A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis

Abstract

Understanding the molecular mechanisms that regulate the corneal epithelial stem cells (CESCs) in maintaining corneal homeostasis remains elusive largely due to the lack of a specific marker for their isolation. This study aims to enrich CESCs from human donor limbal epithelium and to evaluate the level of enrichment based on expression of ΔNp63α, a putative CESC marker. A two-stage enrichment of CESCs was carried out. (a) The limbal basal epithelial cells were isolated by differential enzymatic treatment and five-fold enrichment was achieved from 2% of CESCs present in the total limbal epithelium. The CESCs were quantified on the basis of two parameters-high expression of p63/ABCG2 and nucleus to cytoplasmic (N/C) ratio ≥0.7. (b) Cytospin smears of isolated basal cells were Giemsa stained and cells with N/C ratio ≥0.7 were separated by laser capture microdissection. This strategy resulted in an enrichment of CESCs to 78.57% based on two-parameter analysis using p63 and 76.66% using ABCG2. RT-PCR was carried out for ΔNp63 isoforms (α, β, and γ) and connexin-43, with GAPDH for normalization. The expression of ΔNp63α was restricted to the enriched population of CESCs in contrast to its absence in limbal basal cells with N/C ratio <0.7 and CCECs. The unique expression of ΔNp63α and 5.9-fold reduced connexin-43 expression in the enriched population of CESCs indicates its high purity. Further analysis of these cells will help in elucidating the molecular mechanisms associated with stemness and also in identifying a specific marker for CESCs.

An Insight into the Difficulties in the Discovery of Specific Biomarkers of Limbal Stem Cells

“…Priya et al successfully identified and quantified LSCs based on ABCG2 and N/C ≥ 0.7 [ 69 ]. Additionally, Kasinathan et al established a two-step protocol by combining basal cell isolation and laser capture microdissection (LCM) of small cells with N/C ≥ 0.7 for LSC enrichment, which achieved 76–78% enrichment of LSC from 2% LSCs in total LECs [ 104 ]; Label-retaining cell: bromodeoxyuridine (BrdU) can take the place of thymidine to be incorporated into the replicated DNA during the S-phase of the cell cycle. The BrdU-based “pulse-chase” experiment has been widely applied for the identification of stem cell.…”

“…Priya et al successfully identified and quantified LSCs based on ABCG2 and N/C ≥ 0.7 [ 69 ]. Additionally, Kasinathan et al established a two-step protocol by combining basal cell isolation and laser capture microdissection (LCM) of small cells with N/C ≥ 0.7 for LSC enrichment, which achieved 76–78% enrichment of LSC from 2% LSCs in total LECs [ 104 ];…”

The limbus: Structure and function

“…There have therefore been a number of recent marker-discovery models demonstrating ways in which a more representative LESC pool may be enriched and isolated for study. One such strategy is laser capture microdissection (LCM), in which cells can be selectively removed from tissue section based on features like size and position, providing an enriched pool which can be far more informative for downstream analysis (Bath et al, 2013;Kasinathan et al, 2016;Menzel-Severing et al, 2018;Polisetti et al, 2016). Another approach has been to purify a slow-cycling population of limbal epithelial cells using an inducible transgenic "pulse-chase" murine model, wherein the retention of a GFP label identifies quiescent cells within the tissue for co-expression studies and fluorescenceactivated cell sorting (FACS) for transcriptomic analysis (Sartaj et al, 2017).…”

Hsa-miR-143-3p inhibits Wnt-β-catenin and MAPK signaling in human corneal epithelial stem cells

“…In our previous study, CESCs have been enriched to 80% using a two-step protocol 8 and compared to differentiated central corneal epithelial cells by small RNA sequencing. Six miRNAs- hsa-miR-3168, hsa-miR-21-5p, hsa-miR-143-3p, hsa-miR-150-5p, hsa-miR-10a-5p and hsa-miR-1910-5p were identified to be highly expressed in the enriched CESCs and validated through qPCR.…”