Study On Differentiation Of Murine Cardiac Stem Cells Co-cultrued With Cardiomyocytes

Objective:To investigate whether CSCs can be induced into cardiomyocytes(CM)by Myocardial conditioned medium,whether cellular contact with CM is necessary to CSCs'myocardial differentiation.Methods:1. Myocardial tissue isolated from neonatal mice was cut into 0.5-1mm3 fragments,red cells was washed off by PBS buffer.After mild digestion for 5 min with trypsin,the remaining tissue fragments were seeded and cultured for 11-14 days. Small, phase-bright like cells clusters were massive formed,these kind of primary gener- ation of cells were separated and collected by differential pace digestion.2. The primary cells were sorted with immunobeads to obtain c-Kit+ CSCs.3. With flow cytometry(FCM),Revealing the cell phenotype of c-Kit,Sca-1,CD34,CD45 expressing of the unsorted primary cells,verifying the purity of the sorted c-Kit+ cells.examining the sorted cells'cell cycle4. Using In-site immunofluorescence(IFS) to verify the purity of the sorted c-Kit+ cells.5. There groups were established on the base of sorted c-Kit+ cells.Blank control group,c-Kit+ cells were cultured solo for 21 days using 1% FBS medium.Conditioned medium group,c-Kit+ cells were cultured solo for 21 days using Myocardial conditioned medium(1% FBS medium).Mixed group,c-Kit+ cells labeled with PKH26 or DAPI were co-cultured with CM for 8 days respectively(1% FBS medium).PKH26 labeled cell were undergone Immunohistochemistry in the final,while DAPI labeled cells at the 8th day were separated and pass-generated dispersedly,in the subsequent 2 weeks,Observation were made to spot spontaneous beating events.6. Performing in-site immunohistochemistry in the blank group Conditioned medium group and PKH26 labelling mixed group to detect Troponin-T and connexin43 expression at the final day to assess the CSCs'myocardial differentiation.7. Statistical analysis:Five 200x microscope view were randomly taken from every culture tray,one from the centre,four from the peripheral,Positive or negative cell were summed up,and analyzed by statistical method,the accepted level of significance was P<0.05.Results1. The primary cells mainly expressed c-Kit(44%) and Sca-1(13%),a small ratio(2-3%) of the cell express CD34,CD45.2. The Purity of sorted c-Kit+ cells was approximately 96%,which was consistent with the result of IFS3. The cells of S phase and G2/M phase in the sorted cells were accounted for 34.6%, indicating active proliferation.4. Cells in the blank group were under growth arrest,partially died,Cells in the Conditioned medium group revealed better growth potential,with 2 times better in mean cell density.CSCs in mixed group which contacted with CM were burly in shape,and beat with CM synchronously.Spontaneous beating cells with DAPI were Observed after separation,although small in number(22 spots).5. Myocardial conditioned medium had no effect on inducing CSCs'expression of Troponin-T and connexin43,While CSCs in contact with CM expressed Troponin-T and connexin43 in a shorter period of 8 days. Conclusions1. Myocardial conditioned medium have no effect on conducting CSCs into myocardial differentiation.2. Contact with CM is a potent factor in CSCs'myocardial differentiation.