Stem cells have essential applications in in vitro tissue engineering or regenerative medicine. However, there is still a need to understand more deeply the mechanisms of stem cell differentiation and to optimize the methods to control stem cell function. In this study, we first investigated the activity of DNA methyltransferases (DNMTs) during chondrogenic differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (hBMSCs) and found that DNMT3A and DNMT3B were markedly upregulated during hBMSC chondrogenic differentiation. In an attempt to understand the effect of DNMT3A and DNMT3B on the chondrogenic differentiation of hBMSCs, we transiently transfected the cells with expression vectors for the two enzymes. Interestingly, DNMT3A overexpression strongly enhanced the chondrogenesis of hBMSCs, by increasing the gene expression of the mature chondrocyte marker, collagen type II, more than 200-fold. Analysis of the methylation condition in the cells revealed that DNMT3A and DNMT3B methylated the promoter sequence of early stem cell markers, NANOG and POU5F1(OCT-4). Conversely, the suppression of chondrogenic differentiation and the increase in stem cell markers of hBMSCs were obtained by chemical stimulation with the demethylating agent, 5-azacitidine. Loss-of-function assays with siRNAs targeting DNMT3A also significantly suppressed the chondrogenic differentiation of hBMSCs. Together, these results not only show the critical roles of DNMTs in regulating the chondrogenic differentiation of hBMSCs, but also suggest that manipulation of DNMT activity can be important tools to enhance the differentiation of hBMSCs towards chondrogenesis for potential application in cartilage tissue engineering or cartilage regeneration.

中文翻译：

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基于 DNA 甲基化的人骨髓间充质干细胞/祖细胞软骨分化的调控


干细胞在体外组织工程或再生医学中具有重要的应用。然而，仍然需要更深入地了解干细胞分化的机制并优化控制干细胞功能的方法。在本研究中，我们首先研究了人骨髓间充质干细胞/祖细胞（hBMSC）软骨形成过程中DNA甲基转移酶（DNMT）的活性，发现DNMT3A和DNMT3B在hBMSC软骨形成过程中显着上调。为了了解 DNMT3A 和 DNMT3B 对 hBMSC 软骨形成分化的影响，我们用这两种酶的表达载体瞬时转染细胞。有趣的是，DNMT3A 过表达通过将成熟软骨细胞标志物 II 型胶原蛋白的基因表达增加超过 200 倍，强烈增强了 hBMSC 的软骨形成。对细胞中甲基化状况的分析表明，DNMT3A和DNMT3B甲基化了早期干细胞标记NANOG和POU5F1(OCT-4)的启动子序列。相反，通过用去甲基化剂5-阿扎胞苷进行化学刺激，可抑制hBMSCs的软骨分化并增加干细胞标记物。使用靶向 DNMT3A 的 siRNA 进行的功能丧失检测也显着抑制了 hBMSC 的软骨形成分化。总之，这些结果不仅显示了 DNMT 在调节 hBMSC 软骨形成分化中的关键作用，而且表明操纵 DNMT 活性可以成为增强 hBMSC 向软骨形成分化的重要工具，从而在软骨组织工程或软骨再生中具有潜在应用。