Purchase this article with an account.
S Bhattacharya, JD Jackson, WB Thoreson, C Kuszynski, I Ahmad; Prospectively Enriched Retinal Stem Cells/Progenitors: A Comparison With In Vitro Expanded Retinal Progenitors . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3684.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have recently reported the identification of retinal stem cells/progenitors from fresh embryonic retina (prospective identification) using Hoechst dye efflux assay (Bhattacharya et al, 2001, ARVO Abst, 1059). Here we have compared their characteristics and differentiation potential with that of retinal progenitors expanded in vitro in response to EGF. The objective is to investigate if exposure to mitogen in vitro alters the inherent characteristics and differential potential of neural stem cells/progenitors. Methods: Enrichment of retinal stem cells/progenitors, from fresh E18 retina and EGF-exposed neurospheric culture, as a flow-cytometrically-defined side population (SP) was carried out using Hoechst dye efflux assay as previously described (Goodell et al, J. Exp, Med., 183, 1797-1806). Enrichment of retinal stem cells/progenitors was also carried out by fluorescence activated cell sorting (FACS) using antibodies against Notch1 and the low affinity neurotrophin receptors, p75. Enriched cells were subjected to immunocytochemical and electrophysiological analysis in order to characterize their properties and differentiation potentials. Results: Similar to hematopoietic stem cells, progenitors were present in fresh E18 retinal dissociates that excluded Hoechst 33342 dye and therefore, were enriched as a SP by FACS. The enrichment was verapamil sensitive and SP cells represented 0.01% of total retinal cells. The proportion of cells enriched by FACS using Notch1 and p75 antibodies was larger than those in SP. Retinal progenitors were also enriched as a verapamil sensitive-SP from EGF-exposed neurosphere culture. The proportion of these SP cells represented 1.0% of total cells suggesting a selective expansion of retinal progenitors in the presence of EGF. However, like prospectively identified retinal stem cells/progenitors they expressed Notch1, Nestin, Rx and Chx10 in similar proportions. Similarly, these cells did not express retina specific markers. These cells also differentiated into both neuronal and glial lineages. Conclusion: The retinal stem cells/progenitors enriched from the mitogen-exposed neurospheres appear to be similar in characteristics and differentiation potential to those prospectively identified and therefore may be equally suitable for potential therapeutic purposes. Supported by NEI and Nebraska Research Initiative.
This PDF is available to Subscribers Only