Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114, USA.
Stem Cell Rev Rep. 2012 Sep;8(3):696-705. doi: 10.1007/s12015-011-9319-6.
Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iβ1 and p63 (putative stem cell markers) in all culture conditions. The level of Iβ1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.
角膜缘干细胞缺乏症（LSCD）的可行治疗选择是进行培养的角膜缘或口腔上皮细胞的角膜移植。目前，干细胞与鼠 3T3 饲养层的共培养使上皮结构成为异种移植物。为了克服异种移植相关的潜在风险，我们研究了使用人源性饲养层来体外扩增上皮（干）细胞。将人角膜缘和口腔上皮与鼠 3T3 成纤维细胞、人真皮成纤维细胞（DF）、人间充质干细胞（MSC）和无饲养细胞（NF）共培养。通过相差显微镜监测细胞形态，并通过免疫组织化学、实时 PCR 检测 p63 和 ABCG2（干细胞标志物）以及集落形成效率（CFE）测定评估干细胞特性。免疫组织化学分析在所有培养条件下均检测到 CK3（角膜特异性标志物）和 Iβ1 和 p63（推定干细胞标志物）的阳性染色。与 MSC 或 NF 组相比，在 3T3 饲养层上培养的角膜缘和口腔细胞中 Iβ1 和 p63 的水平明显更高（p<0.01）。这一水平与在 DF 上培养的细胞相当。在 3T3 和 DF 组中，角膜缘和口腔上皮细胞中 p63 和 ABCG2 的表达均明显高于 MSC 或 NF 组（p<0.01）。3T3 和 DF 组之间未检测到统计学差异。在 3T3 成纤维细胞上共培养的角膜缘和口腔细胞的 CFE 与在 DF 上生长的细胞相当，明显高于与 MSC 或 NF 共培养的细胞（p<0.01）。在 DF 饲养层上生长的上皮细胞保持类似于在 3T3 饲养层上生长的细胞的干细胞样表型。总之，DF 在培养无异种生物角膜等效物时为 3T3 饲养细胞提供了有前途的替代品。
