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J. Yang, P. Gu, H. Klassen; CNTF Induces Rapid Expression of Neuronal and Glial Transcripts in Cultured Porcine Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1266.
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Work in rodent and avian models has shown that ciliary neurotrophic factor (CNTF) has a differentiating influence on retinal progenitor cells (RPCs), however, it is less clear whether this is the case in large mammals. CNTF also has a role in retinal neuroprotection and genetically modified RPCs are a potential method of delivery, in which case it is important to understand the influence of this factor on the delivering cell type. Here we examine the impact of exogenous CNTF on the gene expression profile of RPCs derived from the fetal pig.
RPCs originally derived at day-60 gestational age were expanded in proliferation medium containing DMEM/F12 (1:1), N2 supplement, 20 ng/ml EGF and 20 ng/ml bFGF. Medium was changed every 2 days and cells passaged at confluence with trypsin. At passage 4, medium was completely removed and fresh medium containing 10 ng/ml CNTF, without EGF or bFGF, was added to the experimental flasks, whereas non-treated controls received fresh proliferation medium. Medium was changed every 2 days for the following 7 days. On days 0, 1, 3, 5 and 7, total RNA was extracted (Qiagen RNeasy Mini kit) followed by reverse transcription (Omniscriptase Reverse Transcriptase kit, Qiagen) and real-time RT PCR assay (ABI 7500). β-Actin was used as an endogenous control to normalized gene expression.
Cells in the CNTF treatment condition showed notable up-regulation of transcripts for the surface receptor CXCR4 (up to 6.2 fold), rhodopsin (up to 4.5 fold), and GFAP (up to 3.1 fold) together with down-regulation of nestin (up to 3.7 fold). Genes with <2 fold changes included PKC, β3-tubulin, Ki-67, Hes1, Sox2, C-myc, Klf4 and vimentin. Temporal changes in expression were observed and varied by transcript and treatment time point.
Replacement of EGF and bFGF with CNTF rapidly alters the gene expression of porcine RPCs, resulting in rapid down-regulation of the progenitor marker nestin and up-regulation of the migration-associated marker CXCR4 as well as the mature markers rhodopsin and GFAP. These results are consistent with diminution of progenitor status along with induction of differentiation along both neuronal and glial pathways. Any contemplated use of RPCs for intraocular delivery of CNTF will need to take into account the effects of the factor on the carrier cells.
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