| Acute myocardial infarction (AMI) and subsequent heart failure is still an important cause of mortality and morbidity in the world despite recent substantial advances in medical and device therapy. In recent 20 years, cardiac regenerative medicine represented by stem cell therapy has attracted more and more attention and now stem cell therapy has been considered as one of the most promising therapeutic strategies for ischemic heart diseases. Among several cell types used in experimental and clinical studies, bone-marrow derived mesenchymal stem cells seemed to be a preferred cell source for cardiac repair because of their ease of isolation and expansion ex vivo. However, several recent randomized controlled trials suggested that the benefits observed in cardiac function improvement after bone marrow stem cell transplantation appeared to be relatively small. Poor cell survival after transplantation seriously limited the therapeutic efficacy of stem cell therapy for myocardial infarction. For example one study suggested that only 0.44% of implanted cells suvived in the left ventricle 4 days after injection and fewer cells were identified in the myocardium at later time points. Thus, it is critical to promote the resistance of engrafted cells to hostile microenvironment in order to enhance the therapeutic efficacy of stem cell therapy.Hydrogen sulfide (H2S) is a colorless gas with characteristic smell of rotten eggs, which is the third gaseous signaling molecules similar to nitric oxide and carbon monoxide. H2S participates in several physiological process including cytoprotection, vasorelaxation and angiogenesis. Although the cytoprotective effects of H2S have previously been documented in many cell types, it's potential effect on stem cells has not been evaluated until now. Therefore, the purpose of the present study was to investigate whether H2S preconditioning (PC) could promote mesenchymal stem cells'(MSCs) survival and myocardial repair in a rat MI model. Partâ… The isolation, culturing and identification of rat bone marrow derived MSCsObjective:To develop one practical method of the isolation, culture and purification of bone marrow MSCs from the bone marrow of adult rats.Methods:Whole bone marrow adherent cultivation was used to isolate bone marrow MSCs from rats. Then the MSCs were purified and expanded when passaging. The MSCs were identified by detecting the cell surface antigens of CD34 and CD90 by flow cytometry. We also assessed their capacity of differentiation to adipocytes in vitro.Results:Most of primary cells were of shuttle shape, some of irregular shape and grew clonally. These cells grew in parallel or vortex with a similar fibrobalstoid spindle-shaped morphology after several passages. The results of flow cytometry showed that the expression of CD90 and CD34 was 92.4% and 4.36% respectively. These cells could also be induced to differentiate into adipocytes as evidenced by Oil Red staining.Conclusion:Rat bone marrow MSCs can be isolated and expanded in vitro by using whole bone marrow adherent cultivation, and it is an efficient and stable method.Partâ…¡The cytoprotection of H2S on hypoxia induced apoptosis of MSCs and its possible mechanismObjective:To investigate the possible cytoprotective effect of H2S on hypoxia induced apoptosis in MSCs and its underlying mechanism.Methods:To culture MSCs and develop the model of hypoxia induced apoptosis of MSCs in vitro. With the presence or absence of H2S preconditioning, We assessed the hypoxia induced apoptosis by flow cytometry after Annexin V/7-AAD staining and phase contrast microscope after Hochest 33342 staining. We also evaluated the cell viability after hypoxic treatment by the trypan blue dye-exclusion method and Cell Counting Kit 8 assay. Western blots were utilized to determine the intracellular protein levels of pAkt, Akt, pErk1/2, Erk1/2, peNOS, pFox01, pGSK-3β,Fox01. Bcl-2, Bax, and Pim-1, which all participated in the process of apoptosis, were also examined by Western blots. The intracellular level of reactive oxygen species (ROS) was measured using Dihydroethidium by fluorescence microscope. The effect of H2S on MSCs'migration was assessed by scratch test assay. The concentration of VEGF was measured by ELISA to determine the effect of H2S on paracrine function of MSCs.Results:MSCs apoptosis was successfully induced by hypoxia for 6-h in serum- and glucose-free DMEM. We found that the cytoprotective effect of H2S on MSCs was dose-dependent when preconditioning MSCs for 30 min with concentration of H2S ranging from 10 to 300μmol/L. We opted for preconditioning of MSCs with 200μmol/L H2S in the subsequent studies. Preconditioning with H2S could decrease hypoxia-induced apoptosis from 25.45% to 14.09%(P<0.05), as demonstrated by Annexin V/7-AAD staining, and promote cell viability, as shown by Cell Counting Kit-8 assay (52% vs 38%, P<0.05) and trypan blue dye-exclusion method (67% vs 46%, P <0.05). In addition, H2S could also prevent an increased intracellular level of ROS induced by hypoxia. Western blots suggested a significant increase of phosphorylated Akt (pAkt), phosphorylated Erkl/2 (pErkl/2) and pGSK-3βin MSCs after being preconditioned with H2S. H2S could also up-regulate the protein expression of Pim-1 and Bcl-2 and down-regulate the expression of Bax under hypoxic condition. The migration capacity and paracrine effect of MSCs were not affected after H2S treatment.Conclusion:H2S protected MSCs against hypoxia-induced apoptosis. MEK/Erkl/2/GSK-3βand PI3K/Akt pathway might be involved in this cytoprotection. Furthermore, the antioxidant property of H2S might also contribute to its cytoprotective effect on MSCs. Part III The effect of H2S preconditioned MSCs on cardiac function in a rat acute myocardial infarction mode.Objective:To investigate whether preconditioned MSCs with H2S could enhance recovery of myocardial function in vivo.Methods:The MI model was successfully developed through ligation of left anterior descending artery. The male MSCs were used for the following two-part experiments:(1) Gender-mismatched transplantation for quantifying survival of MSCs by real-time PCR:1.0×106 of MSCs (MSCs group) or PC MSCs (H2S group) in 100 u 1 of volume was intramyocardially injected into female recipient hearts (n=3/group); (2) Gender-matched transplantation for cardiac function comparison:1.0×106 of MSCs (MSCs group), PC MSCs (H2S group) or PBS (PBS group) in 100μl of volume was intramyocardially injected into the anterior aspects of the viable myocardium bordering the infarction of male Sprague-Dawley rats hearts immediately after MI with a 30-gauge needle. Cell survival was assessed in a gender-mismatched transplantation MI model by Real-time PCR for sry gene 4 days after transplantation. Cardiac function, left ventricular (LV) remodeling were examined using transthoracic echocardiography. Infarct size was caculated after Masson's staining, and capillary density of peri-infarcted area was examined using an immunohistochemical staining for CD31.Results:Real-time PCR for sry gene 4 days after MI showed that the survival of PC MSCs was 1.64-fold higer than MSCs in infarcted myocardium. Four weeks later,echocardiography indicated improved indices of left ventricular function, including ejection function and fractional shortening in H,S group (LVEF:45.35±2.25%; LVFS:23.51±1.27%) as compared with MSCs group (LVEF:36.92±0.79%; LVFS:18.79±0.47%) and PBS group (LVEF:30.29±1.80%; LVFS:15.02±0.97%). Infarct size in H2S group(30.30±3.37%) was 23.83% smaller than MSCs group(39.78±3.14%). Furthermore, capillary density was highest in H2S group (14.79±2.77 vessels/high power field) as compared with the other groups (9.38±2.52 vessels/high power field in MSCs group and 4.67±1.47 vessels/high power field in PBS group). Conclusion:H2S preconditioning could enhance MSCs'survival in infracted myocardium and promoted their salutary effects on cardiac function after cell transplantation. Enhanced cell survival was the major mechanism of improved cardiac function after transplanting H2S preconditioned MSCs...
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