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R.L. Shearer, J.N. Melvan, C.N. Svendsen, D.M. Gamm; Characterization of Secreted Factors From RPE That Support Growth of Human Retinal Neurospheres . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5742.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize proteinaceous factors present in human fetal RPE conditioned media that enhance the proliferation and survival of late retinal progenitor cells cultured as neurospheres.
Primary monolayer cultures of hfRPE were expanded and maintained in serum–free, defined medium containing DMEM, F12 and B27 supplement. Filtered (0.2 µm) conditioned media (CM) from these cultures was added to retinal neurosphere growth assays with or without the addition of 20 ng/ml EGF and/or 20 ng/ml FGF–2. At confluency, hfRPE cultures were washed and incubated for 24 hours in media without B27, after which time the conditioned media (CM(–B27)) was filtered, concentrated and analyzed via SDS–PAGE and Western blotting. In addition, RPE CM(–B27) was subjected to molecular weight fractionation followed by supplementation with normalized concentrations of B27, EGF and FGF–2. The fractionated RPE CM samples were then used in growth assays to ascertain the approximate size of the RPE factor(s) responsible for the growth–promoting effect of RPE CM on retinal neurospheres. Lastly, the mechanism underlying this effect was investigated using PCR, TUNEL and intracellular phosphorylation assays.
RPE CM greatly enhanced retinal neurosphere growth in a mitogen–dependent manner, whereas unconditioned media containing B27, EGF and FGF–2 failed to support long–term retinal neurosphere growth. SDS–PAGE of RPE CM(–B27) revealed multiple protein bands distributed over a wide size range, some of which were subsequently identified by Western blot analysis. Fractionation experiments further suggested that one or more factors greater than 25 kDa participate in the growth–promoting effect, although smaller molecules could play a lesser role. The absence or removal of RPE CM reduced both mitogen receptor expression and CREB phosphorylation in retinal neurospheres, implying a potential mechanism for the RPE CM effects observed in these cultures.
Fractions of RPE CM containing soluble factors greater than 25 kDa retain the majority of the growth–promoting effect of unfractionated RPE CM. These RPE factor(s) appear to exert their effect through direct or indirect modulation of the EGF and FGF–2 signaling pathways.
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