Experimental Study On The Isolation, Culture, Purification, Biological Characteristics And Induced Differentiation Of Mouse Adult Cardiac Stem Cells

Objective Myocardial infarction induces cardiac dilatation, heart failure and decreasedsurvival rate eventually. The therapeutic methods of myocardial infarction includethrombolysis, percutaneous transluminal coronary angioplasty（PTCA）,coronary arterybypass graft （CABG）,and heart transplantation. Although PTCA and CABG can improveischemia, they can not survive necrotic myocardium. Heart transplanttation can replaceterminal stage failing heart, but insufficient donators and immune reaction have limitedclinical application. Stem cells transplantation therapy may be a new method to treatischemic heart disease. With progress of stem cell research, many questions remainunanswered in this new field. Scientists have been looking for the best stem cell to repairdamaged heart and improve cardiac function. The aim of investigation was to establish amethod for isolating, culturing, purifying adult cardiac stem cells in vitro from mouse,identify the cell surface markers, explore their biological characteristics and theirdifferentiated probability induced by traditional Chinese medicine.Metheds （1）Hearts of C57 mouse were dissociated into single cells suspension bymechanical and enzymatic method, and then were cultured in Dulbecco’s modifiedEagle’s medium and Ham’s F12 （DMEM/ham’s F12） supplemented with 10%FBS,2%B27, 10ng/mlEGF, 20ng/mlbFGF, 10ng/ml CT-1,10ng/mlLIF for expanding in vitro.Proliferation of the cells were observed under the phase contrast microscope.（2）Enrichment of sca-1⁺, c-kit⁺ and sca-1⁺/c-kit⁺ cells was achieved by sorting using themagnetic activated cell sorting （MACS） system. To increase the purity of the cells,magnetic sorting was performed one or twice. The percentage of sca-1⁺,c-kit⁺ andsca-1⁺/c-kit⁺ cells was analyzed by the flow cytometer （FCM）. （3）The cell surface antigens of sca-1, c-kit, sca-1/c-kit, CD34 and lineage mixture （CD3-e, CD-11b, CD45R,Ly-6C/G; TER-119） were observed under laser confocal microscope and analyzed byflow cytometer to identify cardiac stem cells. In order to explore the cardiac stem cellbiological characteristics, we measured cardiac stem cell cycle by the flow cytometer（FCM）, counted cardiac stem cells under phase contrast microscope after culturing andexpanding in vitro, respectively. The cell proliferation curves have been depictedsuccessfully. （4） Sca-1⁺,c-kit⁺ and sca-1⁺/c-kit⁺ cells were indused by Panax notoginoside（100μL/ml）, 5-azacitidine（5-aza, 10μmol/L）, dimethyl sulfoxide minimum （DMSO, 0.8%）for 1 hour, 24 hours and 24 hours. Meanwhile, a blank control group was set to comparethe effect. Immunocytochemical analysis showed the results that sca-1⁺,c-kit⁺ andsca-1⁺/c-kit⁺ cells could express the genes of cardiac transcription factors includingGATA-4 and Nkx2.5.Results （1）Round, bright and small ceils from mouse could be observed under thephase contrast microscope,which proliferated slowly in primary culture, but theyproliferated easily and kept undifferentiation in subculture. （2） Three kinds of cellslabelled with sca-1, c-kit, sca-1/c-kit were sorted by the magnetic activated cell sorting（MACS） system. Flow cytometric analysis revealed that sca-1,c-kit and sca-1/c-kitpositive cells were enriched to 87.4%, 87.8% and 90%, respectively. These cells couldkeep undifferentiation in subculture. （3） The cell surface antigens tested by the flowcytometer （FCM）, laser confocal microscopy and immunofluorescence microscope weresca-1⁺CD34^lowlin^-, c-kit⁺CD34^-lin^-, sca-1⁺c-kit⁺CD34^lowlin^-, respectively. The percentageof G₀/G₁ in sca-1⁺, c-kit⁺ and sca-1⁺/c-kit⁺ analyzed by the flow cytometer （FCM） was71.60%, 42.45%, and 60.44%, respectively. After treatment with Panax notoginoside for1 hour, 5-azacitidine for 24 hours and DMSO for 24 hours, the expression of GATA-4and Nkx2.5 in sca-1⁺,c-kit⁺ and sca-1⁺-c-kit⁺ cells were notably positive in Panaxnotoginoside group,while in 5-aza group and in DMSO group only GATA-4 wereexpressed slightly positive, the expressions of GATA-4 and Nkx2.5 were not detected inblank control group.Conclusion （1）We found round, bright and small cells from adult C57 mouse heart.These cells can proliferate in Dulbecco’s modified Eagle’s medium and Ham’s F12（DMEM/ham’s F 12） supplemented with 10%FBS, 2%B27, 10ng/mlEGF, 20ng/mlbFGF,10ng/ml CT-1,10ng/mlLIF （2） The sca-1⁺,c-kit⁺ and sca-1⁺/c-kit⁺ positive cells can be sorted by magnetic activated cell sorting （MACS） system. （3） Sortting cells can expressstem cell markers and have biology characteristics of stem cell. （4） When treated withPanax notoginoside, cardiac stem cells highly expressed genes of cardiac transcriptionfactors in vitro for a short time. When treated with 5-aza and DMSO, cardiac stem cellsslightly expressed genes of cardiac transcription factors. These results suggest that adultcardiac stem cells exist in the mouse heart which might have the potential ofdifferenciating into cardiomyocytes in vitro. Panax notoginoside can inducedifferentiation of cardiac stem cells into the cells with cardiac characteristics.