Repair mechanisms of bone marrow mesenchymal stem cells in myocardial infarction

The prognosis of patients with myocardial infarction (MI) and resultant chronic heart failure remains extremely poor despite advances in optimal medical therapy and interventional procedures. Animal experiments and clinical trials using adult stem cell therapy following MI have shown a global improvement of myocardial function. Bone marrow-derived mesenchymal stem cells (MSCs) hold promise for cardiac repair following MI, due to their multilineage, self-renewal and proliferation potential. In addition, MSCs can be easily isolated, expanded in culture, and have immunoprivileged properties to the host tissue. Experimental studies and clinical trials have revealed that MSCs not only differentiate into cardiomyocytes and vascular cells, but also secrete amounts of growth factors and cytokines which may mediate endogenous regeneration via activation of resident cardiac stem cells and other stem cells, as well as induce neovascularization, anti-inflammation, anti-apoptosis, anti-remodelling and cardiac contractility in a paracrine manner. It has also been postulated that the anti-arrhythmic and cardiac nerve sprouting potential of MSCs may contribute to their beneficial effects in cardiac repair. Most molecular and cellular mechanisms involved in the MSC-based therapy after MI are still unclear at present. This article reviews the potential repair mechanisms of MSCs in the setting of MI.     

Gene expression profiles following intracoronary injection of mesenchymal stromal cells using a porcine model of chronic myocardial infarction

Background aimsWe evaluated the therapeutic potential of injection of in vitro differentiated bone marrow mesenchymal stromal cells (MSC) using a swine model.Methods and ResultsMyocardial infarction was induced by coronary occlusion. Three groups (n = 5 each) were analyzed: one group received an injection of 17.8 ± 9.3 × 10⁶ 5-azacytidine-treated allogeneic MSC 1 month after infarction; a placebo group received an injection of medium; and controls were kept untreated. After 4 weeks, heart samples were taken from three infarcted areas, interventricular septa, ventricles and atria. Gene expression profiles of genes related to contractility (Serca2a), fibrosis (Col1a1), cardiomyogenesis (Mef2c, Gata4 and Nkx2.5) and mobilization of stem cells (Sdf1, Cxcr4 and c-kit) were compared by quantitative real-time PCR (qRT-PCR). Gene expression profiles varied in different heart areas. Thus Serca2a expression was reduced in infarcted groups in all heart regions except for the left ventricles, where Col1a1 was overexpressed. The expression of genes related to cardiomyogenesis decreased in the infarcted zones and left atria compared with healthy hearts. Interestingly, increased expression of Cxcr4 was detected in infarcted regions of MSC-treated pigs compared with the placebo groupConclusionsInfarction induced changes in expression of genes involved in various biologic processes. Genes involved in cardiomyogenesis were downregulated in the left atrium. The intracoronary injection of MSC resulted in localized changes in the expression of Cxcr4.     

Priming mesenchymal stem cells boosts stem cell therapy to treat myocardial infarction

Cardiovascular diseases are the number one cause of death globally and are projected to remain the single leading cause of death. Treatment options abounds, although efficacy is limited. Recent studies attribute discrete and ephemeral benefits to adult stem cell therapies, indicating the urge to improve stem cell based–therapy. In this study, we show that priming mesenchymal stem cells (MSC) towards cardiomyogenic lineage enhances their beneficial effects in vivo as treatment option for acute phase myocardial infarction. MSC were primed using cardiomyogenic media for 4 days, after which peak expression of key cardiomyogenic genes are reached and protein expression of Cx‐43 and sarcomeric α‐actinin are observed. MSC and primed MSC (pMSC) were characterized in vitro and used to treat infarcted rats immediately after left anterior descending (LAD) occlusion. Echocardiography analysis indicated that MSC‐treated myocardium presented discrete improvement in function, but it also showed that pMSC treatment lead to superior beneficial results, compared with undifferentiated MSC. Seven days after cell injection, MSC and pMSC could still be detected in the myocardium. Connexin‐43 expression was quantified through immunoblotting, and was superior in pMSC, indicating that this could be a possible explanation for the superior performance of pMSC therapy.     

