Dynamics of DNA methylation during osteogenic differentiation of porcine synovial membrane mesenchymal stem cells from two metabolically distinct breeds

Mesenchymal stem cells (MSCs), with the ability to differentiate into osteoblasts, adipocytes, or chondrocytes, show evidence that the donor cell’s metabolic type influences the osteogenic process. Limited knowledge exists on DNA methylation changes during osteogenic differentiation and the impact of diverse donor genetic backgrounds on MSC differentiation. In this study, synovial membrane mesenchymal stem cells (SMSCs) from two pig breeds (Angeln Saddleback, AS; German Landrace, DL) with distinct metabolic phenotypes were isolated, and the methylation pattern of SMSCs during osteogenic induction was investigated. Results showed that most differentially methylated regions (DMRs) were hypomethylated in osteogenic-induced SMSC group. These DMRs were enriched with genes of different osteogenic signalling pathways at different time points including Wnt, ECM, TGFB and BMP signalling pathways. AS pigs consistently exhibited a higher number of hypermethylated DMRs than DL pigs, particularly during the peak of osteogenesis (day 21). Predicting transcription factor motifs in regions of DMRs linked to osteogenic processes and donor breeds revealed influential motifs, including <i>KLF1, NFATC3, ZNF148, ASCL1, FOXI1</i>, and <i>KLF5</i>. These findings contribute to understanding the pattern of methylation changes promoting osteogenic differentiation, emphasizing the substantial role of donor the metabolic type and epigenetic memory of different donors on SMSC differentiation.

间充质干细胞（Mesenchymal stem cells, MSCs）具备向成骨细胞、脂肪细胞或软骨细胞分化的能力，已有研究表明供体细胞的代谢类型会影响成骨过程。目前针对成骨分化过程中的DNA甲基化变化，以及不同供体遗传背景对间充质干细胞分化的影响，相关认知仍较为有限。本研究从两个具有不同代谢表型的猪品种——安格恩马鞍猪（Angeln Saddleback, AS）与德国兰德瑞斯猪（German Landrace, DL）中分离得到滑膜间充质干细胞（synovial membrane mesenchymal stem cells, SMSCs），并对成骨诱导过程中滑膜间充质干细胞的甲基化谱进行了分析。结果显示，成骨诱导的滑膜间充质干细胞组中，绝大多数差异甲基化区域（differentially methylated regions, DMRs）呈低甲基化状态。这些差异甲基化区域在不同时间点分别富集于多种成骨信号通路相关基因，包括Wnt信号通路、细胞外基质（ECM）信号通路、转化生长因子β（TGFB）信号通路以及骨形态发生蛋白（BMP）信号通路。安格恩马鞍猪的高甲基化差异甲基化区域数量始终高于德国兰德瑞斯猪，这一差异在成骨高峰期（第21天）尤为显著。对与成骨过程及供体品种相关的差异甲基化区域进行转录因子基序预测，发现了多个具有调控作用的基序，包括KLF1、NFATC3、ZNF148、ASCL1、FOXI1以及KLF5。本研究结果有助于阐明促进成骨分化的甲基化变化模式，同时强调了供体代谢类型以及不同供体的表观遗传记忆对滑膜间充质干细胞分化的重要作用。