Porcine Bone Marrow Mesenchymal Stem Cells

The Characteristics of RNA m⁶A Modification in pBMSCs and pEFs

Epigenetic reprogramming is essential for SCNT embryo development. The role of RNA N6-methyladenosine (m⁶A) in SCNT embryos has not yet been identified. Zhang's team investigated how RNA m⁶A affects the developmental competency of SCNT embryos derived from porcine bone marrow mesenchymal stem cells (pBMSCs) and porcine embryonic fibroblasts (pEFs).

The researchers compared RNA m⁶A levels between pBMSCs and pEFs by isolating and culturing these cells. Immunofluorescence and ELISA indicated increased RNA m⁶A levels in pBMSCs compared with those in pEFs. qPCR and Western blotting showed that the genes involved in RNA m⁶A methylation were upregulated in pBMSCs (Fig. 1). The difference was further verified by MeRIP-seq. The RNA m⁶A level was strongly correlated with gene expression in the same cell type (Fig. 1F). The m⁶A consensus sequences were GGACU and AAACA for pBMSCs and pEFs, respectively (Fig. 1G), which was similar to the pig RNA m⁶A motif reported previously. RNA m⁶A distribution primarily occurs at transcription start sites and transcription end sites. The RNA m⁶A level was also higher in pBMSCs than that in pEFs (Fig. 1H). The majority of RNA m⁶A sites (~58%) were located in the 3' UTRs, followed by the 5' UTRs (~20%) (Fig. 1I). Genes with RNA m⁶A had lower mRNA levels in pBMSCs compared with those in pEFs (Fig. 1K). KEGG pathway analysis demonstrated that genes with differential methylation levels showed significant enrichment in pathways like Ribosome as well as Proteoglycans in cancer and MAPK signaling pathway among others (Fig. 1M). The results suggested that the difference in RNA m⁶A between pBMSCs and pEFs might affect SCNT embryo development.

SPP1 Affected the Adipogenic Differentiation of pBMSCs

Castration in hog production eliminates boar taint but causes excessive fat accumulation. Xiao's team investigated the role of secreted phosphoprotein 1 (SPP1) in regulating adipose deposition in castrated pigs, using overexpression and interference in porcine bone marrow mesenchymal stem cells (pBMSCs).

To study the role of the SPP1 gene in pBMSCs, they overexpressed it by transfecting PCDH-SPP1 into these cells (Fig. 2A). After 14 days of differentiation, cells with PCDH-SPP1 had fewer lipid droplets and lower levels of adipogenesis markers (PPARγ and FABP4) compared to controls (Fig. 2B, C). This shows that SPP1 overexpression reduces fat formation in pBMSCs. Next, they used three types of SPP1 siRNA to knock down SPP1 in pBMSCs. si-925-SPP1 worked best (Fig. 3A). After 14 days, cells treated with si-925-SPP1 had more lipid droplets and higher levels of PPARγ and FABP4 (Fig. 3B, C). This means that blocking SPP1 promotes fat formation in pBMSCs.