ES Micro-Environment Enhances Stemness and Inhibits Apoptosis in Human Limbal Stem Cells via the Maintenance of Telomerase Activity

Abstract

Our previous work had found that telomerase rejuvenated in the cytoplasm of corneal epithelial cells cultured in embryonic stem cell-conditioned medium, the functional properties of stem-like corneal epithelial cells can be enhanced by co-culturing with embryonic stem cells (ESCs) via activation of the integrinβ1-FAK-PI3K/Akt signaling pathway. The goal of this study was to explore the potential molecular mechanisms of the ES micro-environment that enhance the stem cell-like phenotype and inhibit apoptosis in human limbal stem cells (LSC). The LSC were cultured in different media, either CnT-20 medium or CnT-20 +20% ES culture supernatant (ESC-CM). We observed that LSC cultured in ESC-CM had an increased proliferative capacity, greater serial passage capacity, higher colony-forming efficiency (CFE) and higher levels of stem cell-associated marker than those cultured in CnT-20. Compared with CnT-20, ESC-CM enhanced the undifferentiated status and inhibited apoptosis in the LSC by promoting the maintenance of telomerase activity, which could reduce the generation of reactive oxygen species (ROS), maintain the membrane potential (Δψm) at higher levels and reduce the expression of the p21 protein. Our findings indicated that ESC-CM system induced LSC to maintain a stem cell phenotype and inhibit the process of apoptosis. These effects might partially be achieved via the telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways. This study may have high impact and clinic implication on the expansion of LSC in regenerative medicine, especially for ocular surface reconstruction.

Importance of the stem cell microenvironment for ophthalmological cell-based therapy

“…We have considered two aspects in our studies: (1) cellular microenvironment: embryonic stem cell microenvironment culture systems can make the differentiated corneal epithelial cells, conjunctiva epithelial cells [15] and even human corneal endothelial cells [17] obtain a strong proliferation ability and can be passaged long-term with de-differentiation cell marker expression, normal cell morphology and karyotype, but no tumorigenicity. Our preliminary findings showed that the ES microenvironment may inhibit the apoptosis of the cells by activating telomerase via integrin the b1-FAK-PI3K/Akt, telomerase-p21-mitochondrial axis and FAK/Wnt signaling pathway [18,144] ; and (2) stromal microenvironment: the APCS limbal produced by lipase (not existing protease digestion) [25,30] can retain the normal extracellular matrix, collagen lamellar micro ultrastructure. It can repair and maintain the stemness and proliferation ability of the limbal stem cells after it is transplanted to the limbal stem cell deficiency rabbit model.…”

The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance

“…In recent years, the discovery of telomere-independent effects of TERT on proliferation [87][88][89][90][91][92][93][94][95] and perturbations of resistance to reactive oxygen species damage [96][97][98][99][100] have led to a burgeoning body of evidence that indicates that TERT has many functions outside it's canonical role in telomere length maintenance. 101 Some of these extra-telomeric effects include acting as an RNAdependent RNA polymerase, 102 a mitochondrial reverse transcriptase, 103 interacting with nucleostemin (GNL3), 104 WNT, BRG1, b-catenin, 87,105 NF-kB, 106 and modulating endogenous siRNA 102,107 and miRNA profiles.…”

“…TERT is capable of aiding the maintenance of self-renewal in human limbal and mesenchymal stem cells, increasing the efficiency of directed differentiation, 48,99 enhancing iPSC reprogramming efficiency 40 and activating resting hair follicle stem cells. 90 Several recent reports have shown that TERT is able to interact with BRG1 in a WNT/b-catenin dependent manner.…”

“…[96][97][98][99][100]118,119 It is difficult to unravel the contributions from non-canonical TERT effects versus telomeric effects. The canonical telomere shortening effects generally occur after extended proliferation, and are mediated by telomeric dysfunction.…”

Enhanced functional properties of human limbal stem cells by inhibition of the miR‐31/FIH‐1/P21 axis

“…Studies have shown that LSCs undergo more frequent cell divisions than terminally differentiated corneal epithelial cells (TDCs) and could be cultured ex vivo from limbal tissues (Liu et al. ), which could represent a source of cells for transplantation in the treatment of LSCs deficiency (LSCD) (Rama et al. ; Chae et al.…”