A Protocol For Isolation And Culture Of Bone Mesenchymal Stem Cells From SD Rat Marrow And Experiment In Vitro On Cardiac Differentiation

0Objective: Mesenchymal stem cells are ideal cells for cardical repair. In order to provide enough experiential basis in cardical repair, this research established the technic for isolation and culture SD rat BMSCs,and explored the most effective protocol that bone mesenchymal stem cells differentiate into cardiomyocyte-like cells in vitro,and how to enhance efficienc of myocardialization.Methods:1-. Isolate long bone from4-months rats in sterile conditions. Bone marrow was harvested by flushing with10%FBS, DF-12. SD Rat BMSCs were cultured by whole bone marrow cells adherence method,frequent medium change and differential digestion method, as well as amplified by passage. The structure of BMSCs were observed by Invert Microscope and Switzerland’s Jim dyeing;draw the growth curve for knowing the growth characters;the ability to self-renewal of BMSCs were detected by colony formation assay;molecular marks such as FITC-anti-Mouse/Rat CD29,PE-anti-Rat CD45,PE-anti-mouse/rat CD90.1(Thy-1) were detected by Fluorescence-activated cell sorter.2、P4BMSCs(The percentage of CD29,CD45,CD90were99.9%,0.8%,99.6%)were exposed to different concentrations of5-AZA at5,10,15μmol/L for24h or48h. BMSCs were incubated for total4weeks.The structure of cells after induction were observed by Invert Microscope; the expression of cardiac-specific proteins cTnI were detected by Immunofluorescence.Analyse the difference in efficiency of myocardialization in different concentrations of5-AZAand explore optimal induction conditions for myocardialization in vitro.3、P1BMSCs (The percentage of CD29,CD45,CD90were100%,14.7%,16.5%) and P4 BMSCs(The percentage of CD29, CD45, CD90were99.9%,0.8%,99.6%)were exposed to10μmol/L5-AZA for24h; control groups were exposed to normal medium(without5-AZA).Experiment groups and control groups were incubated for2weeks or4weeks. The structure of cells after induction were observed by Invert Microscope and Switzerland’s Jim dyeing; cardiac-specific proteins cTnl were detect by Immunofluorescence. Analyse difference in efficiency of myocardialization between PI BMSCs and P4BMSCs, and explore optimal induction condition for myocardialization in vitro.Results:1、SD rat BMSCs were suspension growth,round,small,quantity,high refraction. The minority of SD rat BMSCs were adherent after medium change after4hours, and the majority were basically adherent after medium change after24hours. The appearance of adherent cells were polygon, spindle-shape morphology and form small colonies. Colonies increasingly expanded with the days passing. In5days culture became more confluent80%. BMSCs grew quickly after passage and reached80%of confluence within3days. Colony formation assay:colonies efficiency of P1BMSCs and P4BMSCs are10.8%and8.9%. Molecular marks of BMSCs were detected by Fluorescence-activated cell sorter. The percentage of P1BMSCs CD29,CD45,CD90were100%,14.7%,16.5%; the percentage of P4BMSCs CD29, CD45,CD90were99.9%,0.8%,99.6%.2、Proliferation of P4BMSCs were not obviously suppressed after5μmol/L5-AZA for24h or48h,size and shape of P4BMSCs changed indistinctly; proliferation of P4BMSCs were suppressed after10μmol/L5-AZA for24h,20%P4BMSCs died, and others appear oval-shape and have cardiac-fibrosis character.75%P4BMSCs died after10μmol/L5-AZA for48h, or15μmol/L5-AZA for24h or48h, and others stop the growth due to low density.3、P1BMSCs and P4BMSCs were exposed in10μmol/L5-AZA for24h, size and shape of P1BMSCs and P4BMSCs changed distinctly. P1-devived and P4-devived cardiomyocyte-like cells are positive for cTnI. However, P4-devived cardiomyocyte-like cells have"muscle-island" and multi-core muscle tube. Immunofluorescence of P4-devived cardiomyocyte-like cells are stronger than P4-devived cardiomyocyte-like cells.The differentiation proportion of P1BMSCs and P4BMSCs are (2±2)%and (36±3)%. Conclusion:1、Pure bone marrow mesenchymal stem cells which have a good potential of proliferation were harvested by whole bone marrow cells adherence method,frequent medium change and differential digestion method in a short time. The purity of BMSCs gradually increased with the days passing.2、The optimal induction condition for inducing BMSCs into cardiomyocyte-like cells is10μmol/L5-AZA for24h.3、P4BMSCs have a good potential for myocardialization.