Markers Distinguishing Mesenchymal Stem Cells from Fibroblasts Are Downregulated with Passaging
Markers Distinguishing Mesenchymal Stem Cells from Fibroblasts Are Downregulated with Passaging
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DOI：
10.1089/scd.2010.0040
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发表时间：
2011-01-01
影响因子：
4
通讯作者：
Ginis, Irene
中科院分区：
文献类型：
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作者：
Halfon, Svetlana;Abramov, Natalie;Ginis, Irene
塑料粘附的骨髓间充质干细胞（MSC）的扩增导致第5-6代后成骨潜能逐渐丧失。一种解释是MSC培养物被包括成纤维细胞在内的成熟细胞污染。从MSC培养物中鉴定和消除成纤维细胞可以提高MSC产量和分化潜力，并且还可以防止MSC移植后的肿瘤形成。然而，目前还没有特异性标记物可以可靠地区分MSC和成纤维细胞。流式细胞术分析表明，目前用于定义MSC的标志物，如CD 105、CD 166、CD 90、CD 44、CD 29、CD 73和CD 9，也在人皮肤或肺成纤维细胞上表达。但骨髓间充质干细胞中CD 166的表达水平显著高于成纤维细胞，而CD 9的表达水平显著低于成纤维细胞。CD 146仅在MSC中表达。使用小型聚焦微阵列，鉴定了在MSC和成纤维细胞中差异表达的新标志物。实时聚合酶链反应证实，CD 106，整合素α 11，胰岛素样生长因子-2在骨髓间充质干细胞的表达至少比成纤维细胞高10倍，而基质金属蛋白酶1和基质金属蛋白酶3的表达几乎低100倍。流式细胞术和免疫组化结果显示，肿瘤坏死因子-α可上调MSCs细胞表面CD 106蛋白的表达，但对成纤维细胞无明显影响。比较第2代和第6代MSC培养物中常用和新鉴定的MSC标志物的表面表达，表明第6代MSC中CD 106（有和无肿瘤坏死因子-α处理）、整合素α 11和CD 146下调，CD 9上调;而所有其他标志物均未发生变化。新鉴定的在基因和蛋白质水平上在MSC和成纤维细胞中具有稳健表达差异的标记物可用于扩增、冷冻保存、基因转染和其他操作后的MSC培养物的质量控制。
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.