No abstract

A method for isolating adult human bone marrow mesenchymal stem cells (MSCs) was established, and the ability of human MSCs to differentiate into cells with characteristics of cardiomyocytes in vitro was investigated. Selected MSC surface antigens were analyzed by flow cytometry. The MSCs at Passage 2 were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. Characteristics of the putative myogenic cells were determined by immunohistochemistry and transmission electron and confocal microscopies. The expression of myogenic specific genes was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR, and DNA sequencing. The MSCs were spindle-shaped with irregular processes and were respectively positive for CD(13), CD(29), CD(44), CD(71) and negative for CD(3), CD(14), CD(15), CD(33), CD(34), CD(38), CD(45), and HLA-DR. The myogenic cells differentiated from MSCs were positive for beta-myosin heavy chain (beta-MHC), desmin, and alpha-cardiac actin. When the myogenic cells were stimulated with low concentration of K(+) (5.0 mM), an increase in intracellular calcium fluorescence was observed. Myofilament-like structures were observed in electron micrographs of the differentiated myogenic cells. The mRNAs of beta-MHC, desmin, alpha-cardiac actin, and cardiac troponin T were highly expressed in the myogenic cells. These results indicate that 5-azacytidine can induce human MSCs to differentiate in vitro into cells with characteristics commonly attributed to cardiomyocytes. Cardiomyocytes cultured from bone marrow sources are potentially valuable for repairing injured myocardium.

Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. In this study human MSCs were successfully isolated from the umbilical cords. The research characteristics of these cells, e.g., morphologic appearance, surface antigens, growth curve, cytogenetic features, cell cycle, differentiation potential and gene expression were investigated. After 2weeks of incubation, fibroblast-like cells appeared to be dominant. During the second passage the cells presented a homogeneous population of spindle fibroblast-like cells. After more than 4months (approximately 26 passages), the cells continued to retain their characteristics. Flow cytometry analysis revealed that CD29, CD44, CD95, CD105 and HLA-I were expressed on the cell surface, but there was no expression of hematopoietic lineage markers, such as CD34, CD38, CD71 and HLA-DR. Chromosomal analysis showed the cells kept a normal karyotype. The cell cycle at the third passage showed the percentage of G(0)/G(1), G(2)/M and S phase were 88.86%, 5.69% and 5.45%, respectively. The assays in vitro demonstrated the cells exhibited multi-potential differentiation into osteogenic and adipogenic cells. Both BMI-1 and nucleostemin genes, expressed in adult MSCs from bone marrow, were also expressed in umbilical cord MSCs. Here we show that umbilical cords may be a novel alternative source of human MSCs for experimental and clinical applications.

Mesenchymal stem cells (MSCs), which can be isolated from umbilical cords and induced to differentiate into multiple cell types in vitro, represent an ideal source for cell and gene therapy. MSCs are typically expanded in culture prior to their therapeutic application. However, similar to other types of stem cell, MSCs undergo senescence following a certain number of cell expansion passages in vitro, and eventually stop proliferating. The objective of the present study was to measure the changes that occur over successive passages of MSCs during long‑term in vitro culture, and to detect the effect of aging on MSC morphology, phenotype, proliferation, cell cycle, differentiation, intracellular reactive oxygen species (ROS) levels and gene expression. To understand the importance of oxidative stress in the aging of adult stem cells, the current study established a cell model of H2O2‑induced MSC premature senescence. Analysis of the biological characteristics of human umbilical cord MSCs during replicative and premature senescence revealed the importance of extrinsic factors in the aging of stem cells, particularly ROS. The findings of the present study suggest that cellular senescence, a state of irreversible growth arrest, can be triggered by ROS. Thus, it is important to improve the extrinsic culture environment of MSCs to retain the phenotype of expanded cells and delay the process of senescence prior to their clinical application.

The hepatitis B virus (HBV) X protein (HBx) has a key role in the molecular pathogenesis of HBV-related hepatocellular carcinoma (HCC). However, the mechanism of HBx-mediated hepatocarcinogenesis remains to be elucidated. In this study, we aimed to better understand the effects of HBx on gene-expression profiles that participate in hepatocarcinogenesis and the mechanism by which HBx regulates these genes. Differentially expressed genes between L02-HBx and L02-Vector control cells were identified by microarray and validated using quantitative real-time PCR. HBx upregulates 456 genes and downregulates 843 genes, including programmed cell death 4 (PDCD4). PDCD4 was downregulated in clinical HCC specimens and the downregulation of PDCD4 in HCC is correlated with HBx. Furthermore, overexpression experiments in HCC cells proved that PDCD4 has strong tumor-suppressive effects both in vitro and in vivo, and may induce cell apoptosis to suppress the development of HCC. HBx induces expression of DNA methyltransferases (DNMTs), but failed to change the methylation status of the PDCD4 promoter. HBx downregulates PDCD4 expression at least partially through miR-21. Taken together, this study reported for the first time that HBx downregulates PDCD4 and upregulates miR-21 expression. The overexpression of PDCD4 could suppress tumorigenicity. The deregulation of PDCD4 by HBx through miR-21 represents a potential novel mechanism of the downregulation of PDCD4 in HBV-related HCC and provides new insights into HCC development.