Rapidly constructed scaffold-free embryonic stem cell sheets for ocular surface reconstruction

Abstract

This study is an extension of our previously published work demonstrating the generation of corneal epithelial sheets using optimal centrifugation procedure. In this study, we investigated the therapeutic potential of embryonic stem cells (ESCs) for ocular surface reconstruction in rabbits with limbal stem cell deficiency (LSCD) using the centrifugation method. Rabbits with LSCD were assigned to two groups: 30 LSCD rabbits were treated with scaffold-free embryonic stem cells sheets (SESCS) and six LSCD rabbits received no treatment (control group). The two groups were followed up for 15 days using slit lamp observation. Cytokeratin K3, mucin 5AC, and OCT-4 were used to evaluate the re-epithelialization of the cornea. Fluorescent DiIC18(3)-DS dye was used to trace the transplanted cells. Atomic force microscopy (AFM) was used to investigate the cultured epithelial cells of the SESCS-treated group. The SESCS transplant facilitated the reconstruction of the ocular surface in 75% of the treated animals. Conjunctivalization and neovascularization were observed in the control group. The SESCS group was K3 positive and MUC5AC negative, and OCT-4 was observed on the re-epithelialized corneal epithelium. Labeled SESCS cells were detected in vivo at 15 days post-transplant. Using AFM, three different types of cultured cells were identified in the rabbit corneal epithelium of the SESCS treatment group. The SESCS were demonstrated to reconstruct the ocular surface in rabbits with LSCD. ES cells differentiated into corneal epithelial cells when in direct contact with the stroma and thus can serve as a cell source in corneal tissue engineering.

Therapeutic Effect of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Experimental Corneal Failure Due to Limbal Stem Cell Niche Damage

“…The expression of the corneal epithelial cell marker CK3 increased in the limbus of the partial LSCD model, indicating the possibility that limbal epithelial stem cells were differentiating and migrating from the limbus to the central cornea during the restoration of the corneal epithelium. In contrast, the expression of CK3 decreased in the cornea of the untreated total LSCD model in agreement with the data published by other authors using different LSCD models .…”

“…Neovascularization and corneal opacification were more severe than in the partial LSCD, and they developed over the first 3 weeks after the creation of the injury, as other authors have noted . Moreover, the epithelial defects underwent partial recovery during the first 2 weeks and were maintained during the follow‐up period, as reported by others . In two of the six untreated rabbits, a thick conjunctival invasion occurred on the corneal surface, indicating that the total LSCD model was more severe than the partial LSCD model, as we expected.…”

Application of the cell sheet technique in tissue engineering

“…Such ES-derived cardiac cell sheets may be a promising tool for the development of bioengineered myocardium. Zhang et al (52) used ESC sheets for ocular surface reconstruction in rabbits with limbal stem cell deficiency using the centrifugation method. The cell sheet transplant facilitated reconstruction of the ocular surface in 75% of the treated animals.…”

Células madre Mesenquimales de tejido adiposo como tratamiento de la superficie ocular en la deficiencia de células madre limbares

“…Por lo tanto, para asegurar la protección del trasplante frente al parpadeo las tarsorrafias fueron mantenidas durante 2 semanas. Como en nuestro caso, el grupo de Zhang et al 204 señaló que la mayoría de las MA se reabsorben por completo durante los 10 primeros días tras la realización del trasplante. En cambio, en otro trabajo, 202 describen que la MA se reabsorbe entre la segunda y la quinta semana post-trasplante.…”

“…En algunos trabajos llevados a cabo por otros grupos de investigación, también se ha observado una gran recuperación del defecto epitelial durante las 2 primeras semanas después de la creación de la lesión, pero sin llegarse a reepitelizar por completo. 167,203,204 Por otro lado, en 2 de los 6 conejos en los que se desarrolló el SIL total, se produjo una invasión del tejido conjuntival hacia el centro corneal, signo que no fue observado en el modelo de SIL parcial. Este dato corroboró que el modelo de SIL total desarrollado fue más severo que el modelo de SIL parcial.…”