Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derived from the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary.

中文翻译：

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使用基于TWIST1 RNA的探针对活的间充质干细胞进行分选取决于孵育时间和摄取能力。

骨髓来源的间充质基质细胞（BMSC）是基于细胞的组织工程疗法特别感兴趣的多能祖细胞。但是，限制其临床用途的一个缺点是它们的异质性。在过去的几十年中，人们为基于细胞表面标记物选择BMSC亚群付出了巨大的努力，但是，对于使用哪种标记物获得最佳的组织再生BMSCs仍未达成共识。寻找替代品时，我们决定专注于一种基于探针的方法，即SmartFlare技术，用于检测活细胞中的细胞内mRNA。这项技术不需要固定细胞，而使我们能够根据基因表达将活细胞分类为功能不同的种群。然而，由于这项技术是可用的，因此人们争论探针是否能特异性识别其靶标mRNA。我们验证了TWIST1探针，并证明了它能特异性​​识别BMSC中的TWIST1。但是，探针浓度，孵育时间和细胞摄取的差异会严重影响信号的特异性。此外，我们发现，与源自相同初始BMSC群体的TWIST1低表达细胞相比，TWIST1高表达细胞具有更高的扩增速率。SmartFlare探针可识别其靶基因，但是对于每种探针和协议，必须对细胞类型进行验证。孵育时间和细胞摄取会强烈影响信号特异性。此外，我们发现，与源自相同初始BMSC群体的TWIST1低表达细胞相比，TWIST1高表达细胞具有更高的扩增速率。SmartFlare探针可识别其靶基因，但是对于每种探针和协议，必须对细胞类型进行验证。孵育时间和细胞摄取会强烈影响信号特异性。此外，我们发现，与源自相同初始BMSC群体的TWIST1低表达细胞相比，TWIST1高表达细胞具有更高的扩增速率。SmartFlare探针可识别其靶基因，但是对于每种探针和协议，必须对细胞类型进行验证。