Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells
Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells
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DOI：
10.1007/s10059-011-2277-7
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发表时间：
2011-08-01
影响因子：
3.8
通讯作者：
Kassem, Moustapha
中科院分区：
文献类型：
--
作者：
Andersen, Ditte C.;Kortesidis, Angela;Kassem, Moustapha
人类间充质干细胞（hMSC）目前被引入用于细胞治疗，然而，在临床使用之前，需要对天然和分化的MSC特异性的抗体来对其进行鉴定。在此，通过用hMSC免疫小鼠，并通过使用一组随后的筛选方法，开发了针对MSC表面蛋白的高质量抗体。流式细胞术分析显示，83.5%、1.1%和8.5%的hMSC原代培养物分别为STR 0 -1和DJ 3、9和18中任一者的双阳性。然而，三种DJ抗体均不允许作为单一试剂从BMMNC富集克隆原性hMSC。使用质谱分析，我们鉴定出DJ 3识别的抗原为CD 44，而DJ 9和DJ 18分别识别HLA-DRB 1和胶原VI。所鉴定的蛋白质在整个体外成骨和成脂分化过程中高度表达。有趣的是，未分化的细胞显示胶原VI的唯一细胞质分布模式，然而，在成骨和成脂分化后，其改变为细胞外基质外观。与此相关，我们发现与STR 0 - 1（+/-）/胶原VI（+）hMSC相比，STR 0 - 1（+/-）/胶原VI（-）分选的hMSC含有更少的分化的碱性磷酸酶（+）细胞，表明细胞膜上的胶原VI专门限定分化的MSC。总之，我们已经产生了一组用于表征MSC的高质量抗体，此外，我们的结果可能表明，DJ 18产生的针对胶原VI的抗体可用于培养的未分化MSC的阴性选择。
Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.