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.

被认为不适合移植的角膜脱细胞和再细胞化的能力可能会增加可用移植物的数量。去细胞角膜（DCs）可能为细胞粘附和分化提供自然的微环境。尽管如此，迄今为止，尚无研究评估其作为诱导干细胞分化为角膜细胞的基质的功效。本研究旨在比较NaCl和NaCl加核酸酶方法使整个人角膜脱细胞的效率，并研究整个DC的上皮基底膜（EBM）对人胚胎干细胞（hESCs）分化为干细胞的能力的影响。在动物无血清分化培养基中培养角膜上皮样细胞。由于层粘连蛋白是EBM的主要成分，我们还研究了其对hESCs分化的影响。通过组织学，电子显微镜，DNA定量，免疫荧光和核染色研究获得的脱细胞效率和细胞外基质（ECM）的完整性。通过免疫荧光和RT-qPCR分析评估hESCs接种到DC或层粘连蛋白包被的孔的EBM上时分化为角膜上皮样细胞的能力。单独的NaCl处理（没有核酸酶）不足以去除细胞成分，而NaCl加核酸酶处理导致有效的脱细胞作用和ECM的保存。与诱导在层粘连蛋白上分化的细胞不同，在DC上分化的hESCs表达高水平的角膜上皮特异性标志物角蛋白3和角蛋白12。首次证明，脱细胞基质对hESCs向角膜上皮样细胞的分化具有积极作用。这样的策略支持了人类DC和hESC在角膜上皮组织工程中的潜在应用。