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G.V. Hegde, J. James, A.V. Das, S. Bhattacharya, X. Zhao, I. Ahmad; Characterization of extrinsic factor(s) that influences the differentiation of late retinal progenitors/stem cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5412.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The retinal stem cells/progenitors are multi–potent, and the decision taken by the progenitors to differentiate along a particular lineage depends on local cell–cell interactions. Recently, we have shown that factor(s) present in the retina representing the early stage of histogenesis can influence the differentiation of late retinal progenitors into retinal ganglion cells (RGCs) and rod photoreceptors (James et al., 2003; J Neurosci. 23(23): 8193–8203). To understand the nature of extrinsic factor(s), we have begun characterization of activities, in the conditioned medium (CM) of early retinal cells, that regulate neurogenesis. Methods: Retinal cells obtained from E3 chick retina, representing early stage of histogenesis, were cultured to obtain CM containing differentiation–regulating activities. Neurospheres were obtained from E18/PN1 rat retina as previously described (James et al., 2003), and cultured in the presence of size fractionated, heat treated and untreated CM. Differentiation of late retinal progenitors into rod photoreceptors or RGCs was evaluated by immunocytochemical and RT–PCR analysis. Results: In the presence of E3 chick retinal CM, a subset of late retinal progenitors expressed the regulators of RGC differentiation, Ath5 and Brn3b, and acquired RGC phenotype, as ascertained by the expression of Islet1, RPF–1 and Thy1. In contrast, the differentiation of late retinal progenitors into rod photoreceptors was suppressed in the presence of E3 chick retinal CM, as ascertained by the expression of opsin, rhodopsin kinase and arrestin. Culturing of late retinal progenitors in the presence of size fractionated CM demonstrated that the activity influencing differentiation was present in the fraction higher than 30kDa. This activity was lost when fractions were heat–treated. Conclusions: The activities present in E3 chick retinal CM that positively and negatively influences the differentiation of late retinal progenitors into RGCs and rod photoreceptors, respectively, is present in the protein fraction higher than 30kDa and is heat labile.
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