Omoto Masahiro, Miyashita Hideyuki, Shimmura Shigeto, Higa Kazunari, Kawakita Tetsuya, Yoshida Satoru, McGrogan Michael, Shimazaki Jun, Tsubota Kazuo
Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
Invest Ophthalmol Vis Sci. 2009 May;50(5):2109-15. doi: 10.1167/iovs.08-2262. Epub 2009 Jan 10.
To report the efficacy of human bone marrow-derived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets.
Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in alpha-modified Eagle's medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithelial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air-liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cytokeratin 3 (K3), K15, p63alpha, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and transplanted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochemistry against K3 and K4.
MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63alpha, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits.
MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.
报告人骨髓间充质干细胞作为可移植角膜上皮细胞片培养的饲养层细胞来源的有效性。
人骨髓间充质干细胞（骨髓贴壁干细胞；MASC）在含10%血清的α-改良伊格尔培养基中培养，并用丝裂霉素C处理。通过逆转录-聚合酶链反应确认MASC中细胞因子的表达。将人角膜缘上皮细胞与MASC或3T3饲养层细胞共培养以比较集落形成效率（CFE）。将角膜缘上皮细胞在MASC或3T3饲养层细胞上于气液界面培养以使其分层，并用抗细胞角蛋白3（K3）、K15、p63α和ABCG2的免疫组织化学方法分析分层的上皮片。用MASC饲养层细胞培养兔角膜缘上皮细胞片并移植到角膜缘缺陷兔的眼表。用裂隙灯显微镜观察上皮移植物4周，然后通过组织学以及抗K3和K4的免疫组织化学进行评估。
MASC饲养层细胞表达角质形成细胞生长因子、肝细胞生长因子和N-钙黏蛋白。人角膜缘上皮细胞在MASC和3T3饲养层组中的CFE相似。用表达K3、K15、p63α和ABCG2的MASC饲养层细胞成功培养出分层的细胞片。移植的上皮片在角膜缘缺陷兔中再生出角膜表型症状。
MASC来源的饲养层细胞适用于上皮片的工程构建，避免了使用潜在有害的异种饲养层细胞。
