## 4 Discussion  
Histone modification and DNA methylation constitute major mechanisms that are responsible for epigenetic regulation of gene expression during development and differentiation [52; 53; 54]. Among various histone modifications, acetylation is known to impact the physical properties of chromatin and there is significant evidence that histone acetylation is important in nucleosome assembly and chromatin folding [55]. It was shown that lysine charge neutralization achieved through histone tail acetylation reduces inter-nucleosomal interactions, and results in the unfolding of the chromatin fiber. It is now widely acknowledged that acetylation promotes the opening of the chromatin structure by interfering with the interactions between nucleosomes and releasing the histone tails from the linker DNA. HDAC inhibitors are small molecules that can promote either self-renewal, differentiation, or reprogramming of cells under certain conditions. In the past decade, due to their role in epigenetic regulation, gene expression profiles, and signal transduction pathways, HDAC inhibitors have been widely used to convert the state of cells into other lineages. It has been reported that VPA, which is a HDAC inhibitor, promotes chromosomal remodeling and maintains acetylated histones as an \"open form\" of the chromatin structure by acting as an inhibitor of HDAC [26; 49; 50; 51], thereby enhancing reprogramming of somatic cells into iPSCs and other lineages. Previous reports have shown that treatment of VPA arrests cellular growth [51; 56]. Similar to the effect of VPA in osteogenic medium, increasing the concentration of VPA in complete medium showed increased growth inhibition of cells (Figure 2c). However, the extent of the growth inhibition was less profound in complete medium than in osteogenic medium, which is considered to be because the fibroblasts in osteogenic medium supplemented with VPA underwent a shift from a proliferative state to a cellular reprogramming process. In addition, treatment of VPA in complete medium resulted in no observable difference in cellular morphology and calcium deposition (data not shown), whereas significant changes were observed in osteogenic medium with VPA (Figure 1b). These results suggest that osteogenic medium is a key factor for the direct conversion of fibroblasts into osteogenic lineage, and VPA acts as an enhancer in the reprogramming process. Previously, it has been shown that VPA promotes cell fate determination via activation of the ERK pathway [57] and suppression of STAT3 and HDAC3 pathways [58]. In particular, VPA was used as a substitute for c-myc in reprogramming [26]. The treatment of VPA increased the number of iPSC colonies by more than 50-fold, and the reprogrammed iPSCs resembled ESCs with regard to their morphology, pluripotency and gene expression profiles [26]. Recently, it was demonstrated that VPA is a potential inducer of osteogenesis in mouse mesenchymal stem cells (MSCs) [59]. Molecular analysis showed upregulation of Runx2, osteoblast-related differentiation markers, and bone-related transcription factors in VPA-treated mouse MSCs. In vitro analysis using ARS and Von Kossa staining results showed an increase in the deposition of calcium phosphate in VPA-treated mouse MSCs and confirmed osteoblast differentiation and mineralization. It is considered that VPA sets somatic cells in a transition state before they continue and complete the reprogramming process. In addition, it has been reported that VPA promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) by alleviating the adverse effect of glucocorticoids on BMSC proliferation, apoptosis and osteogenic differentiation [60]. In a previous study, expression levels of Runx2 and other osteogenic markers were analyzed as early markers for the differentiation of MSCs into osteogenic lineage [61]. It was noticeable that Runx2 and other osteogenic markers were increased simultaneously.