An improved transplantation strategy for mouse mesenchymal stem cells in an acute myocardial infarction model

To develop an effective therapeutic strategy for cardiac regeneration using bone marrow mesenchymal stem cells (BM-MSCs), the primary mouse BM-MSCs (1(st) BM-MSCs) and 5(th) passage BM-MSCs from β-galactosidase transgenic mice were respectively intramyocardially transplanted into the acute myocardial infarction (AMI) model of wild type mice. At the 6(th) week, animals/tissues from the 1(st) BM-MSCs group, the 5(th) passage BM-MSCs group, control group were examined. Our results revealed that, compared to the 5(th) passage BM-MSCs, the 1(st) BM-MSCs had better therapeutic effects in the mouse MI model. The 1(st) BM-MSCs maintained greater differentiation potentials towards cardiomocytes or vascular endothelial cells in vitro. This is indicated by higher expressions of cardiomyocyte and vascular endothelial cell mature markers in vitro. Furthermore, we identified that 24 proteins were down-regulated and 3 proteins were up-regulated in the 5(th) BM-MSCs in comparison to the 1(st) BM-MSCs, using mass spectrometry following two-dimensional electrophoresis. Our data suggest that transplantation of the 1(st) BM-MSCs may be an effective therapeutic strategy for cardiac tissue regeneration following AMI, and altered protein expression profiles between the 1(st) BM-MSCs and 5(th) passage BM-MSCs may account for the difference in their maintenance of stemness and their therapeutic effects following AMI.     

Lipopolysaccharide preconditioning enhances the efficacy of mesenchymal stem cells transplantation in a rat model of acute myocardial infarction

Background  

Mesenchymal stem cells (MSCs)-based regenerative therapy is currently regarded as an alternative approach to salvage the acute myocardial infarcted hearts. However, the efficiency of MSCs transplantation is limited by lower survival rate of engrafted MSCs. In previous study, we found that 1.0 μg/ml Lipopolysaccharide (LPS) could protect MSCs against apoptosis induced by oxidative stress and meanwhile enhance the proliferation of MSCs. Therefore, in the present study, we firstly preconditioned MSCs with 1.0 μg/ml LPS, then transplanted MSCs into ischemic myocardium, and observed the survival and cardiac protective capacity of MSCs in a rat model of acute myocardial infarction. Furthermore, we tried to explore the underlying mechanisms and the role of Toll-like receptor-4 (TLR4) in the signal pathway of LPS-induced cardiac protection.     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.     

Insulin-like growth factor 1 improves the efficacy of mesenchymal stem cells transplantation in a rat model of myocardial infarction

Background Previous study demonstrated the improvement of cardiac function was proportional to the number of cells implanted. Therefore, increasing cell survival in the infarcted myocardium might contribute to the improvement of the functional benefit of cell transplantation. Methods and results MSCs were treated with IGF-1 in vitro and infused into the acute myocardial infarction rats via the tail vein. After treatment of MSCs with IGF-1 for 48 h, flow cytometric analysis showed marked enhancement of expression of CXCR4 in the cell surface. After 4 weeks of transplantation, we found 1) a greater number of engrafted MSCs arrived and survived in the peri-infarct region; 2) TnT protein expression and capillary density were enhanced; 3) LV cavitary dilation, transmural infarct thinning, deposition of total collagen in the peri-infarct region and cardiac dysfunction were attenuated. Conclusion 1) IGF-1 treatment has time-dependent and dose-dependent effects on CXCR4 expression in MSCs in vitro. 2) IGF-1 improves the efficacy of MSCs transplantation in a rat model of myocardial infarction mainly via enhancement of the number of cells attracted into the infarcted heart. These findings provide a novel stem cell therapeutic avenue against ischemic heart disease.     

