Abstract
The objective of the study was to elucidate the microRNA (miRNA) profile of an enriched human corneal epithelial stem cell (CESC) population in comparison to differentiated central corneal epithelial cells (CCECs) by small RNA sequencing. The CESCs were enriched by differential enzymatic treatment to isolate the basal limbal epithelial cells followed by laser capture microdissection of cells with nucleus to cytoplasm ratio ≥0.7, from donor tissues. Small RNA sequencing was carried out using Illumina NextSeq. 500 platform and the validation of differentially expressed miRNAs by quantitative real-time PCR (qPCR) and locked nucleic acid miRNA in-situ hybridization (LNA-ISH). The sequencing identified 62 miRNAs in CESCs and 611 in CCECs. Six miRNAs: hsa-miR-21-5p, 3168, 143-3p, 10a-5p, 150-5p and 1910-5p were found to be significantly upregulated in enriched CESCs, which was further confirmed by qPCR and LNA-ISH. The expression of hsa-miR-143-3p was exclusive to clusters of limbal basal epithelial cells. The targets of the upregulated miRNAs were predicted to be associated with signaling pathways -Wnt, PI3K-AKT, MAPK and pathways that regulate pluripotency of stem cells, cell migration, growth and proliferation. Further studies are essential to elucidate their functional role in maintenance of stemness. The findings of the study also hypothesize the inherent potential of hsa-miR-143-3p to serve as a biomarker for identifying CESCs.
Highlights
Even though it is well accepted that the limbus is the site of stem cells for corneal epithelial homeostasis[10,11,12], the regulatory mechanism governing the maintenance of these corneal epithelial stem cell (CESC) is still not clear
The entire quantity of total RNA isolated from enriched CESCs was used for library preparation as the concentration was lesser than the detection limit in the Qubit analysis
The sequencing generated more than 11 million reads in CESCs and central corneal epithelial cells (CCECs)
Summary
Even though it is well accepted that the limbus is the site of stem cells for corneal epithelial homeostasis[10,11,12], the regulatory mechanism governing the maintenance of these CESCs is still not clear. The importance of miRNAs as a potential epigenetic regulator of stem cell potency, proliferation, differentiation and survival in embryonic stem cells[13,14], induced pluripotent stem cells[15,16] and adult tissue resident stem cells like human skin/hair follicle[17] has been reported. It is crucial to www.nature.com/scientificreports make use of a highly enriched CESCs in order to identify the profile of miRNA specific to adult tissue resident stem cells. In this study we have made use of the strategy we have developed earlier[7,18] to enrich CESCs to the extent of 80% and this enriched population are known to express stem cell markers ΔNp63α and ABCG218. It was possible to identify a specific profile of miRNA, significantly up-regulated in CESCs compared to differentiated CCECs19.
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