Hsa-miR-150-5p inhibits Wnt-ß-catenin signaling in human corneal epithelial stem cells.

Purpose: In our earlier study, we identified hsa-miR-150-5p as a highly expressed miRNA in enriched corneal epithelial stem cells (CESCs). In this study, we aimed to understand the molecular regulatory function of hsa-miR-150-5p in association with the maintenance of stemness in CESCs. Methods: The target mRNAs of hsa-miR-150-5p were predicted and subjected to pathway analysis to identify targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-150-5p mimic, inhibitor, or scrambled sequence using Lipofectamine 3000. The transfected cells were analyzed to determine (i) their colony-forming potential; (ii) the expression levels of stem cell (SC) markers/transcription factors (ABCG2, NANOG, OCT4, KLF4, and ΔNp63), the differentiation marker (Cx43), and the hsa-miR-150-5p predicted targets (JARID2, INHBA, AKT3, and CTNNB1) by qPCR; and (iii) the expression levels of ABCG2, p63α, Cx43, JARID2, AKT3, p-AKT3, ß-catenin, and active ß-catenin by immunofluorescence staining and/or western blotting. Results: The ectopic expression level of hsa-miR-150-5p increased the colony-forming potential (8.29% ± 0.47%, p < 0.001) with the ability to form holoclone-like colonies compared with the control (1.8% ± 0.47%). The mimic-treated cells had higher expression levels of the SC markers but reduced expression levels of Cx43 and the targets of hsa-miR-150-5p that are involved in the Wnt-ß-catenin signaling pathway. The expression levels of ß-catenin and active ß-catenin in the inhibitor-transfected cells were higher than those in the control cells, and the localized nuclear expression indicated the activation of Wnt signaling. Conclusions: Our results indicate a regulatory role for hsa-miR-150-5p in the maintenance of CESCs by inhibiting the Wnt signaling pathway.

Functional characterization of adult human trabecular meshwork stem cells.

We earlier identified native human trabecular meshwork stem cells (TMSCs) based on two-parameters- high ABCG2 expression and high nucleus to cytoplasmic ratio. The TMSCs also expressed p75 and AnkyrinG. Based on the high expression of ABCG2 and p75, the TMSCs were identified to be located in the Schwalbe's line region of the TM. In continuation, the current study aimed at elucidating the functional characteristics of human TMSCs. Upon culturing, only a small proportion of TM cells (0.96 ± 0.21% in <30 years) expressing stem cell markers ABCG2 and p75 adhered to the culture dish. This proportion significantly reduced with ageing (0.32 ± 0.23% in 30-60 years and 0.35 ± 0.04% in >60 years). Characterization of the primary TM cultures identified 7.00 ± 1.80% of stem cells with label retaining property. Further, cultured cells had the ability to form TM spheres (0.82 ± 0.23%) which consisted of high ABCG2 and p75 positive cells. Upon dexamethasone induction, 86.00 ± 14.87% and 64.60 ± 7.24% of the cells derived from the TM spheres expressed myocilin and exhibited cross linked actin networks respectively, indicating differentiation of the TMSCs in the sphere to TM cells. In addition, the sphere derived TM cells also possessed phagocytic potential (13.28 ± 3.30%) equivalent to primary TM cells (16.33 ± 4.04%) which was evident upon internalization of zymosan particles. In conclusion, this study has established that a proportion of cultured TM cells had the label retaining property as well as sphere forming ability of adult stem cells. Thus, these results confirm the presence of adult stem cells in the human TM that might be responsible for the maintenance of tissue homeostasis.

Age-related reduction in the functional properties of adult stem cells located in the peripheral region of human retinal pigment epithelium.

