Sequential changes in the gene expression profile of murine retinal progenitor cells during the induction of differentiation

摘要

Purpose: Following transplantation,cultured retinal progenitor cells (RPCs) integrate into the diseasedhost retina and exhibit morphologies and markers indicative of localcellular phenotypes. In vitro analysis of cultured RPCs allows detailedexamination of marker gene expression during the initial phase ofdifferentiation and can provide insight into the variables influencingthis process. Methods: Using cultured murine RPCs,this study compares the effects of fetal bovine serum (FBS) with thoseof ciliary neurotrophic factor (CNTF), individually or in combinationwith epidermal growth factor (EGF). Differentiation was assessed by wayof the relative expression of 17 genes using quantitative PCR (qPCR) atfive time points over a seven-day period. Results: Both CNTF and FBS rapidlyaltered the gene expression of RPCs, with very marked upregulation ofglial fibrillary acidic protein (GFAP; FBSCNTF) and markeddown-regulation of the proliferation marker Ki-67,consistent with the induction of differentiation. The evidence supportsa preponderantly pro-glial influence for both the FBS and CNTF,however, neuronal markers were also upregulated to a lesser extent.Immunocytochemistry confirmed subpopulations labeling with neuronalmarkers, including rhodopsin. In the presence of sustained EGFstimulation, the differentiating influences of both FBS and CNTFremained perceptible as transient peaks of relative gene expression,but were markedly diminished overall. Conclusions: This study shows that it ispossible to compare the relative efficacy of in vitro differentiationprotocols using murine RPCs and qPCR. The differentiating influences ofboth serum and CNTF were confirmed, but shown to be powerfullymoderated by EGF. This suggests that EGF withdrawal is the dominantfeature of these differentiation protocols and that exposure to eitherserum or CNTF is insufficient to irreversibly commit a cultured RPCpopulation to terminal differentiation unless accompanied byconcomitant cessation of mitogenic stimulation.