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Indumathi Mariappan, Mangalangi Udayakumar, Savitri Maddileti; Characterization of Human Ciliary Pigmented Epithelial (CPE) Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1194.
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To culture and characterize the human ciliary pigmented epithelial (CPE) cells and to assess their potential to differentiate into retinal cell types.
Total RNA was isolated from uncultured CPE cells, retinal pigmented epithelial (RPE) cells and the neuro-retinal tissues of individual human cadaveric donor eyeballs. Semi-quantitative, RT-PCR profiling was done to compare the relative expression levels of Pax6, Nestin, Chx10, Otx2, Mitf, Crx, BMP7, LEF1 and Gli1. Adherent cultures of CPE was established using basal medium containing 10% FBS. Suspension cultures of neurospheres were done using neural induction medium (NIM: Basal medium containing 1% N2 supplement, 10 ng/ml bFGF and 100 ng/ml Noggin). Differentiation was induced using retinal differentiation medium (RDM: Basal medium containing 1% N2 supplement, 2% B27 supplement, 10 ng/ml bFGF) and the cells were assessed for marker expression by immunocytochemistry.
Freshly isolated, uncultured CPE cells expressed Mitf and BMP7 at similar levels as that of RPE cells and also expressed Chx10 at similar levels as that of neuro-retinal cells. This suggests that the CPE cells share some common genetic program with both the neuro-retinal and RPE cells. In adherent cultures, the pigmented cells of the ciliary epithelium adhered and underwent cell proliferation, which gradually lost their pigmentation upon expansion. The CPE cells also formed growing neurospheres in suspension cultures within 7-10 days at an efficiency of about 0.01%. While majority of the cells in the neurospheres (>90%) expressed Nestin and βIII-tubullin, only 10% of the cells were proliferating and expressed Ki67. Upon retinal differentiation, about 60% of the cells expressed the retinal stem cell marker, Rx, 30% cells expressed the neuro-retinal progenitor marker, Chx10 and only a rare population of cells (<5%) expressed the photoreceptor marker, rhodopsin. While the sub-cellular localization of Rx was normal and found to be in the nucleus, Chx10 was restricted to the cytosol, suggesting its inactive state.
CPE cells share a common gene expression signature with the neuro-retinal and RPE cells and a rare population of CPE cells retains the potential to proliferate and differentiate into retinal cells. However, the abnormal sub-cellular localization of Chx10 and the downstream signaling might contribute to the limited retinal potential of CPE cells.
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