Integrated analysis of DNA methylome and transcriptome reveals the differences in biological characteristics of porcine mesenchymal stem cells

Abstract

Background: Bone marrow (BM) and umbilical cord (UC) are the main sources of mesenchymal stem cells (MSCs). These two MSCs display significant differences in many biological characteristics, yet the underlying regulation mechanisms of these cells remain largely unknown.

Results: BMMSCs and UCMSCs were isolated from inbred Wuzhishan miniature pigs and the first global DNA methylation and gene expression profiles of porcine MSCs were generated. The osteogenic and adipogenic differentiation ability of porcine BMMSCs is greater than that of UCMSCs. A total of 1979 genes were differentially expressed and 587 genes were differentially methylated at promoter regions in these cells. Integrative analysis revealed that 102 genes displayed differences in both gene expression and promoter methylation. Gene ontology enrichment analysis showed that these genes were associated with cell differentiation, migration, and immunogenicity. Remarkably, skeletal system development-related genes were significantly hypomethylated and upregulated, whereas cell cycle genes were opposite in UCMSCs, implying that these cells have higher cell proliferative activity and lower differentiation potential than BMMSCs.

Conclusions: Our results indicate that DNA methylation plays an important role in regulating the differences in biological characteristics of BMMSCs and UCMSCs. Results of this study provide a molecular theoretical basis for the application of porcine MSCs in human medicine.

Keywords: Bone marrow; DNA methylation; Inbred Wuzhishan miniature pigs; Mesenchymal stem cells; Umbilical cord.

Fig. 1

Isolation and identification of porcine BMMSCs and UCMSCs. A The fibroblast-like morphology of porcine MSCs. B FCM analysis of surface markers expressed on MSCs. Fluorescence in the range of M1 was considered an indicator that cells were recognized by the directed antibody. Autofluorescence intensity was less than 10¹; cells will fluorescence below this threshold were considered negative. C Osteogenic and adipogenic differentiation potential of porcine BMMSCs and UCMSCs. Calcium deposits in osteocytes and lipid droplets in adipocytes were stained red with Alizarin Red and Oil Red O, respectively. Scale bars, 50 μm

Fig. 2

DNA methylome and transcriptome maps of porcine MSCs. The distribution of DNA methylation and levels of gene expression throughout the pig chromosomes were determined. To compare DNA methylation and transcription levels in BMMSCs and UCMSCs, read depths were normalized to the average number of reads in each sample. A 1-Mb sliding window was used to smooth the distribution. Repeat elements, CGI length, gene density, CpG number, and CpGo/e ratio were all calculated in the 1-Mb sliding window

Fig. 3

DNA methylation distribution around gene bodies and flanking regions in porcine MSCs. The 2-kb regions upstream and downstream of TSSs and TTSs, respectively, were split into 20 non-overlapping windows, and the body of each gene was split into 40 equal windows. Average alignment depth was calculated for each window. The Y-axis is the average read depth for each window

Fig. 4

Promoter methylation and transcriptional repression in porcine MSCs. A The number of gene promoters and/or gene bodies showing methylation modifications in BMMSCs. B The number of gene promoters and/or gene bodies showing methylation modifications in BMMSCs. C Comparison of expression between genes showing promoter and/or gene body methylation. D Comparison of expression between genes with promoter CGIs and genes without promoter CGIs. (E) Comparison of expression between genes with different methylation levels at promoter CGIs

Fig. 5

GO functional enrichment analysis of DEGs in BMMSCs and UCMSCs. A–B The top 10 biological process terms significantly enriched for hypermethylated (A) and hypomethylated (B) genes in UCMSCs compared to those in BMMSCs. C, D The top 10 biological process terms significantly enriched for upregulated (C) and downregulated (D) genes in UCMSCs compared to those in BMMSCs

Fig. 6

RNA-seq and MeDIP-seq data validation by RT-qPCR and Sequenom MassARRAY, respectively. The expression and promoter methylation levels of three representative genes (HOXB5, FGF21, and CYP26A1) were validated by RT-qPCR and Sequenom MassARRAY, respectively. A HOXB5, B FGF21, and C CYP26A1. The expression levels of these three genes in BMMSCs and UCMSCs are shown in the left panel. Error bars denote standard errors of means (* represents P < 0.05, *** represents P < 0.001). The right panel shows the Sequenom MassARRAY results. Each dot corresponds to one CpG position in the genome sequence. The colored bar summarizes the methylation level at that position, with blue indicating methylation (100%) and yellow indicating a lack of methylation (0%). Both analyses were performed with three biological replicates for each MSC. Results of the validation of other DEGs or differentially methylated promoter regions are shown in Additional file: Table S3