PURPOSE: Adult stem cells (SCs) with self-renewal and multilineage potential have been reported upon culturing human retinal pigment epithelial (RPE) cells. The current study aimed to identify the location of SCs in human RPE and to elucidate the age-related changes. METHODS: Peripheral, equatorial, and central RPE cells from donors of three age groups were analyzed for their sphere-forming, clonal, and label-retaining cell properties. Furthermore, native human RPE flatmounts were immunostained for SC and proliferating cell markers. RESULTS: Cells with higher sphere-forming and clonal ability were identified only in young donors (<30 years) and were restricted to the periphery. Upon culturing, cells from peripheral and equatorial regions had the label-retaining cell (LRC) property. With aging, the LRCs were restricted to the periphery and were reduced. In young donors, Ki67 + proliferating cells were not observed in native RPE. However, such cells were observed in the peripheral RPE of older donors correlating with the need for regeneration. The native RPE cells were negative for SC marker expression. CONCLUSION: The above findings highlighted the presence of SCs with the ability to proliferate in the peripheral RPE and a reduction in these functional properties of SCs with aging.

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

Our previous study demonstrated hsa-miR-143-3p as one of the highly expressed miRNAs in enriched corneal epithelial stem cells (CESCs). Hence this study aims to elucidate the regulatory role of hsa-miR-143-3p in the maintenance of stemness in CESCs. The target genes of hsa-miR-143-3p were predicted and subjected to pathway analysis to select the targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-143-3p mimic, inhibitor or scrambled sequence using Lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential, (ii) expression of stem cell (SC) markers/ transcription factors (ABCG2, NANOG, OCT4, KLF4, ΔNp63), (iii) differentiation marker (Cx43), (iv) predicted five targets of hsa-miR-143-3p (DVL3, MAPK1, MAPK14, KRAS and KAT6A), (v) MAPK signaling regulators and (vi) Wnt-ß-catenin signaling regulators by qPCR, immunofluorescence staining and/or Western blotting. High expression of hsa-miR-143-3p increased the colony forming potential (10.04 ± 1.35%, p < 0.001) with the ability to form holoclone-like colonies in comparison to control (3.33 ± 0.71%). The mimic treated cells had increased expression of SC markers but reduced expression of Cx43 and hsa-miR-143-3p targets involved in Wnt-ß-catenin and MAPK signaling pathways. The expression of ß-catenin, active ß-catenin and ERK2 in hsa-miR-143-3p inhibitor transfected cells were higher than the control cells and the localized nuclear expression indicated the activation of Wnt and MAPK signaling. Thus, the probable association of hsa-miR-143-3p in the maintenance of CESCs through inhibition of Wnt and MAPK signaling pathways was thus indicated.

Reduction in trabecular meshwork stem cell content in donor eyes with primary open angle glaucoma.

We previously identified and characterized human trabecular meshwork stem cells (TMSCs) based on high expression of ABCG2/p75 positivity and high nucleus to cytoplasmic ratio. These TMSCs expressing high ABCG2 and p75 were located to the insert region of the human TM. Additionally, we demonstrated an age-related reduction in the TMSC content which was significantly associated with TM cell loss. In continuation, this study was aimed to determine the TMSC content in glaucomatous donor eyes wherein a drastic reduction in TM cellularity has already been reported. Anterior segments from known glaucomatous (n = 6) and age-matched normal (n = 8) donors were dissected into four quadrants. A minimum of three sections from each quadrant were used for histopathological analysis as well as immunostaining. Analysis of hematoxylin and eosin-stained sections from glaucomatous tissues revealed a decrease in total TM cellularity, thickening of trabecular beams, fusion of trabeculae, absence of patent Schlemm's canal compared to age-matched controls. In addition, the TM thickness at various positions of the meshwork and the coronal as well as the meridional diameters of the Schlemm's canal were observed to be significantly reduced in glaucomatous eyes. Further, sections from both the groups were immunostained for universal stem cell marker ABCG2 and neural crest derived stem cell marker p75. The images were acquired using Leica SP8 confocal microscope. Quantification of total TM cellularity based on nuclear counterstain (mean ± SD) using ImageJ identified 69.33 ± 12.77 cells/section in control eyes. In glaucomatous donors, the TM cellularity was found to be reduced significantly to 41.83 ± 9.0 (p = 0.0007). In addition, a reduction in the percentage of TMSCs (cells with high ABCG2 expression and p75 positivity) was evident in glaucomatous donors (0.14 ± 0.17%) compared to age-matched controls (4.73 ± 5.46%) (p = 0.064). Thus, the present study confirmed the significant decline in TM cellularity and a reducing trend in the TMSC content, though this reduction was non-significant in glaucomatous donor eyes. Further studies are essential to elucidate the role of TMSCs in the pathogenesis of primary open angle glaucoma.

