Fig. 3: MSCs from healthy donors recapitulate DNA methylation changes observed in MSCs from MM patients upon interacting with MM plasma cells.

A Scheme depicting workflow (left panel) and sorting strategy (right panel) for selecting CD13 + MSCs after 14 days of co-culture with the MM.1S cell line. B Gene expression analysis of IL1B, IL6, and HGF, normalized against RPL38, of the hMSC-TERT cell line (HD-MSC) co-cultured with the MM.1S cell line for 14 days. Expression levels were normalized against MSCs in monoculture. Data are represented as the mean ± SEM from three independent experiments. C ALP activity and matrix mineralization were assessed in differentiated osteoblasts from HD-MSCs in the presence (HD-OB + CM-MM) or absence (HD-OB) of conditioned media from the MM.1S cell line (CM-MM). Representative images of each experimental condition are shown. Barplot represents the mean ± SEM from six independent experiments, and a paired two-tailed t-test was performed to evaluate statistical significance (***p < 0.005). D Heatmap showing differentially methylated CpG sites (eBayes-moderated paired t-test *p < 0.05) in sorted HD-MSCs (three independent donors) in monoculture (HD1-3) or co-cultured with the MM.1S cell line (HD1-3MM) for 14 days that overlaps with previously identified DMPs. The color scale from blue to red represents low to high methylation levels. E GO enrichment analysis of DMPs in HD-MSCs co-cultured with the MM.1S cell line overlapping with myeloma-associated DMPs analyzed using the GREAT online tool. p-values were calculated using a binomial test. F Bar plots showing β-values obtained from the DNA methylation array presented in D, representing mean and ±SEM of three independent experiments. G DNA methylation and H gene expression levels of DMPs validated in HD-MSCs monoculture (blue) or co-cultured with MM.1S (orange) or RPMI-8226 (red) cell lines as indicated. Gene expression data were normalized against RPL38 and all data were normalized against HD-MSC monoculture. Statistical significance was calculated using paired one-tailed Student t-tests and bar plots represent mean ± SEM of 3–5 independent experiments (*p-value < 0.05, **p-value < 0.01, and ***p-value < 0.005).