Concept and biological characteristics of BMSCs
Friedenstein et al[10] first proposed the concept of BMSCs and reported that these cells possess the capacity to transform into bone and cartilage. Recently, under different induced conditions, this kind of cells can be differentiated into mature interstitial cells, called “mesenchymal stem cells” or “mesenchymal progenitor cells”[11-12].
In the present study, we detected the phenotype characteristics of BMSCs and demonstrated CD44-positive reaction, indicating that cell surface contains hematopoietic cell antigen phenotype CD44 but not CD34, which is consistent with previous findings[13]. Under the effects of corresponding induction factors, BMSCs cultured in vitro can differentiate towards different lineages
The mechanism of inductors and the significance of combined induction
Induction mechanism of 5-azacytidine
The mechanism underlying 5-azacytidine promotion of myogenic differentiation of mesenchymal stem cells remains unclear. Konieczny et al[14] reported that there is a determinant site of myogenic differentiation at stem cell genes. Transcriptional activity does not exist in methylated determinant site, but it exists in demethylated determinant site after induction of 5-azacytidine at a sufficient concentration, leading to expression of some a specific protein and regulation of myogenic differentiation of stem cells. The transcription function of many genes is closely related to pyrimidine methylation. During the process of cell division, methylation always exists owing to the effect of methyltransferase, and 5-azacytidine influences gene expression and subsequent cell differentiation because it inhibits DNA methylation. However, 5-azacytidine influences the expression of some but not all genes. The underlying mechanism remains poorly understood.
Significance of combined induction of myogenic differentiation factors and 5-azacytidine
5-azacytidine can induce BMSCs to differentiate into cardioblasts[15-18]. Myogenic differentiation factors are the sponsor of skeletal cell differentiation and activate the expression of myogenin, myosin, myoglobin and desmin, which are the earliest markers of myogenic differentiation[19-20]. At the same time, myosin and desmin are the members indicative of myogenic differentiation in the microfilament and intermediate filament families, respectively[21]. IGF1, with the homology to insulin, is a kind of polypeptide that exhibits insulin-like anabolic action, promotes cell and tissue growth, and contributes to mitosis of various tissues and cells. TGF-β1 shows multiple biological effects. It is one of the primary growth factors that regulate the proliferation and oriented differentiation of BMSCs, and inhibits the biological activity of many inflammatory media. In addition, it is also a strong immunosuppressive agent, which makes allogeneic cell transplantation more safe[15]. The present study used various cytokines and 5-azacytidine to successfully induce BMSCs differentiation into skeletal muscle cells.
Mechanism underlying in vitro induced differentiation of skeletal muscle cells
There are two possible regulation factors during the process of BMSCs differentiation into skeletal muscle cells. One is that when local muscle is injured, many phenotypic border cells exert great non-hematopoietic potential, and the other one is that heterokaryon formation via nucleus fusion results in rearrangement of nuclei, indicating that the surrounding environment can influence the final fate of BMSCs[22]. BMSCs cannot be effectively enriched in traumatic muscle tissue, but the repair of muscle tissue requires the regulation of BMSCs, as validated in non-radiated rats[23]. The traumatic affected limb requires more X-ray examinations to make stem cells in the circulation not to accommodate in the region of muscle tissue defects, which avoids the formation of “cell pools”[24]. Therefore, only transplantation of stem cells cultured in vitro into the region of skeletal muscle defects can contribute to repair occurrence. The present study altered the growth environment of BMSCs through the use of drugs and cytokines, and induced BMSCs to differentiate into skeletal muscle cells. At 3 weeks after induction, polykaryotic sarcotubule structure was observed, which is consistent with a previous report[25]. Immunohistochemical staining also confirmed the skeletal muscle cells. It is to be regretted that BMSCs help myogenic differentiation, but the precise differentiation procedures at the molecular level remain poorly understood.
