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Purpose: Till date there is no exclusive marker for human corneal epithelial stem cells (CESCs). In this study, our strategy is to combine high expression of ABCG2, a putative SC marker with high N/C ratio to develop a specific method for identification of CESCs., Methods: Limbal/corneal epithelial cells (LECs/CECs) were isolated from cadaver eyes by enzymatic treatment and the cytospin smears/cryosections were immunostained for ABCG2, p63, or connexin-43 (Cx-43) and counterstained with propidium iodide/DAPI. Analysis was carried out for (1) ABCG2 expression, N/C ratio, (2) p63, ABCG2, N/C ratio, and (3) ABCG2, Cx-43, N/C ratio using confocal/fluorescence microscopy., Results: ABCG2 was highly expressed by cells in the basal and suprabasal layers of limbus. The SCs identified on the basis of our earlier method using high levels of p63 expression along with high (>0.7) N/C ratio were strongly positive for ABCG2 and negative for differentiation marker Cx-43. High expression of ABCG2 was also observed in cells with high p63 expression and low (<0.7) N/C ratio; however, they were positive for Cx-43. Combining high ABCG2 expression with large N/C ratio in fluorescence microscopy identified 5.3 ± 3.5% LECs as putative SCs and this was confirmed by confocal microscopic analysis., Conclusions: We demonstrate the importance of combining ABCG2 expression with N/C ratio for identification and quantification of human CESCs. This method will have application in evaluating the role of niche cells/factors in the maintenance of stemness., (Copyright © 2012 Wiley Periodicals, Inc.)

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. [ABSTRACT FROM AUTHOR]

Corneal ulcers caused by Pseudomonas aeruginosa may lead to severe visual disability due to impaired bacterial clearance from corneal tissues. Our purpose was to study the role of autophagy in the intracellular clearance of P. aeruginosa from human corneal epithelial cells (HCET) and its regulation by the bacterial type III secretion system (T3SS) toxins. Nine different corneal ulcer isolates of P. aeruginosa , PAO1 and T3SS mutants of PAO1 were used to infect HCET cells. Induction of autophagy (Immunofluorescence and Western blot) and pro-inflammatory gene expression (real time PCR) by P. aeruginosa and the role of autophagy in intracellular bacterial clearance were studied in the context of T3SS genotypes. The clinical isolates and PAO1 induced autophagy irrespective of the T3SS genotype, whereas the T3SS mutants were relatively defective in inducing autophagy and becn1 gene expression. External induction of autophagy significantly reduced the intracellular load of P. aeruginosa strains that were associated with worst clinical outcomes. The T3SS negative isolate and PAO1Δ exoST were less sensitive to pharmacological modulation of autophagy and had relatively higher replication potential, suggesting a possible mechanism of bacterial survival in the absence of T3SS toxins. Overall, our results highlight a selective role for autophagy in bacterial clearance from corneal epithelial cells and emphasize the pro-autophagic role of bacterial toxins in the context of corneal ulcers. [ABSTRACT FROM AUTHOR]