Cotransplantation of human umbilical cord-derived mesenchymal stem cells and umbilical cord blood-derived CD34+ cells in a rabbit model of myocardial infarction

The objective of the study is to investigate the effect of hypoxic preconditioning on the immunomodulatory properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and the effect of cotransplantation of hUC-MSCs and human umbilical cord blood (hUCB)-derived CD34⁺ cells in a rabbit model of myocardial infarction. hUC-MSCs with or without hypoxic preconditioning by cobalt chloride were plated in a 24-well plate, and then cocultured with hUCB-CD34⁺ cells and PBMCs for 96 h at 37 °C in a 5 % CO₂ incubator. For the negative control, hUC-MSCs were omitted. The groups were divided as follows: A1 = HP-MSCs + hUCB-CD34⁺ cells + PBMC, A2 = hUC-MSCs + hUCB-CD34⁺ cells + PBMC, Negative Control = hUCB-CD34⁺ cells + PBMC. Culture supernatants of each group were collected, and the IL-10 and IFN-γ levels were measured by ELISA. A rabbit model of MI was established using a modified Fujita method. The animals were then randomized into three groups and received intramyocardial injections of 0.4 ml of PBS alone (n = 8, PBS group), hUC-MSCs in PBS (n = 8, hUC-MSCs group), or hUC-MSCs + CD34⁺ cells in PBS (n = 8, Cotrans group), at four points in the infarct border zone. Echocardiography was performed at baseline, 4 weeks after MI induction, and 4 weeks after cell transplantation, respectively. Stem cell differentiation and neovascularization in the infracted area were characterized for the presence of cardiac Troponin I (cTnI) and CD31 by immunohistochemical staining, and the extent of myocardial fibrosis was evaluated by hematoxylin and eosin (H&E) and Masson’s trichrome. IFN-γ was 27.00 ± 1.11, 14.20 ± 0.81, and 7.22 ± 0.14 pg/ml, and IL-10 was 31.68 ± 3.08, 61.42 ± 1.08, and 85.85 ± 1.80 pg/ml for the Control, A1 and A2 groups, respectively, which indicated that hUCB-CD34⁺ cells induced immune reaction of peripheral blood mononuclear cells, whereas both hUC-MSCs and HP-MSCs showed an immunosuppressive effect, which, however, was attenuated by hypoxic preconditioning. The Cotrans group had less collagen deposition in the infarcted myocardium and better heart function than the hUC-MSCs or PBS group. The presence of cTnI-positive cells and CD31-positive tubular structures indicated the differentiation of stem cells into cardiomyocytes and neovascularization. The microvessel density was 12.19 ± 3.05/HP for the hUC-MSCs group and 31.63 ± 2.45/HP for the Cotrans group, respectively (P < 0.01). As a conclusion, both hUC-MSCs and HP-MSCs have an immunosuppressive effect on lymphocytes, which, however, can be attenuated by hypoxic preconditioning. Cotransplantation of hUC-MSCs and hUCB-CD34⁺ cells can improve heart function and decrease collagen deposition in post-MI rabbits. Thus, a combined regimen of hUC-MSCs and hUCB-CD34⁺ cells would be more desirable than either cells administered alone. This is most likely due to the increase of cardiomyocytes and enhanced angiogenesis in the infarcted myocardium.     

Injectable collagen scaffold promotes swine myocardial infarction recovery by long-term local retention of transplanted human umbilical cord mesenchymal stem cells

Stem cell therapy is an attractive approach for recovery from myocardial infarction(MI) but faces the challenges of rapid diffusion and poor survival after transplantation. Here we developed an injectable collagen scaffold to promote the long-term retention of transplanted cells in chronic MI. Forty-five minipigs underwent left anterior descending artery(LAD) ligation and were equally divided into three groups 2 months later(collagen scaffold loading with human umbilical mesenchymal stem cell(hUMSC) group, hUMSC group, and placebo group(only phosphate-buffered saline(PBS) injection)). Immunofluorescence staining indicated that the retention of transplanted cells was promoted by the collagen scaffold. Echocardiography and cardiac magnetic resonance imaging(CMR) showed much higher left ventricular ejection fraction(LVEF) and lower infarct size percentage in the collagen/hUMSC group than in the hUMSC and placebo groups at 12 months after treatment. There were also higher densities of vWf-, α-sma-, and cTnT-positive cells in the infarct border zone in the collagen/cell group, as revealed by immunohistochemical analysis, suggesting better angiogenesis and more cardiomyocyte survival after MI. Thus, the injectable collagen scaffold was safe and effective on a large animal myocardial model, which is beneficial for constructing a favorable microenvironment for applying stem cells in clinical MI.     

