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

Abstract

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 KLF1, NFATC3, ZNF148, ASCL1, FOXI1, and KLF5. 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.

Keywords: DNA methylation; Epigenetic pattern; Mesenchymal stem cells; Osteogenic differentiation; Pig breeds.

Figure 1.

(a) Gene expression level of three DNMTs of SMSCs grown in growth medium (Con) and osteogenic medium (Ost) over 21 days. (b) Gene expression level of three DNMTs of SMSCs derived from as and DL pigs. (c) Genic annotation of CpG sites. (d) Genomic annotation of CpG sites to DNA-methylation environments. (e) Principal component analysis (PCA) of methylation data of CpG sites. (f) Venn diagram showing unique and overlapping DMR-associated genes between control SMSCs and osteogenic induced SMSCs at each time point. (g) Venn diagram showing unique and overlapping DMR-associated genes between as and DL prior to and after osteogenic differentiation at each time point.

Figure 2.

(a) Volcano plots of DMRs between control SMSCs and osteogenic induced SMSCs at each time point. (b) Volcano plots of DMRs between AS and DL prior to and after osteogenic differentiation at each time point.

Figure 3.

(a) Methylation heatmap of 209 DMRs with relative methylation difference ≥ 30% in the promoter or gene body regions between control SMSCs and osteogenic induced SMSCs at each time point. (b) Methylation heatmap of 143 DMRs with relative methylation difference ≥ 30% in the promoter region between AS and DL prior to and after osteogenic differentiation at each time point.

Figure 4.

(a) BP and KEGG pathway enrichment analysis of DMRs-associated genes between control SMSCs and osteogenic induced SMSCs at each time point. (b) BP and KEGG pathway enrichment analysis of DMRs-associated genes between AS and DL prior to and after osteogenic differentiation at each time point. (c) IPA canonical pathway analysis of DMRs-associated genes from 9 significant time-series profiles detected by STEM.

Figure 5.

(a) Genes shown negative correlation between DNA methylation and gene expression and its enriched pathways. (b) Top 5 negative correlations between methylation levels and gene expression in the comparisons of control SMSCs and osteogenic induced SMSCs. (c) Top 5 negative correlations between methylation levels and gene expression in the comparisons of SMSCs from as and DL. (d) Most frequently enriched TF binding motifs in promoter DMRs.