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.

Purpose: Using in vivo confocal microscopy, we established that unique hyperreflective structures in the anterior limbal stroma of healthy individuals represent the limbal stromal niche. The aim of this study was to characterize the limbal stromal microarchitecture in patients with limbal stem cell deficiency (LSCD). Methods: After obtaining informed consent, 10 patients with LSCD and 3 with macular corneal dystrophy were recruited. In vivo confocal imaging of the limbus and cornea of the affected and normal eyes was performed using an HRT III laser scanning microscope, beyond the epithelium deep into the stroma. Results: In the case of LSCD, the limbal epithelium was replaced by conjunctival epithelium. A large number of inflammatory and dendritic cells were identified along with blood vessels from the epithelium to deep stromal layers. The unique hyperreflective niche structures were replaced by homogenously bright fibrous structures in all eyes with total LSCD. In patients with partial LSCD, even the clinically defined normal limbus had fibrotic stroma. In a patient with focal LSCD, only the affected limbal stroma remained fibrotic, whereas the adjacent clinically normal limbus had the unique hyperreflective structures. Although the opaque corneal stroma appeared bright because of proteoglycan deposition, it was possible to identify the normal limbal epithelial and stromal architecture in macular corneal dystrophy. Conclusions: In the case of LSCD, the limbal stromal niche was replaced by bright fibrotic structures indicating persistence of damage several months after injury. Further studies are required to characterize the sequential events occurring in the anterior limbal stroma after injury using this noninvasive method.

Fibrotic cataracts, posterior capsular opacification (PCO), and anterior subcapsular cataracts (ASC) are mainly attributed to the transforming growth factor-β (TGFβ)-induced epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs). Previous investigations from our laboratory have shown the novel role of non-canonical TGFβ signaling in the progression of EMT in LECs. In this study, we have identified YAP as a critical signaling molecule involved in lens fibrosis. The observed increase in nuclear YAP in capsules of human ASC patients points toward the involvement of YAP in lens fibrosis. In addition, the immunohistochemical (IHC) analyses on ocular sections from mice that overexpress TGFβ in the lens (TGFβ^tg) showed a co-expression of YAP and α-SMA in the fibrotic plaques when compared to wild-type littermate lenses, which do not. The incubation of rat lens explants with verteporfin, a YAP inhibitor, prevented a TGFβ-induced fiber-like phenotype, α-SMA, and fibronectin expression, as well as delocalization of E-cadherin and β-catenin. Finally, LECs co-incubated with TGFβ and YAP inhibitor did not exhibit an induction in matrix metalloproteinase 2 compared to those LECs treated with TGFβ alone. In conclusion, these data demonstrate that YAP is required for TGFβ-mediated lens EMT and fibrosis.

Purpose: To characterize the microarchitecture of anterior limbal stroma in healthy individuals using in vivo confocal microscopy (IVCM) and to correlate it with mesenchymal stem cells (MSCs), a component of the limbal niche. Methods: The corneal side of the superior limbus was scanned in 30 eyes of 17 normal subjects beyond the basal epithelium, deep into the stroma using an HRT III laser scanning microscope. The IVCM findings were correlated with the immunohistochemical features of MSCs in the anterior limbal stroma. Results: Clusters of hyperreflective structures were observed in the anterior limbal stroma, subjacent to the basal epithelium (depth, 50.2 ± 8.7 μm to 98 ± 12.8 μm), but not in the corneal stroma. The structures showed unique morphology compared with epithelial cells, keratocytes, neurons, and dendritic cells. In parallel, confocal analysis of immunostained sections showed clusters of cells, double positive for MSC-specific markers (CD90 and CD105) in the anterior limbal stroma at a depth of 55.3 ± 12.7 μm to 72 ± 37.6 μm. The organization and distribution of the MSC clusters locates them within the hyperreflective region in the anterior limbal stroma. Conclusions: The hyperreflective structures, demonstrated for the first time in the human anterior limbal stroma, probably represent an important component of the limbal niche. Our approach of in vivo imaging may pave the way for assessing the limbal stromal health.

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.

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.