5 - Azacytidine In The Bone Marrow Mesenchymal Stem Cell Differentiation Induced Mechanism Of Cardiomyocyte-like Cells And Autologous Bone Marrow Mononuclear Cell Transplantation For Acute Myocardial Infarction In Patients With Clinical Observation

Stemcells have become main sources of cell transplantation for treating cardiac disease. Embryonic stemcells transplantation is preclued for such problems as moral and ethical issues and immunological rejections. While stemcells from mature tissues ,that is somatic stemcells, especially mesenchymal stemcells(MSCs) can resolve above difficulties and may be the current preferred stem cell model for cellular theraputic development.MSCs lies in bone marrow and they can support hematopoietic stem cells . Recent evidence suggests that MSCs have the potential to differentiate into skeletal myoblasts, cardiacmyocyte cells, endothelial,liver, neural and smooth muscle cells. We call this potential 'transdifferentiation'. MSCs can be isolated, expanded in culture, and characterized in vitro and in vivo .In our previous studies, BMSCs, by morphologic changes and immunocytochemical staining such as sarcomeric actin and connexin-43, had been successfully induced to cardiomyocytes in vitro with induction of 5-azacytide. However, the specific induction mechanism is not clear yet and not to be reported at home and abroad. Therefore, the purpose of the present study was as follows: (1) To study hMSCs biological characteristics (2) to investigate the 5-azacytidine induction mechanics in hMSCs differentiating into cardiomyocyte-like CellsMaterials and methods:1.hMSCs( human mesenchymal stemcells)Isolation,cultivation and identification:Bone marrow (10ml) was aspirated from ilium of MI or cardiomyopathy patients . Mononuclear bone marrow cells were isolated by Ficoll density seperation on Lymphocyte Seperation Medium . Cells isolated from bone marrow in 5ml DMEM-LG medium, with 15% fetal bovine serum, penicillin G(100U/ml) and streptomycin(100ug/ul), were then introduced into plastic culture dishes(Falcoon 3002) and incubated with 95% air and 5.0% CO₂ at 37℃. The medium was completely changed 24 hours later in order to discard nonadherent cells and subsequently replaced every 3 days. Cultured BMSCs were observed under contrast-phase microscope to assess the level of expansion. When cultured cells reached about 80% confluence, the culture medium was discarded and washed three times by PBS. Then the adherent cells were released from the dishes with 0.25% trypsin and passage at ratio 1:2.The twice-passaged cells were washed with PBS 3 times and were digested with trypsin to single-cell supernatant. Cells were centrifuged at 1500rpm for 15 minutes to drop upper clear liquid and then were washed twice or three times with PBS. Cells were incubated for 30 minutes at room temperature separately stained with monoclonal antibodies against CD34, CD45, HLA-DR, CD31, vWF, CD29, CD44, CD105 or CD166 and were washed and suspended in PBS for cytometry analysis. 2.hMSCs differentiation into cardiomyocyte-like cells and Immunohistochemical Examinations:After the twice-passaged cells became nearly confluent, 5-azacydine was added into the culture medium at a final concentration of 10 mol/L for 24 hours and then washed with PBS .Cells were fixed and identified by immunocytochemical staining for sarcomeric actin and connexin43.3.DNA purification -. Primer design and methylation analysis:Puregene DNA purification kit ( Gentra systems company) was used andfollowed the provided steps; Cells were centrifuged at 15,000Xg for 5 seconds topellet cells. Remove supernatant ,vortex and add 30014 cell lysis solution and mix.Then add 1.5H1 RNase A solution to the cell lysate ,mix and incubate at 37°C for 60 minutes. Cool sample, precipitate protein and then DNA, add 50W DNAHydration Solution to hydrate DNA. Store DNA at 4°C. We entered web http://www.methdb.de/,searched CpG island through software CpG island Searcher , then used Meth Primer to design the primers we needed and sent to Biological Engineering Company to synthesize.DNA was bisulphate modified and we performed Methylation Specific PCR using MehyEasy? kit.(Human Genetic Signatures Pty Ltd,Australia).The kit provided positive primer control. We used water instead of DNA model as negative control. Check the products by electrophoresing on a 6% polyacrylamide gel.Results:1. hMSCs biological characteristics and after-differentiation Immunohistochemical identification:After 24 hours of planting of cell suspensions, the cells adherent to culture plate showed oblate morphology. After 2-3 days, adherent cells showed spindle-shaped with protrusions, after ahout 7 days, attached cells covered about 80% of culture plate, showed like fibroblast. Cytometry analysis showed that hMSCs were uniformly positive for CD29, CD44, CD105 , CD166 and negative for CD34, CD45 , CD31, vWFand HLA-DR. After treatment by 5-AzaC, part cells were slenderer and longer than before and formed myotube-like structure to connect each other. But spontaneous beats were not found. And immunohistochemical examinations showed the cells express sarcomeric actin and connexin43. Although hMSCs morphology and structure were same with cardiomyocytes, we were not clear whether they had the entire same function as cardiomyocytes, so we call them cardiomyocyte-like cells.2. DNA methylation analysis:Electrophoresing analysis showed that dring MSP , CpG sites of sarcomeric actin gene could product PCR bands before 5-aza treatment and not after treatment, andthere was no band in connexin-43 and CD105 before and after induction. Conclusions:1. hMSCs were positive forCD29, CD44, CD105 , CD166 and negative for HSCs surface markers such as CD34, CD45 and endothelial phenotype CD31, vWF and HLA-DR.2. hMSCs were treated with 5-azacydine and they were found to express cardiac-specific proteins including sarcomeric actin and connexin-43 not present before induction. As one of the demethylating agents, 5-AzaC was a cytosine analog, it could cause extensive demethylation of 5-methylcytosine residues and reduce DNA methyltransferase activity in the cells. DNA methylation went against gene expression while the low methylation level of promoter was positive related with transcription activity.l. 5-AzaC inhibted sarcomeric actin gene methylation thus activated gene transcription. 2.Connexin-43 gene's own methylation status did not change before and after induction, its specific expression after induction might due to the unmethylating of its transcription factor affected by 5-azacytidine. 3. In the control of CD105 gene expression, the gene's own methylation status did not play a major role , while its related transcription factor played more important role. And the mechanisms needed to be further studied.