The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.
Ocular surface reconstruction is essential for treating corneal epithelial defects and vision recovery. Stem cell-based therapy demonstrates promising results but requires further research to elucidate stem cell survival, growth, and differentiation after transplantation in vivo. This study examined the corneal reconstruction promoted by EGFP-labeled limbal mesenchymal stem cells (L-MSCs-EGFP) and their fate after transplantation. EGFP labeling allowed us to evaluate the migration and survival rates of the transferred cells. L-MSCs-EGFP seeded onto decellularized human amniotic membrane (dHAM) were transplanted into rabbits with a modeled limbal stem cell deficiency. The localization and viability of the transplanted cells in animal tissue were analyzed using histology, immunohistochemistry, and confocal microscopy up to 3 months after transplantation. EGFP-labeled cells remained viable for the first 14 days after transplantation. By the 90th day, epithelialization of the rabbit corneas reached 90%, but the presence of viable labeled cells was not observed within the newly formed epithelium. Although labeled cells demonstrated low survivability in host tissue, the squamous corneal-like epithelium was partially restored by the 30th day after transplantation of the tissue-engineered graft. Overall, this study paves the way for further optimization of transplantation conditions and studying the mechanisms of corneal tissue restoration.
Highlights. Notch signaling is known to be important regulator of endothelium homeostasis and cardiovascular disease. Particularly, Notch seems to be associated with pathological changes in endothelium epigenome although no such Notch effects have been found. We have discovered that activation of Notch signaling alters histone 1 repertoire in the human endothelial cells and this is the first example of epigenomic Notch targets.Aim. The disturbance of blood flow and alteration of physiological shear stress is one of the main reasons for endothelial dysfunction. Mechanosensitive and dosedependent Notch pathway is assumed to be an important player of endothelial dysfunction progression, but the molecular mechanisms of the influence of Notch dysregulation on endothelium are still not understood. In particular, there is no data about possible targets of Notch in the endothelial epigenome.Methods. Here we focused on the analysis of changes in histone code of human umbilical vein endothelial cells (HUVEC) after activation of Notch. For this purpose, we transduced cells by lentiviruses with construction for Notch 1 intracellular domain (N1ICD) overexpression or by empty vector (control). Then we isolated histone enriched fraction and secretome proteins and performed their shotgun proteomics analysis on timsToF Pro instrument. Proteomics data are available via ProteomeXchange with identifier PXD032978.Results. We found the shift in proteomics profile of HUVEC caused by Notch activation and, particularly, the increase in the levels of N-terminal acetylated forms of histone 1: H1-0, H1-3, H1-4, H1-5, H1-10. We also found changes in the cell secretome profile which are associated with the decrease in proangiogenic effect of HUVEC secretome.Conclusion. Our data identified epigenomic Notch targets and we assume that changes in H1 repertoire might be associated with cardiovascular disease progression in vivo.
Objective: to conduct an experimental study on the properties of three different types of synthetic polyester matrices, to carry out their comparative assessment, and to identify the optimal carrier for the cultivation and transplantation of limbal stem cells while eliminating limbal insufficiency. Materials and methods. Transparency, mechanical properties (strength, relative elongation at break, and elastic modulus), biocompatibility with corneal cell cultures were assessed, and duration of matrix biodegradation in vivo were studied. Results. In the course of our study, the optical and mechanical properties of matrices, made of polylactide-glycolide (PLG), polylactide-caprolactone (PLC) and poly-E-caprolactone (PCL), were studied. It has been experimentally shown that limbal stem cells of humans and rabbits, as well as human corneal epithelial cells, adhered to the surface of all types of the studied matrices. During the cultivation process, they retained the typical structure of the actin cytoskeleton, along with the ability to proliferate and migrate. Differences in the interaction of different cell cultures with different types of carriers were revealed. The biodegradation time of 5 μm thick PLC matrices was about 30 days. Conclusion. Our results obtained implied the possibility of using 5 µm thick PLC matrices as a carrier for cultured limbal stem cells.
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