Adropin-based dual treatment enhances the therapeutic potential of mesenchymal stem cells in rat myocardial infarction

Both weak survival ability of stem cells and hostile microenvironment are dual dilemma for cell therapy. Adropin, a bioactive substance, has been demonstrated to be cytoprotective. We therefore hypothesized that adropin may produce dual protective effects on the therapeutic potential of stem cells in myocardial infarction by employing an adropin-based dual treatment of promoting stem cell survival in vitro and modifying microenvironment in vivo. In the current study, adropin (25 ng/ml) in vitro reduced hydrogen peroxide-induced apoptosis in rat bone marrow mesenchymal stem cells (MSCs) and improved MSCs survival with increased phosphorylation of Akt and extracellular regulated protein kinases (ERK) l/2. Adropin-induced cytoprotection was blocked by the inhibitors of Akt and ERK1/2. The left main coronary artery of rats was ligated for 3 or 28 days to induce myocardial infarction. Bromodeoxyuridine (BrdU)-labeled MSCs, which were in vitro pretreated with adropin, were in vivo intramyocardially injected after ischemia, following an intravenous injection of 0.2 mg/kg adropin (dual treatment). Compared with MSCs transplantation alone, the dual treatment with adropin reported a higher level of interleukin-10, a lower level of tumor necrosis factor-α and interleukin-1β in plasma at day 3, and higher left ventricular ejection fraction and expression of paracrine factors at day 28, with less myocardial fibrosis and higher capillary density, and produced more surviving BrdU-positive cells at day 3 and 28. In conclusion, our data evidence that adropin-based dual treatment may enhance the therapeutic potential of MSCs to repair myocardium through paracrine mechanism via the pro-survival pathways.Subject terms: Cell death, Heart stem cells     

Autophagy in fate determination of mesenchymal stem cells and bone remodeling

Mesenchymal stem cells (MSCs) have been widely exploited as promising candidates in clinical settings for bone repair and regeneration in view of their self-renewal capacity and multipotentiality. However, little is known about the mechanisms underlying their fate determination, which would illustrate their effectiveness in regenerative medicine. Recent evidence has shed light on a fundamental biological role of autophagy in the maintenance of the regenerative capability of MSCs and bone homeostasis. Autophagy has been implicated in provoking an immediately available cytoprotective mechanism in MSCs against stress, while dysfunction of autophagy impairs the function of MSCs, leading to imbalances of bone remodeling and a wide range of aging and degenerative bone diseases. This review aims to summarize the up-to-date knowledge about the effects of autophagy on MSC fate determination and its role as a stress adaptation response. Meanwhile, we highlight autophagy as a dynamic process and a double-edged sword to account for some discrepancies in the current research. We also discuss the contribution of autophagy to the regulation of bone cells and bone remodeling and emphasize its potential involvement in bone disease.     

Dihydroxy-cholecalciferol stimulates adipocytic differentiation of porcine mesenchymal stem cells

Dihydroxy-cholecalciferol [1,25(OH)2D3] has been shown to have pleiotropic effects on the differentiation of mesenchymal stem cells (MSC) based on species and culture conditions. We have examined the effects of 1,25(OH)2D3 on the differentiation of porcine MSC under culture conditions designed to promote proliferation in order to attempt to mimic the conditions in young, rapidly growing animals. The MSC were isolated from bone marrow of a young pig and grown in basal media (BM) containing DMEM+10% fetal bovine serum and antibiotics. Cells received either BM, BM+10(-8) M 1,25(OH)2D3 or BM+10(-7) M 1,25(OH)2D3 with complete media changes every 3 days for a total of 12 days of culture. On days 3, 6, 9 and 12, viable cell numbers were determined, and samples were collected for gene expression analysis and cytochemical staining. There was a treatment-based reduction in cell numbers on 6, 9 and 12 days (P<.05). The concentrations of mRNAs encoding peroxisome proliferator-activated receptor gamma, lipoprotein lipase, and adipocyte-binding protein 2 were increased (P<.05) in a manner indicative of adipocytic differentiation by treatment with 1,25(OH)2D3 in a dose-dependent manner. However, the mRNA levels of osteocalcin, a late stage marker of osteoblastic differentiation, was also increased (P<.05) by treatment with 1,25(OH)2D3. An increased percentage of lipid filling, based on Oil Red O staining, and decreased alkaline phosphatase activity, was also seen with 1,25(OH)2D3 treatment. These data suggest that 1,25(OH)(2)D(3) stimulates the differentiation of porcine MSC towards an adipocytic phenotype.     

