Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
Department of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan,
Cells Tissues Organs. 2019;207(3-4):115-126. doi: 10.1159/000502885. Epub 2019 Oct 1.
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.
干细胞在体外组织工程或再生医学中有重要的应用。然而，仍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。在这项研究中，我们首先研究了 DNA 甲基转移酶（DNMTs）在人骨髓间充质干细胞/祖细胞（hBMSCs）向软骨分化过程中的活性，发现 DNMT3A 和 DNMT3B 在 hBMSC 软骨分化过程中显著上调。为了了解 DNMT3A 和 DNMT3B 对 hBMSC 软骨分化的影响，我们用两种酶的表达载体瞬时转染细胞。有趣的是，DNMT3A 的过表达强烈增强了 hBMSC 的软骨生成，使成熟软骨细胞标志物胶原 II 基因的表达增加了 200 多倍。对细胞中甲基化状态的分析表明，DNMT3A 和 DNMT3B 甲基化了早期干细胞标志物 NANOG 和 POU5F1（OCT-4）的启动子序列。相反，用去甲基化剂 5-氮杂胞苷化学刺激可抑制 hBMSC 的软骨分化，并增加干细胞标志物。针对 DNMT3A 的 siRNA 的功能丧失实验也显著抑制了 hBMSC 的软骨分化。总之，这些结果不仅表明 DNMTs 在调节 hBMSC 向软骨分化中的关键作用，还表明操纵 DNMT 活性可以成为增强 hBMSC 向软骨分化的重要工具，用于软骨组织工程或软骨再生的潜在应用。
