Histone Deacetylase 1 Regulates The Transdifferentiation Of Bone Marrow Mesenchymal Stem Cells Into The Cardiac Phenotype

ObjectiveTo investigate the characteristics of histone deacetylase 1 during the transdifferent- -iation of bone mesenchymal stem cells (BMSCs) into myocardial-like cells in the microenvironment provided by cardiomyocytes(CM), and initially explore the acetylation mechanism involved in the transdifferentiation of adult stem cells.Methods1. BMSCs from Sprague-Dawley (SD) rats were isolated and cultured in vitro by using the whole bone marrow adherence method. When 80-90% confluency, BMSCs were digested by trypsin- EDTA(0.25%:0.05%). Cell morphology was observed under the inverted phase contrast microscope. At the third passage, surface markers of BMSCs were determined by flow cytometry. And BMSCs were induced to differentiate into osteoblasts and adipocytes. Osteogenic ability was examined by alizarin red staining. Adipogenic ability was measured by oil red O staining.2. 293A cells were cultured. When 50-70% confluency,cells were digested by 0.25% trypsin and then passage cultured in a ratio of 1:10-1:20. 3.When 80-90 % confluency , enhanced green fluorescent protein adenovirus (Ad-EGFP) was transfected into 293A cells with MOI 100 for virus amplification. After 3 days, cells showed cytopathic effect (CPE) and were collected.4. Ad-EGFP was transfected into BMSCs with MOI 0, 10, 50, 100, 200, 400. The trans- -fection efficiency was examined by flow cytometry, and the optimum MOI was obtained.5. Neonatal rat myocardial cells (cardiomyocytes, CM) were isolated, cultured. The expression of cardiac troponin T(CTnT) was identified by immunofluorescence staining after 72 h.6. BMSCs which were marked by EGFP co-cultured with CM in a ratio of 1:1.7. According to co-culture time, we divided six groups. Namely, BMSCs group (control group) ,co-culture for 3 days, 6 days, 9 days, 12 days-group, respectively, cardiomyocyte (CM) group. BMSCs which expressed enhanced green fluorescent protein (EGFP-positive BMSCs) were sorted from the co-culture system by FACS.8. The expression of cardiac troponinT (CTnT) in EGFP-positive BMSCs was exami- -ned by Immunofluorescence staining.9. The mRNA expression of histone deacetylase 1(HDAC1)was assessed by FQ-PCR.10. The protein expression of HDAC1 was assessed by Western Blot.11. Data was analyzed using SPSS 13.0 statistical software. P<0.05 indicated the diff- -erence Statistically significant.Rusults1.The primary cells and the passage cells were mostly fusiform in shape, and were similar to fibroblasts. At the third passage, BMSCs strongly expressed the surface markers of stromalcells: CD29 (99.86%), CD44 (99.28%), CD90 (99.74%) , but were negative for CD34(3.04%), CD45(1.14%), which were the surface markers of hematopoietic cells. Following 21 days of osteogenic induction, cell alizarin red staining showed that alizarin red was positive in osteoblasts. Following 2 weeks of adipogenic induction, oil red O staining showed that red lipid droplets existed in adipocytes.2.After adherence, 293A cells were fibroblast-like. Ad-EGFP was transfected into 293A cells. Then cells became round and detached on the third day, like grapes gathered. 293A cells all emitted green fluorescence under fluorescence microscope.3.EGFP was expressed after 24 h. With MOI 10-100, the transfection efficiency was less than 60%,and was 80-85% with MOI 400, but the pathological effects (CPE) appeared. With MOI 200, the transfection efficiency was 80 % without any significant morphological changes.4. Cardiomyocytes were isolated and cultured. After 24 h, cells showed triangle or flat irregular forms and began the voluntary contraction. The positive rate of cardiac troponinT (CTnT) was above 90%by immunofluorescence staining.5. After co-cultured for 2-3 days, the spindle BMSCs gradually shortened, thickened. CTnT can be detected on the 4th day after co-culture. The transdifferentiation of BMSCs into spontaneously beating cells was not observed. The EGFP+BMSCs sorted by FACS had positive rate of above 97%.6. HDAC1 mRNA was expressed in each group cells and showed a downward trend with co-culture time. P<0.05. HDAC1 mRNA of BMSCs group was about 14 times as much as that of CM group.7. Contrast to the control group (BMSCs group), the protein of HDAC1 in other groups was significantly reduced. The protein expression of HDAC1 also showed a downward trend with co-culture time. P<0.05. The protein of HDAC1 of BMSCs group was about 8 times as much as that of CM group.Conclusion HDAC1 played a negative role in the transdifferentiation of bone marrow mesenchymal stem cells into the cardiac phenotype.