Abstract
Purpose: :
To better understand the differentiation of retinal progenitor cells, sequential quantitative assessment of 17 transcripts was performed on murine retinal progenitor cells (RPCs) cultured under 2 established differentiation conditions.
Methods: :
Passage-60 RPCs, originally isolated from the neural retina of postnatal day-1 GFP-transgenic mice, were seeded as a single cell suspension in proliferation medium containing DMEM/F12 (1:1), N2 supplement and 20 ng/ml EGF, then incubated for 48 hours at 37ºC. Medium was then removed and fresh medium without EGF and containing 10 ng/ml ciliary neurotrophic factor (CNTF) or 10% fetal bovine serum (FBS) was added to the experimental flasks, whereas controls received standard proliferation medium. Medium was changed every 2-3 days and cells harvested on days 0, 1, 3, 5 and 7 for total RNA isolation followed by quantitative PCR (qPCR).
Results: :
An increased proportion of cells with small somata and long, thin processes was observed in CNTF treated cultures, whereas FBS resulted in more cells with large, polygonal morphology. For both conditions, qPCR showed dramatic up-regulation of GFAP (>100 fold), together with substantial (>2 fold) increases in vimentin, Hes5, Mash1, DCX and rhodopsin. Other genes increasing included Sox2, β-tubulin3 and Map2. Expression of Ki-67 decreased sharply (>10 fold) with both treatments. Other genes decreasing were Hes1 and CRALBP. With CNTF, Chx10 and recoverin also increased (13 fold, 1.5 fold), while Pax6 decreased. With FBS, Notch1 increased (1.5 fold) and recoverin decreased. The temporal profile of expression changes varied by transcript and treatment condition.
Conclusions: :
Both CNTF and FBS rapidly alter the gene expression of RPCs, with massive up-regulation of GFAP and marked down-regulation of Ki-67, consistent with the induction of differentiation. Detailed profile analysis revealed that increased GFAP was not associated with co-induction of the mature Mueller cell marker CRALBP but rather concomitant up-regulation of vimentin and multiple markers of neuronal lineage, including rhodopsin, within the cultured RPC population. Increased expression of Chx10 following CNTF treatment is consistent with the known ability of this agent to induce bipolar cell differentiation.
Keywords: regeneration • retina • transplantation