Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza", C.so della Repubblica 79, 04100, Latina, Italy.
Stem Cell Rev Rep. 2013 Dec;9(6):847-57. doi: 10.1007/s12015-013-9451-6.
Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.
通过在 3T3-J2 成纤维细胞上体外扩增角膜缘干细胞（LSCs）来实现角膜上皮再生已经揭示了一些局限性，主要是由于角膜微环境没有得到适当的复制，从而影响了长期结果。对于用于扩增 LSCs 的饲养细胞以及影响人饲养细胞作用的机制的深入了解尚未完全阐明。我们最近开发了一种标准化的方法来扩增人 Tenon's 成纤维细胞（TFs）。在这里，我们旨在研究 TFs 是否可作为 LSCs 的饲养细胞使用，表征共培养物的表型并评估人分泌的可溶性因子。TFs 可作为替代人饲养层的假说尚未得到探索。从上角膜缘活检中分离出 LSCs，将其与 TFs、3T3-J2 或真皮成纤维细胞（DFs）共培养，然后通过免疫荧光（p63α）、集落形成效率（CFE）测定和 qPCR 分析一组假定的干细胞和角膜上皮分化标志物（KRT3）。共培养物上清液被筛选出一组可溶性因子。结果表明，与 DFs（p = 0.032）和 3T3-J2（p = 0.047）相比，在 TFs 上共培养的 p63α（+）LSCs 的百分比显著更高。有趣的是，与 3T3-J2 和 DFs 相比，TFs 上共培养的 LSCs 的 CFE 和 ΔNp63α 的 mRNA 表达水平均显著更高（p < 0.0001），并且可溶性因子（IL-6、HGF、b-FGF、G-CSF、TGF-β3）的水平也显著更高。因此，TFs 可以作为 3T3-J2 和 DFs 的替代饲养层，为 LSCs 培养提供合适的微环境。
