Abstract：
Keratocytes are the main cellular components of the corneal stroma. This cell is quiescent and cannot be cultured easily. The aim of this study was to investigate differentiate human adipose mesenchymal stem cells (hADSCs) into corneal keratocyte cells by combining natural scaffolds and conditioned medium (CM) and evaluating their safety in the rabbit\'s cornea. Keratocytes were cultured in an optimal culture medium and this medium was collected and kept as a CM. hADSCs were cultured on the decellularized human small incision lenticule extraction (SMILE) lenticule (SL), amniotic membrane (AM), and collagen-coated plates, and were exposed to keratocyte-CM (KCM) for 7, 14, and 21 days. Differentiation was evaluated using Real-time PCR and immunocytochemistry (ICC). hADSCs were cultured on the SL scaffolds and implanted in the corneal stroma of 8 New Zealand male rabbits. Rabbits were followed for 3 months and the safety was evaluated by clinical and histological variables. Real-time PCR results showed a significant increase in the expression of keratocyte-specific markers on the 21 day of differentiation compared to the control group. ICC also confirmed the induction of differentiation. Implantation of SLs containing differentiated cells in the cornea of animals showed no serious complications including neovascularization, corneal opacity, inflammation, or signs of tissue rejection. Furthermore, the evaluation of the presence of keratocyte-like cells after three months in the rabbit stroma was confirmed by Real-time PCR and immunohistochemistry (IHC) analysis. Our results showed that combination of combination of corneal extracellular matrix and KCM can induced keratocytes differentiation of hADSC and can be introduced as a alternative method to supply the required keratocytes in corneal tissue engineering.
摘要：
角膜细胞是角膜基质的主要细胞成分。该细胞是静止的并且不能容易地培养。本研究的目的是研究通过结合天然支架和条件培养基（CM）将人脂肪间充质干细胞（hADSCs）分化为角膜角膜细胞，并评估其在兔角膜中的安全性。在最佳培养基中培养角质细胞,收集该培养基并作为CM保存。hADSCs在脱细胞的人小切口微透镜提取（SMILE）微透镜（SL）上培养，羊膜（AM），和胶原蛋白涂层板，并暴露于角膜细胞-CM（KCM）7、14和21天。使用实时PCR和免疫细胞化学(ICC)评价分化。将hADSC在SL支架上培养并植入8只新西兰雄性兔的角膜基质中。家兔随访3个月，并通过临床和组织学变量评估安全性。实时PCR结果显示，与对照组相比，在分化21天，角膜细胞特异性标志物的表达显着增加。ICC也证实了分化的诱导。在动物的角膜中植入含有分化细胞的SL显示没有严重的并发症，包括新生血管形成，角膜混浊,炎症,或组织排斥反应的迹象.此外，通过Real-timePCR和免疫组织化学(IHC)分析证实了兔基质中3个月后角膜样细胞的评估.我们的结果表明，角膜细胞外基质和KCM的组合可以诱导hADSC的角膜细胞分化，可以作为一种替代方法来提供角膜组织工程中所需的角膜细胞。
