A novel tissue-engineered corneal epithelium based on ultra-thin amniotic membrane and mesenchymal stem cells

Abstract Purpose: Currently, in vitro cultured corneal epithelial transplantation is effective for treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. This study aimed to effectively simulate the limbal stem cells (LSCs) microenvironment by inoculating mesenchymal stem cells (MSCs) on the basal surface of an ultra-thin amniotic membrane (UAM) to develop an improved tissue-engineered corneal epithelial carrier. Methods: Traditional AM was modified, and MSCs were inoculated into the UAM stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier. The tissue-engineered corneal epithelium structure and rabbit LSCD model corneas cultured on different carriers were observed through hematoxylin and eosin staining. Cell phenotypes were evaluated through fluorescence staining, WB, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Results: Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. Conclusions: The UAM-MSCs vector we constructed can better simulate the limbal microenvironment, and the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype.