Mouse Bone Marrow Mesenchymal Stem Cell Subsets Differentiation Of Cardiac-like Cells

Background Since1976, Friedenstein for the first time provides more direct evidence toprove that the precursor cells in bone marrow stromal cells.1984, Owen first defined theadherent growth of bone marrow mononuclear cells for bone marrow mesenchymal stemcells (BMSC).1987, Friedenstein et al also found that, under certain conditions, theadherent bone marrow mononuclear cells cultured in plastic Petri dishes differentiate intoosteoblasts、chondrocytes、adipocytes and myoblasts,and amplified20-30generations stillmaintain differentiation potential。1981Martin first isolated embryonic stem cells frommouse embryos, stem cell transplantation is the rapid development. In2003, Food andDrug Administration (FDA) first approved the autologous bone marrow stem celltransplantation for treatment of myocardial infarction disease. All over the world havecarried out the study of stem cell therapy. At present, bone marrow mesenchymal stemcells are used the most widely. China has carried out studies of bone marrow mesenchymalstem cells to treat liver, pancreas, nerves, heart and other organ lesions. And published alots of researches and bone marrow mesenchymal stem cells to treat autoimmune diseaseshave been clinical stage. But undeniable bone marrow mesenchymal stem cells have somedegree of treatment effect, but far short of expectations. In particular, many basic andclinical trials showed that stem cell transplantation can repair the damage myocardial,promote angiogenesis, reduce the myocardial infarct size and improve heart function. But,there is a dispute to what extent bone marrow mesenchymal stem cells improve heartfunction on different research institutions. It may be due to bone marrow mesenchymalstem cells are a group of polyclonal populations of cells, different clonal have differentfunctions or different differentiation direction.Find the different functions subsets is a urgent mission. In2002, Daniele Torella, first isolate an inherent cardiac stem cells frommouse heart, thus break a concept of the myocardial cells are immortalized cells. Furtherstudies showed that its surface differentiation antigens, can be divided into six subsets andsix subsets cells can differentiation into myocardial cells directly. And cardiac stem cellsderived from bone marrow stem cell pool. On the basis of these studies, we consider usinghave been known differentiation antigen of mouse cardiac stem cells, sorting mouse bonemarrow mesenchymal stem cells and found directed to the cardiac differentiation of bonemarrow mesenchymal stem cells clone subsets.Part I Murine bone marrow mesenchymal stem cells were isolated andculturedObjective Bone marrow mesenchymal stem cells (Bone Mesenchymal Stem CellsBMSCs) isolated from mouse bone marrow. Experimental platform is builted for furthercell sorting.Methods whole bone marrow culture. The beads and adherent purify and passage.Fat bone and Cartilage differentiation experimental testing cell differentiation function.Identification of cell surface antigen expression by flow cytometry.Results In vitro culture of primary mouse bone marrow mesenchymal stem cellsafter (7-10) days80%-90%confluence.3rd passage bone marrow mesenchymal stem cellsnegative selected by CD11b beads and continued to culture to7passage. Identification offlow cytometry: the results showed that over90%of mouse bone marrow mesenchymalstem cells express CD29(cell integrin molecules), CD34(progenitor cell surfacedifferentiation antigen) and CD44(adhesion molecule); but did not express thehematopoietic progenitor cell surface marker CD11b、MRD-1、ABCG2.7th passageBMSCs in G0/G1phase cells was73.23%, G2phase cells was0%, S phase cells (G3) was0%, the cells in a quiescent, comply with the stem cell characteristics.Conclusion mouse whole bone marrow culture. The beads and adherent purify andpassage can effectively culture mouse bone marrow mesenchymal stem cells for stem cell subsets sorting.Part II The establishment of mouse myocardial infarction modelObjective Tracheal intubation thoracotomy direct vision complete mouse coronaryanterior descending artery (LAD) ligation, and the establishment model of mousemyocardial infarction.Methods C57BL/6male mice,20, weight:16-20(17.8±2.2) g were randomlydivided into experimental group and control group (sham-operation group). Experimentalgroup,10, tracheal intubation, thoracotomy direct vision mouse coronary anteriordescending artery (LAD) ligation.Results After ligation, the limb leads (I or aVL), Q-wave (>1mv). chest leadelectrocardiogram sum R wave is less than10mv. In lead I and aVL R wave absence. LeadII ST segment elevation of more than0.2mv. After4weeks, review of echocardiography:compare with the control group (sham-operation group), Experimental group, mice leftventricular end diastolic diameter (LVEDd) and left ventricular end systolic diameter(LVEDs) increased significantly (P<0.01), left ventricular ejection fraction (LVEF) and leftventricular fractional shortening index (FS) significantly reduced in myocardial infarctionmodel group (P <0.01, P <0.01). Two-dimensional: left ventricular cavity to expand, theleft ventricular anterior wall segments movement weakened, disappear.Conclusion Tracheal intubation thoracotomy direct vision complete mouse coronaryanterior descending artery (LAD) ligation, model of mouse myocardial infarction can besuccessfully established.Part III Mouse bone marrow mesenchymal stem cell subsets sortingObjective According to test results of flow cytometry seventh passages the mousebone marrow mesenchymal stem cells by magnetic bead sorting purpose of subsets cells.Methods To7th passages mouse bone marrow mesenchymal stem cells though CD45+columns, sorting to get the CD45+cell population and CD45-cell population.CD45-cell though CD31+columns, sorting the SCA-1+/CD45-/CD31-and the SCA-1+/CD45-/CD31+two groups cells. And then the CD45+cell though CD31+columns, sortingthe SCA-1+/CD45+/CD31+and the SCA-1+/CD45+/CD31-, two groups cells.Results The final the SCA-1+/CD31-/CD45-cell count:7.8×106; the SCA-1+/CD31+/CD45-cell count:3×106; SCA-1+/CD31+/CD45+cell count:2×105; SCA-1+/CD31-/CD45+cell count:5×105. Magnetic bead sorting was approximately60%. Cells ingood condition, and culture three weeks, the number of various subsets cells up to:2×107.Conclusion Magnetic bead can sorting mouse bone marrow mesenchymal stemcells. cell yield is acceptabl and minor damage. Meet the further research.Part IV Murine bone marrow mesenchymal stem cells different subsets directeddifferentiation into cardiomyocytes, anti-apoptotic, homing capacityObjective Explore murine bone marrow mesenchymal stem cells subsets thecardiac differentiation, anti-apoptosis and homing ability.Methods First of all murine bone marrow mesenchymal stem cells subsets directlyco-cultured with the myocardial cells and5-aza-2’-deoxycytidine (5-aza-2’-deoxycytidine)induction method contrast to outgoing myocardial directional differentiationcapacity.Next, the mouse bone marrow mesenchymal stem cell subsets and unsorted groupinto hypoxia (5%), serum-free culture environment24hours using flow cytometry subsetsand unsorted group apoptosis rate.And transwell chamber Shop into the Matrigel testsubsets and unsorted group of stem cells homing ability.Murine bone marrowmesenchymal stem cells four sub-groups and a unsorting group were injected into themyocardial infarction model in mice at48hours,96hours and seven days ofechocardiography and small animal in vivo imaging various subsets of cells differences inthe body.Results And co-cultured myocardial cells and in5-aza-cytidine induction subsetsin mouse bone marrow mesenchymal stem cells express the cardiac myocyte-specific antigen.PCR shows that SCA-1+/CD45+/CD31+subsets with myocardial cells co-culturedto express the cardiac myocyte-specific antigen gene and early myocardial promoterGATA-4,, NKx2.5are higher than the other subsets.In vitro using flow cytometry subsetsand unsorted group of stem cell anti-apoptotic ability show of SCA-1+/CD45+/CD31+anti-apoptotic capacity is the strongest. Transwell chamber confirmedSCA-1+/CD45+/CD31+the strongest cell homing ability.Echocardiography suggestionstem cells were injected48hours,96hours and7days of the SCA-1+/CD45+/CD31+subsets of cardiac function improved significantly.Vivo imaging of small animals Tip:stem cells were injected48hours,96hours and7days: the SCA-1+/CD45+/CD31+subsets in mean fluorescence intensity higher than the other subsets.In situ immunohistochemical detection of various subsets of stem cells were injected96hours MImyocardium, visible local cardiac stem cells.Subsets of cells to mobilize the capacity ofcardiac stem cells:SCA-1+/CD45+/CD31+> SCA-1+/CD45-/CD31+>SCA-1+/CD45-/CD31-> SCA-1+/CD45+/CD31-.Conclusion Directed differentiation of myocardium the SCA-1+/CD45+/CD31+sub-populations, anti-apoptosis, homing are better than the other subsets andnon-sorting group. The in vivo experiments show that after stem cell subsets are injectedaccordance with the hemodynamic completed in the first initial distribution, and thencomplete their homing. In vivo experiments show that the SCA-1+/CD45+/CD31+improvement of cardiac function, homing, anti-apoptotic ability is better than the othersubsets and unsorted groups and match the in vitro experiments. This test confirmed thatthe mouse bone marrow mesenchymal stem cell subsets can mobilize in cardiac stem cells.Speculated that bone marrow mesenchymal stem cells to treat myocardial infarction, earlymobilization of the body’s own cardiac stem cells through paracrine. Treatment ofadvanced primarily through the stem cell transdifferentiation to complete the repair. Andvarious subsets of cells mobilization of cardiac stem cells have different ability.PartVI Murine bone marrow mesenchymal stem cell subsets microarray analysisObjection Explore the biological performance of4subsets of mouse bone marrowmesenchymal stem from the molecular level. Methods Using gene chip technology complete the Agilent mouse whole genome4*44k a transcriptome-chip.Result There are four-fold change (Logarithmic is2fold change), that the genesdifferentially expressed between any two groups. A total of5619transcripts differentiallyexpressed. After GO and Pathway analysis of5619transcription complete a clusteranalysis.There are172genes expression four times on cardiovascular developmentalbetween SCA-1+/CD45+/CD31+and SCA-1+/CD45+/CD31-、SCA-1+/CD45-/CD31+,SCA+/CD45-/CD31-. There are152genes expression four times oncell migration between SCA-1+/CD45+/CD31+and SCA-1+/CD45+/CD31-、SCA-1+/CD45-/CD31+,SCA+/CD45-/CD31-. There are277genes expression four times oncell apoptotic between SCA-1+/CD45+/CD31-and SCA-1+/CD45+/CD31+、SCA-1+/CD45-/CD31+，SCA+/CD45-/CD31-. Further involved in the migration Genes toexpress the highest and lowest gene cluster visible: SCA-1+/CD45+/CD31+compared withother groups were significantly reduced on Madcam1, Spag9, Rap2a, I116, Ednrb gene.The ednrb, Dcx, Rasgrf1, Srgap1genes, the SCA-1+/CD45+/CD31+compared with othergroups was significantly increased. Nrcam, Co111a1, Prrx1genes were significantlyreduced the SCA-1+/CD45+/CD31+compared with other groups. Further involved incardiovascular development genes highest and lowest gene cluster can be seen:SCA-1+/CD45+/CD31+was significantly increased compared with other groups express onRapgef1, Dsc2Nrcam, Pr17d1Itga4, Pcsk5Scma3c, My13, Myo18b, Chi31lgenes.Nrcam, Co111a1, Prrx1genes the SCA-1+/CD45+/CD31+compared with othergroups was significantly reduced. Further involved apoptosis-related gene expression of inthe highest and lowest gene cluster can be seen: Lcn2、Egr3、Pik3r3、Mdrn4、Cul7、Fcerlg、Map3kg genes, the SCA-1+/CD45+/CD31-significantly higher than the other groupexpression. Park2、Prkcb、Frzb、Dmpk genes, the SCA-1+/CD45-/CD31-compared withother groups were significantly reduced. Together in cluster analysis and Network resultson the SCA-1+/CD45+/CD31+migration of other groups of genes visible the Dcx, andMADCAM1both the intersection description of its function and expression of the migration and other subsets.They are key genes. Togetherin cluster analysis and Networkresults on the SCA-1+/CD45+/CD31+migration of other groups of genes visible the Dcx,and MADCAM1both the intersection description of its function and expression of themigration and other subsets.They are key genes.Togetherin cluster analysis and Networkresults on the SCA-1+/CD45+/CD31+cardiovascular development gene other groups ofgenes visible: SETD2, NCL, EPOR, Rock2of genes is key genes. SCA-1+/CD45+/CD31-andother groups, cluster analysis of apoptotic genes and Network results to compare visiblethe two no intersection, considering the above-mentioned differences in gene expressionwas4-fold, solid-based Network results visible: NFkB1, RB1, E2F1is key genes.Concluse: Further clear murine bone marrow mesenchymal stem cells as a polyclonalgroups. Regulation genes of developmental of the circulatory system SETD2、NCL、EPOR、Rock2lead to the SCA-1+/CD45+/CD31+cells compare to the other three groups ofcells has significant difference.Regulation genes of cell migration Dcx and MADCAM1lead to the SCA-1+/CD45+/CD31+cells compare to the other three groups of cells hassignificant difference.Regulation genes of cell apoptosis NFkB1、RB1、E2F1lead to theSCA-1+/CD45+/CD31+cells compare to the other three groups of cells has significantdifference.