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Kapil Bharti, Janine Davis, Barbara Corneo, Sally Temple, Sheldon Miller; Use of an iPS Reporter Cell Line Expressing RPE-Specific GFP For Improving iPS Cell to RPE Differentiation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2691.
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The use of induced pluripotent stem (iPS) cell-derived retinal pigment epithelium (RPE) for cell-based therapy requires growing cells using current Good Manufacturing Protocols. The currently available RPE differentiation protocols are not optimized for this purpose. The aim of this study is to develop and characterize an iPS reporter cell line for optimizing RPE differentiation.
A lentivirus construct containing a constitutively expressed red fluorescent protein (RFP) and an RPE-specific green fluorescent protein (GFP) was transduced into a fibroblast- derived iPS cell line. Four independently transduced RFP-positive colonies were differentiated into RPE using existing protocols and GFP expression was monitored. The ability of GFP-positive cells to differentiate into RPE cells was assessed by gene expression, imaging, FACS, and physiology assays.
GFP-positive cells were observed in differentiating iPS cell cultures 4-7 days after addition of ACTIVIN A. The GFP expression peaked at around 10-12 days when GFP-positive cells started to attain an epithelial morphology and initiate pigmentation. GFP positive-and negative-cells were separated by FACS and analyzed for RPE-specific and fibroblast gene expression using qRT-PCR analysis. As compared to the GFP-negative cells, GFP-positive cells showed several fold higher expression of early RPE transcription factors MITF, PAX6, OTX2, and TCFEC, pigmentation enzymes including TYROSINASE, TYRP, DCT, PMEL17, and OCA2, junctional protein CLAUDIN19, channel proteins TRPM1 and TRPM3, and the visual cycle protein RLBP1. In comparison, the expression of fibroblast genes, such as SNAIL1 and 2, was much lower in GFP-positive cells. This reporter line was further used to optimize the differentiation process and obtain significantly higher number of pigmented cells from iPS cells.
Expression and imaging analyses of the GFP-positive versus GFP-negative cells confirmed the specificity of the GFP reporter line. GFP expression marks iPS cells that initiate an RPE-differentiation program. This reporter line will be useful for several applications including high throughput screens to optimize iPS cell to RPE differentiation, genomic and proteomic analysis of differentiating RPE cells, and in tracking of RPE cells transplanted in pre-clinical animal models.
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