Division of Ophthalmology, Tri-Service General Hospital, Songshan Branch, Taipei, Taiwan, ROC; Department of Ophthalmology, Tri-Service General Hospital & National Defense Medical Center, Taipei, Taiwan, ROC.
Department of Ophthalmology, Tri-Service General Hospital & National Defense Medical Center, Taipei, Taiwan, ROC; Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, ROC.
J Chin Med Assoc. 2017 Sep;80(9):595-607. doi: 10.1016/j.jcma.2017.01.008. Epub 2017 May 18.
Mesenchymal stem cells (MSCs) are important for regenerative medicine. Limbal fibroblasts (LFs), present in the corneal limbus, have been shown to possess MSC characteristics, and can differentiate into other cell types. The current study sought to investigate the effect of microgravity on the proliferation and differentiation of LFs, and identify culture conditions to obtain a high proportion of LFs possessing MSC characteristics.
A rotary cell culture system was used to generate microgravity. Cellular proliferation and MSC marker (CD14, CD45, CD90, CD105, and SSEA4) expression were evaluated by WST-1 test and flow cytometry, respectively. Differentiation of LFs into adipocytes, osteocytes, and chondrocytes was examined. The effects of LF-conditioned medium on limbal stem cell differentiation were assessed.
The cellular proliferation rates under microgravity were significantly lower than those under normal gravity (0.44 vs. 0.18 at 24 h, and 0.70 vs. 0.44 at 48 h, both P ≤ 0.004). Higher proportions of cells expressed CD90 (95.33% vs 81.69%), CD105 (95.32% vs 87.96%), and SSEA4 (68.26% vs 26.13%) under microgravity than under normal gravity. The differentiation potential of LFs was more prominent under microgravity. The LF-conditioned medium attenuated the differentiation of limbal corneal epithelial stem cells.
Under microgravity, LFs showed a higher proportion of MSC characteristics and were easily induced into different linage cells. Culture in a microgravity environment may allow harvesting a greater number of MSC-like LFs for stem cell therapy in ocular surface reconstruction.
间充质干细胞（MSCs）对于再生医学非常重要。角膜缘中的角膜缘成纤维细胞（LFs）已被证明具有 MSC 特征，并能分化为其他细胞类型。本研究旨在探讨微重力对 LFs 增殖和分化的影响，并确定获得具有 MSC 特征的 LFs 高比例的培养条件。
采用旋转细胞培养系统产生微重力。通过 WST-1 试验和流式细胞术分别评估细胞增殖和 MSC 标志物（CD14、CD45、CD90、CD105 和 SSEA4）的表达。观察 LFs 向脂肪细胞、成骨细胞和软骨细胞的分化情况。评估 LF 条件培养基对角膜缘干细胞分化的影响。
微重力下的细胞增殖率明显低于正常重力下的增殖率（24 h 时分别为 0.44 和 0.18，48 h 时分别为 0.70 和 0.44，均 P ≤ 0.004）。微重力下的细胞表达 CD90（95.33% vs 81.69%）、CD105（95.32% vs 87.96%）和 SSEA4（68.26% vs 26.13%）的比例明显高于正常重力下的比例。微重力下 LFs 的分化潜能更为明显。LF 条件培养基减弱了角膜缘上皮干细胞的分化。
在微重力下，LFs 表现出更高比例的 MSC 特征，并且容易被诱导分化为不同的细胞谱系。在微重力环境下培养可能会收获更多类似 MSC 的 LFs，用于眼表重建中的干细胞治疗。
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