Characterization of age-related macular degeneration in Indian donor eyes.

Purpose: The purpose of this study was to test the reliability of fundus stereomicroscopy in postmortem eyes to assign severity of age-related macular degeneration (AMD) using the Minnesota grading and confirmation by histology using Alabama and Sarks grading scales and to assess the incidence of AMD pathology in donor eyes from a South Indian population. Methods: Eyes (199) from 153 donors (55-95 years) after obtaining fundus images were processed for histology. Fundus images were graded according to the Minnesota grading system based on drusen size, area of depigmentation, and atrophy. At least one eye from each donor displaying the AMD phenotypes were subjected to histological examination. The fundus grading was correlated with histology and the stages of AMD assigned for early AMD by the Alabama AMD grading system and for both early and advanced AMD by the Sarks classification. Results: Stereoscopic examination of the fundus found that 10 of the 153 donors had features of early AMD and 3 advanced AMD. Following histological examination, one of the early AMD eyes was reclassified as advanced AMD. Early AMD features that were observed on histology included soft drusen (>63 µm), basal laminar deposits, photoreceptor outer segment degeneration, disorganization of retinal pigment epithelium (RPE), Bruch's membrane thickening. Advanced AMD features observed in histology are extensive atrophy of RPE, choroidal neovascularization and disciform scar formation. . Conclusion: Identification of either early or advanced AMD using stereomicroscopic assessment (SMA) showed high sensitivity and specificity. However, misclassification between AMD stages can occur when only SMA is used.

MicroRNA Profiling of Highly Enriched Human Corneal Epithelial Stem Cells by Small RNA Sequencing.

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.

Identification, quantification and age-related changes of human trabecular meshwork stem cells.

BACKGROUND: Loss of cells in the human trabecular meshwork (TM) has been reported with ageing and in glaucoma. This study aims to identify, quantify and determine the age-related changes of human TM stem cells (TMSCs). METHODS: Isolation of TM cells/ paraffin sectioning was carried out using human corneoscleral rings and whole globes. The TM cells/ sections were immunostained for the stem cell markers ATP-binding cassette protein G2 (ABCG2), nerve growth factor receptor p75 and AnkyrinG (AnkG). Images were acquired using Leica SP8 confocal microscope. The isolated cells were analyzed for two parameters- ABCG2 expression and nucleus to cytoplasmic ratio (N/C ratio). The total number of TM cells and those positive for ABCG2 and p75 in each section were quantified. Spearman rank order correlation was used to determine the association between age and the cell counts. RESULTS: The TMSCs were identified based on two parameters- high ABCG2 expression and high N/C ratio > 0.7. These stem cells were also positive for p75 and AnkG. The TMSC content based on the two parameters was 21.0 ± 1.4% in < 30 years age group, 12.6 ± 6.6% in 30-60 years and 4.0 ± 3.5% in > 60 years. The stem cells with high ABCG2 and p75 expression were restricted to the Schwalbe's line region of the TM. A significant correlation was observed between the reduction in TMSC content and TM cell count during ageing. CONCLUSION: The human TMSCs were identified and quantified based on two parameter analysis. This study established a significant association between age-related reduction in TMSC content and TM cell loss.

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

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, ß, 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.