Induction of Corneal Epithelial Differentiation of Induced Pluripotent and Orbital Fat-Derived Stem Cells Seeded on Decellularized Human Corneas.

Up to 40% of donor corneas are deemed unsuitable for transplantation, aggravating the shortage of graft tissue. In most cases, the corneal extracellular matrix is intact. Therefore, their decellularization followed by repopulation with autologous cells may constitute an efficient alternative to reduce the amount of discarded tissue and the risk of immune rejection after transplantation. Although induced pluripotent (hiPSCs) and orbital fat-derived stem cells (OFSCs) hold great promise for corneal epithelial (CE) reconstruction, no study to date has evaluated the capacity of decellularized corneas (DCs) to support the attachment and differentiation of these cells into CE-like cells. Here, we recellularize DCs with hiPSCs and OFSCs and evaluate their differentiation potential into CE-like cells using animal serum-free culture conditions. Cell viability and adhesion on DCs were assessed by calcein-AM staining and scanning electron microscopy. Cell differentiation was evaluated by RT-qPCR and immunofluorescence analyses. DCs successfully supported the adhesion and survival of hiPSCs and OFSCs. The OFSCs cultured under differentiation conditions could not express the CE markers, TP63, KRT3, PAX6, and KRT12, while the hiPSCs gave rise to cells expressing high levels of these markers. RT-qPCR data suggested that the DCs provided an inductive environment for CE differentiation of hiPSCs, supporting the expression of PAX6 and KRT12 without the need for any soluble induction factors. Our results open the avenue for future studies regarding the in vivo effects of DCs as carriers for autologous cell transplantation for ocular surface reconstruction.

Epithelial basement membrane of human decellularized cornea as a suitable substrate for differentiation of embryonic stem cells into corneal epithelial-like cells.

The ability to decellularize and recellularize the corneas deemed unsuitable for transplantation may increase the number of available grafts. Decellularized corneas (DCs) may provide a natural microenvironment for cell adhesion and differentiation. Despite this, no study to date has evaluated their efficacy as a substrate for the induction of stem cell differentiation into corneal cells. The present study aimed to compare the efficiency of NaCl and NaCl plus nucleases methods to decellularize whole human corneas, and to investigate the effect of epithelial basement membrane (EBM) of whole DCs on the ability of human embryonic stem cells (hESCs) to differentiate into corneal epithelial-like cells when cultured in animal serum-free differentiation medium. As laminin is the major component of EBM, we also investigated its effect on hESCs differentiation. The decellularization efficiency and integrity of the extracellular matrix (ECM) obtained were investigated by histology, electron microscopy, DNA quantification, immunofluorescence, and nuclear staining. The ability of hESCs to differentiate into corneal epithelial-like cells when seeded on the EBM of DCs or laminin-coated wells was evaluated by immunofluorescence and RT-qPCR analyses. NaCl treatment alone, without nucleases, was insufficient to remove cellular components, while NaCl plus nucleases treatment resulted in efficient decellularization and preservation of the ECM. Unlike cells induced to differentiate on laminin, hESCs differentiated on DCs expressed high levels of corneal epithelial-specific markers, keratin 3 and keratin 12. It was demonstrated for the first time that the decellularized matrices had a positive effect on the differentiation of hESCs towards corneal epithelial-like cells. Such a strategy supports the potential applications of human DCs and hESCs in corneal epithelium tissue engineering.

The proteolytic fraction from Vasconcellea cundinamarcensis accelerates wound healing after corneal chemical burn in rabbits.

INTRODUCTION: Chemical ocular burns are among the most frequently eye-related injuries, which require immediate and intensive evaluation and care since they may lead to potential complications such as superinfection, corneal perforation, and blindness.Vasconcellea cundinamarcensis, a species from Caricaceae family, contains highly active proteolytic enzymes in its latex that show healing activity in animal models bearing lesions of different etiologies. METHODS: We evaluate the ocular toxicity of the proteolytic fraction from V. cundinamarcensis (P1G10) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hen's Egg Test-Chorioallantoic Membrane test. The corneal healing property of P1G10 was studied by the ethanol-chemical burn in the rabbit's eyes. RESULTS: P1G10 is safe for ocular administration, except when administrated at 10µg/mL. P1G10 at 1µg/mL accelerates the corneal re-epithelization achieving complete wound closure after 72h of chemical burn. Also, P1G10 modulated the inflammatory response and controlled the arrangement of collagen fibers in the stroma, demonstrating its potential corneal healing properties. CONCLUSIONS: Our work was the first one to evaluate the ophthalmic application of P1G10. Here we demonstrated that P1G10 is suitable for ocular administration and it has a promising corneal healing activity which may emerge as a new pharmacological tool to the development of a new drug for ocular surface chemical injuries in the future.