cell culture appeared to be specific to ngn2, as two other bHLH genes, ath3 and ath5, which are known to participate in retinal neurogenesis, did not do so under similar conditions. This might reflect the differences among the different bHLH genes in their respective functions during retinal development. Conclusion: Ngn2 can induce the formation of neural spheres from non-neural RPE cell cultures. The neural spheres can be subcultured and may have the potential to differentiate into various types of retinal neurons. Our study suggests that it may be possible to derive proliferative retinal progenitor cells from RPE cell culture using ngn2 as a molecular trigger. Currently, we are studying how much the cells in the neural spheres behave like retinal stem cells.
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