Objective To analyze the changes of gene expression profiles during the process that human bonemarrow mesenchymal stem cells (hBMSCs) are induced to differentiate into cardiomyogenic cells with 5-azacytidine (5-aza). Methods hBMSCs were isolated from marrow of obsolete ribs and induced with 5-aza. Then immunocytochemicalstaining was used to detect the expressions of α-actin, cardiac troponin T (cTnT), and connexin 43, and the percentage ofcTnT positive cells was tested with flow cytometry. In the process of differentiation, variation of gene expression was screenedwith Genechi ps Operating System of human gene expression profiles. And the differentially expressed genes were functionallyanalyzed and hierarchical clustered. Results When BMSCs were induced in vitro with 5-aza, part of the cells turnedinto myogenic cells morphologically. Before induction, immunocytochemical staining for α-actin and cTnT showed sl ightpositive and for connexin 43 showed negative. While after 3 weeks of induction, immunocytochemical staining for α-actin,cTnT, and connexin 43 showed all positive. With flow cytometry, the percentage of cTnT positive cells was 7.43% ± 0.02%before induction, but it was 49.64% ± 0.05% after induction. During differentiation, 1 814 differentially expressed geneswere reported by gene chi ps. Of them, 647 genes were divided into 5 groups with hierarchical clustering. They had variousbiological functions, involving signal transduction, cell metabol ism, prol iferation, differentiation, development, andtopogenesis. Conclusion hBMSCs can differentiate into cardiomyogenic cells with the induction of 5-aza in vitro. Multi plegenes related with signal transduction, transcri ption, and growth factors are involved during this process.
ObjectiveTo analyze the expression profile changes of osteogenic-related genes during spontaneous calcification of rat bone marrow mesenchymal stem cells (BMSCs). MethodsBMSCs were isolated from 3-day-old healthy Sprague Dawley rats;cells at the 4th generation were used to establish the spontaneous calcification model in vitro. Spontaneous calcification process was recorded by inverted phase contrast microscope observation and alizarin red staining after 7 and 14 days of culture. For gene microarray analysis, cell samples were collected at 0, 7, and 14 days after culture; the differentially expressed genes were analyzed by bioinformatics methods and validated by real-time quantitative PCR (RT-qPCR) assay. ResultsRat BMSCs calcified spontaneously in vitro. When cultured for 7 days, the cells began to aggregate and were weakly positive for alizarin red staining. After 14 days of culture, obvious cellular aggregation and typical mineralized nodules were observed, the mineralized nodules were brightly positive for alizarin red staining. A total of 576 gene probe-sets expressed differentially during spontaneous calcification, corresponding 378 rat genes. Among them, 359 gene probe-sets expressed differentially between at 0 and 7 days, while only 13 gene probe-sets expressed differentially between at 7 and 14 days. The 378 differentially expressed genes were divided into 6 modes according to their expression profiles. Moreover, according to their biological functions, differentially expressed genes related to bone cell biology could be classified into 7 major groups:angiogenesis, apoptosis, bone-related genes, cell cycle, development, cell communication, and signal pathways related to osteogenic differentiation. In cell cycle group, 12 down-regulated genes were linked with each other functionally. Matrix metalloproteinase 13 (Mmp13), secreted phosphoprotein 1 (Spp1), Cxcl12, Mmp2, Mmp3, Apoe, and Itga7 had more functional connections with other genes. The results of genes Spp1, Mgp, Mmp13, Wnt inhibitory factor 1, Cxcl12, and cyclin A2 by RT-qPCR were consistent with that of gene microarray. ConclusionThe first 7 days after rat BMSCs were seeded are a key phase determining the fate of spontaneous calcification. Multiple genes related with cell communication, bone-related genes, cell cycle, transforming growth factor-β signaling pathway, mitogen-activated protein kinase signaling pathway, and Wnt signaling pathway are involved during spontaneous calcification.