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Jodhbir Mehta, Adrian Cheong, Gary Peh, William Sun; Identification of New markers of Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2198.
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There are no specific markers of human corneal endothelial cells (HCEC). Currently, the identification of HCEC in culture relies mainly on expression of pump or tight junction markers.The aim of this study is to describe new HCEC markers.
Isolated human corneal DM-HCEC and remaining stroma from corneal donors were homogenized individually for RNA sequencing. RNA-seq libraries were prepared using AB protocol for non-barcoded libraries and SOLiD fragment library for barcoded library. The results were processed using ABI Bioscope. Gene expression was measured by counting the number of reads mapping uniquely to both strands of each gene footprint. Results were verified by quantitative PCR, BD lyoplate screening, immunofluorescence and flow cytometry using cultivated primary HCEC isolated and grown to the third passage.
RNA-seq showed 5 genes that were over expressed in the HCEC-DM compared to stromal fibroblasts, GPC4, CNTN6, SLC9A7, PVRL3, and SLC4A4. The resulting over-expression of these genes was confirmed on comparing qPCR data from cultured HCEC and stromal fibroblasts. BD lyoplate identified CD104 and CD200 as potential markers. On immuno-histochemistry anti-GPC4, anti-CD200 and anti-SLC4A cell-surface antibodies clearly stained the the endothelial layer specifically. In a population of pre-CMFDA labelled stromal fibroblasts and HCEC, mixed in a 1:1ratio fluorescent-activated cell sorting (FACS) showed a 96% recovery of HCEC when sorted with anti-GPC4 and a 79.8% with anti-CD200.
By RNA sequencing verified on qPCR and immunohistochemistry we have identified two novel cell surface antigens on human corneal endothelial cells. These markers may be used to aid cell purification during harvesting or in the identification of cultured HCEC to ensure quality control of cultured cells.
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