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Iqbal Ahmad, Sowmya Parameswaran; Retinal Progenitors Are Stem Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2243.
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It has been observed that the enriched retinal progenitors fail the critical stem cell test, i.e., they do not display self-renewing capacity. Here, we demonstrate in vitro that retinal progenitors can self-renew but this property is non-cell autonomous, requiring interactions with other cell types.
Retinal cell dissociates from embryonic day (E) E18 rat embryos were cultured in the presence EGF /EGF + endothelial cell conditioned medium (ECCM) at different densities to generate primary, secondary, and tertiary neurospheres. Resulting neurospheres were pulsed with BrdU to estimate the number of proliferating cells and shifted to differentiation conditions (absence of mitogens+ECCM and substitution of FBS) to determine whether or not progenitors posses parental multipotentiality. Secondary neurospheres were subjected to limiting dilution analysis (LDA) to determine the frequency of stem cells that generated colonies at clonal density.
Primary neurospheres were detected at plating density of 2X10^5 cells/cm^2 in the presence of EGF. In contrast, addition of ECCM led to the generation of neurospheres at a density that was 100 fold lower (~2X10^3 cells/cm^2), and at which neurospheres were not detected in the presence of EGF alone. Neurospheres were relatively larger and cells therein generated secondary and tertiary neurospheres in the presence of ECCM. Cells in secondary neurospheres gave rise to neurons and glia in a proportion similar to those in primary neurospheres, thus demonstrating self-renewal. LDA demonstrated that the frequency of a single limiting cell type for the generation of neurospheres at clonal density was 0.078%. Results regarding putative factors that facilitate self-renewal of retinal progenitors will be presented.
Enriched retinal progenitors possess the cardinal feature of stem cells, i.e., they can self renew but in cooperation with other cell types. This non-cell autonomous property highlights the role of the microenvironment (niche) in the maintenance of retinal stem cells in vivo.
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