Involvement of DNA Methylation in Bone Marrow-derived Stromal Cell Differentiation

Despite the interest in tissue regeneration using bone marrow-derived stromal cells (BMSCs), mechanisms of BMSCs differentiation, particularly involvement of epigenetic events such as DNA methylation is poorly understood. Objectives: To identify down-regulating genes during osteogenic or neurogenic differentiation and to investigate the possibility that differentiation of BMSCs involves DNA methylation. Methods: BMSCs isolated from adult mice were cultured for 8 days in non-induced medium (control medium: 15% FBS/a-MEM), osteogenically-induced medium (dexamethasone, b-glycerophosphate and ascorbic acid/control medium), and neurogenically-induced medium (B27 and BDNF/Neurobasal medium). Phenotypic profiles of the cells under each condition were determined by RT-PCR and extracellular matrix (ECM)/bone-related genes specific microarray analysis, and expression levels of DNA methyltransferase 1 (Dnmt1; DNA methylation pattern maintenance-type DNA methyltransferase), DNMT3a, and Dnmt3b (de novo DNA methyltransferases) mRNAs were evaluated by semi-quantitative RT-PCR. Genomic DNA was isolated from non-induced and osteogenically-induced BMSCs, bisulfite converted and analyzed the methylation status of nestin gene promoter region by methylation-specific PCR. Results: Neuronally guided BMSCs down-regulated sets of ECM/bone-related gene expressions during differentiation, as determined by microarray analysis. RT-PCR analysis demonstrated that expression of neuronal marker genes, including nestin and beta-tubulin III was silenced in osteogenically guided BMSCs. Non-induced and osteogenically-induced BMSCs constantly expressed Dnmt1 mRNA. Neurogenically-induced BMSCs clearly increased expression of Dnmt3a and Dnmt3b mRNAs. In contrast, little expression of these genes was observed in non-induced and osteogenically-induced BMSCs. Methylation-specific PCR showed that non-methylated DNA-specific primers yielded bands in both non-induced and osteogenically-induced BMSCs, whereas methylated primers yielded them only in non-induced BMSCs, indicating that the examined nestin promoter region is de-methylated during osteogenic differentiation. Conclusion: These results suggest that osteogenic and neurogenic differentiation of BMSCs down-regulates sets of gene expressions, and epigenetic events, such as DNA methylation and/or demethylation may be involved in part in the gene silencing during the differentiation.