Ezura Yoichi, Sekiya Ichiro, Koga Hideyuki, Muneta Takeshi, Noda Masaki
Department of Molecular Pharmacology, Tokyo Medical and Dental University, Tokyo, Japan.
Arthritis Rheum. 2009 May;60(5):1416-26. doi: 10.1002/art.24472.
Human synovium-derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium-derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis.
Human synovium-derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real-time reverse transcription-polymerase chain reaction. Methylation status was also examined in human articular cartilage.
Bisulfite sequencing analysis indicated that 10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG-rich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture.
The DNA methylation levels of CpG-rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium-derived MSCs.
人滑膜来源的间充质干细胞（MSCs）能够高效分化为成熟软骨细胞。有研究表明，DNA甲基化是调节人类软骨形成的一种机制；然而，与软骨分化相关基因的甲基化状态尚不清楚。本研究的目的是调查人滑膜来源的MSCs在实验性软骨形成过程中的CpG甲基化状态，以期用于骨关节炎的潜在治疗。
将人滑膜来源的MSCs进行软骨球培养3周。在分化前后，通过亚硫酸氢盐测序分析10个候选基因（SOX9、RUNX2、CHM1、FGFR3、CHAD、MATN4、SOX4、GREM1、GPR39和SDF1）启动子中12个区域的甲基化状态。通过实时逆转录-聚合酶链反应分析这些基因的表达水平。还检测了人类关节软骨中的甲基化状态。
亚硫酸氢盐测序分析表明，无论基因表达水平如何，在培养3周前后，所分析的11个富含CpG的区域中有10个在人祖细胞中处于低甲基化状态。SOX9、RUNX2、CHM1、CHAD和FGFR3在分化后表达增加，其甲基化状态持续较低；GREM1和GPR39在软骨形成后表达下降，其甲基化状态也较低。一个富含CpG的区域甲基化差异的例外情况是在SDF1的1 kb上游序列中，其表达在分化后下降。矛盾的是，在软骨球培养3周后，该区域的高甲基化状态降低。
在人滑膜来源的MSCs软骨形成过程中，与软骨细胞表型相关基因的富含CpG启动子的DNA甲基化水平大多保持较低。
