Teva Pharmaceutical Industries, Rehovot, Israel .
Stem Cells Dev. 2011 Jan;20(1):53-66. doi: 10.1089/scd.2010.0040. Epub 2010 Oct 26.
Expansion of plastic-adherent bone marrow-derived mesenchymal stem cells (MSCs) results in gradual loss of osteogenic potential after passage 5-6. One explanation is contamination of MSC cultures with mature cells including fibroblasts. Identification and elimination of fibroblasts from MSC cultures could improve MSC yield and differentiation potential and also prevent tumor formation after MSC transplantation. However, no specific markers currently exist that can reliably discriminate between MSCs and fibroblasts. Flow cytometry analysis demonstrated that markers currently used to define MSCs, such as CD105, CD166, CD90, CD44, CD29, CD73, and CD9, are also expressed on human skin or lung fibroblasts. However, the level of expression of CD166 was significantly higher and that of CD9 was significantly lower in MSCs than in fibroblasts. CD146 was expressed only in MSCs. Using small focused microarrays, new markers differentially expressed in MSCs and fibroblasts were identified. Real-time polymerase chain reaction confirmed that expression of CD106, integrin alpha 11, and insulin-like growth factor-2 in MSCs was at least 10-fold higher than in fibroblasts; whereas expression of matrix metalloproteinase 1 and matrix metalloproteinase 3 was almost 100-fold lower. Flow cytometry and immunostaining demonstrated that CD106 protein expression on cell surface could be upregulated in MSCs but not in fibroblasts by the treatment with tumor necrosis factor-alpha. Comparison of surface expression of commonly used and newly identified MSC markers in MSCs cultures of passage 2 and passage 6 demonstrated that CD106 (with and without tumor necrosis factor-alpha treatment), integrin alpha 11, and CD146 were downregulated in MSCs of passage 6, and CD9 was upregulated; whereas all other markers did not change. Newly identified markers that have robust differences of expression in MSCs and fibroblasts on gene and protein level could be used for quality control of MSC cultures after expansion, cryopreservation, gene transfection, and other manipulations.
贴壁生长的骨髓间充质干细胞（MSC）在传代 5-6 代后会逐渐丧失成骨潜能。一种解释是 MSC 培养物中存在成熟细胞（包括成纤维细胞）的污染。从 MSC 培养物中鉴定和去除成纤维细胞可以提高 MSC 的产量和分化潜能，并防止 MSC 移植后形成肿瘤。然而，目前还没有可靠区分 MSC 和成纤维细胞的特异性标记物。流式细胞术分析表明，目前用于定义 MSC 的标记物，如 CD105、CD166、CD90、CD44、CD29、CD73 和 CD9，也在人皮肤或肺成纤维细胞上表达。然而，CD166 的表达水平在 MSC 中明显高于成纤维细胞，CD9 的表达水平在 MSC 中明显低于成纤维细胞。CD146 仅在 MSC 中表达。使用小型聚焦微阵列，鉴定了 MSC 和成纤维细胞中差异表达的新标记物。实时聚合酶链反应证实，MSC 中 CD106、整合素α11 和胰岛素样生长因子-2 的表达至少是成纤维细胞的 10 倍；而基质金属蛋白酶 1 和基质金属蛋白酶 3 的表达则几乎降低了 100 倍。流式细胞术和免疫染色表明，肿瘤坏死因子-α处理可使 MSC 表面 CD106 蛋白表达上调，但不能使成纤维细胞上调。比较第 2 代和第 6 代 MSC 培养物中常用和新鉴定的 MSC 标记物的表面表达，发现 CD106（有或无肿瘤坏死因子-α处理）、整合素α11 和 CD146 在第 6 代 MSC 中下调，CD9 上调；而其他所有标记物均未改变。在基因和蛋白水平上，在 MSC 和成纤维细胞中表达差异明显的新标记物可用于 MSC 培养物在扩增、冷冻保存、基因转染和其他操作后的质量控制。
