| Mesenchymal stem cells (MSCs), a population of adult stem cells, are mainly located in bone marrow stroma. They are nonhematopoietic stem cells with pluripotency. Although the percentage of MSCs in adult bone marrow cells is very small, the multipotential of these cells, their characteristics of easy isolation and culture, as well as high ex vivo expansive potential make them an attractive therapeutic tool capable of playing a role in a wide range of clinical application. MSCs retain the ability to differentiate into various types of tissue cells and provide stem cells for the regeneration of various tissues including bone, cartilage, muscle and myocardium.MSCs were isolated and cultured from adult human, embryonic and porcine bone marrow and their basic biological characteristics were investigated. MSCs comprised a single phenotypic population and displayed a fibroblast-like morphology. After culturing for three days, MSCs began to adhere to culture flask and displayed spindle shape in culture. After ten days, it became confluent and fibroblast-like. After primary culture for ten passages, approximately (1~5) 109 and (5~10) 1012 cells were respectively obtained from adult human and embryonic bone marrow MSCs. The morphology of the three kinds of MSCs was similar.These expanded and attached MSCs were positive for CD13 CD29 CD44 CD71 and negative for GD14 CD33 CD34 CD38 CD45 HLA-DR, which were detected by Flow cytometer(FCM). Ten passages culture for adult human MSCs , 15 passages for embryonic MSCs and more than 40 passages for porcine MSCs were accomplished. The results of DNA contents and growth curve showed that porcine MSCs had stronger self-renewal capacity than that of adult human MSCs and embryonic MSCs. Karyotype of these MSCs did not change after long-time culture.These cells were positive for a -NAE, PAS and negative for POX, SB, ALP in the histochemical analysis. These results have demonstrated that MSCs have strong self-renewal capacity and can be cultured for long time.The adult human MSCs at passage 2 were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. The differentiated MSCs were spindle shaped with irregular processes and formed myotube after two weeks' treatment. The myogenic cells differentiated from MSCs were positive for beta-myosin heavy chain (beta-MHC), desmin and alpha-cardiac actin.Approximately 30% of all the remaining adherent cells had enlarged and assumed ball-like or stick-like morphologies and differentiated into a cardiomyocyte phenotype in vitro. Myofilament-like structures were observed in electron micrographs of the differentiated myogenic cells. RT-PCR, real-time quantitative PCR showed that mRNAs of beta-MHC, desmin, alpha-cardiac actin and cardiac troponin T were highly expressed in the myogenic cells. DNA sequencing results confirmed that the human cardiac troponin T and desmin genes amplified by PCR were identical to their respective reference sequences provided by GeneBank (X74819; NM 001927). The myogenic cells displayed both spontaneous rhythmic Ca2+ fluxes and KC1 induced Ca2+ fluxes when observed under confocal laser scanning microscope. The KCl-stimulated Ca2+ fluxes peaked rapidly and gradually declined thereafter. These results indicate that 5-azacytidine can induce human MSCs to differentiate into cells with characteristics commonly attributed to cardiomyocytes in vitro. Cardiomyocytes derived from bone marrow sources are potentially valuable for repairing injured myocardium.Differentiation of embryonic bone marrow MSCs into neural cells were induced with -mercaptoethanol. Neuron-specific enolase(NSE), neuronfilament 200(NF200), glial fibrilary acidic protein(GFAP) were positive in the differentiated cells detected by immuneohistochemistry. The mRNAs of NSE, NF200, GFAP in these cells were expressed higher than those of control group. The results showed that embryonic bone marrow MSCs treated with - mercaptoethanol in vitro could differentiate into neural cells.Porcine bone marrow MSCs were treated with ascorbic...
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