Pluripotent stem cell-derived mesenchymal stromal cells improve cardiac function and vascularity after myocardial infarction

Background aimsMesenchymal stromal cells (MSCs) have been shown to improve cardiac function after injury and are the subject of ongoing clinical trials. In this study, the authors tested the cardiac regenerative potential of an induced pluripotent stem cell-derived MSC (iPSC-MSC) population (Cymerus MSCs) in a rat model of myocardial ischemia-reperfusion (I/R). Furthermore, the authors compared this efficacy with bone marrow-derived MSCs (BM-MSCs), which are the predominant cell type in clinical trials.MethodsFour days after myocardial I/R injury, rats were randomly assigned to (i) a Cymerus MSC group (n = 15), (ii) a BM-MSC group (n = 15) or (iii) a vehicle control group (n = 14). For cell-treated animals, a total of 5 × 10⁶ cells were injected at three sites within the infarcted left ventricular (LV) wall.ResultsOne month after cell transplantation, Cymerus MSCs improved LV function (assessed by echocardiography) compared with vehicle and BM-MSCs. Interestingly, Cymerus MSCs enhanced angiogenesis without sustained engraftment or significant impact on infarct scar size. Suggesting safety, Cymerus MSCs had no effect on inducible tachycardia or the ventricular scar heterogeneity that provides a substrate for cardiac re-entrant circuits.ConclusionsThe authors here demonstrate that intra-myocardial administration of iPSC-MSCs (Cymerus MSCs) provide better therapeutic effects compared with conventional BM-MSCs in a rodent model of myocardial I/R. Because of its manufacturing scalability, iPSC-MSC therapy offers an exciting opportunity for an “off-the-shelf” stem cell therapy for cardiac repair.     

Transplantation of mesenchymal stem cells overexpressing IL10 attenuates cardiac impairments in rats with myocardial infarction

Mesenchymal stem cell (MSC) has been well known to exert therapeutic potential for patients with myocardial infarction (MI). In addition, interleukin‐10 (IL10) could attenuate MI through suppressing inflammation. Thus, the combination of MSC implantation with IL10 delivery may extend health benefits to ameliorate cardiac injury after MI. Here we established overexpression of IL10 in bone marrow‐derived MSC through adenoviral transduction. Cell viability, apoptosis, and IL10 secretion under ischemic challenge in vitro were examined. In addition, MSC was transplanted into the injured hearts in a rat model of MI. Four weeks after the MI induction, MI, cardiac functions, apoptotic cells, and inflammation cytokines were assessed. In response to in vitro oxygen‐glucose deprivation (OGD), IL10 overexpression in MSC (Ad.IL10‐MSC) enhanced cell viability, decreased apoptosis, and increased IL10 secretion. Consistently, the implantation of Ad.IL10‐MSCs into MI animals resulted in more reductions in myocardial infarct size, cardiac impairment, and cell apoptosis, compared to the individual treatments of either MSC or IL10 administration. Moreover, the attenuation of both systemic and local inflammations was most prominent for Ad.IL10‐MSC treatment. IL10 overexpression and MSC may exert a synergistic anti‐inflammatory effect to alleviate cardiac injury after MI.     

Timing of transplantation of autologous bone marrow derived mesenchymal stem cells for treating myocardial infarction

It is still unclear whether the timing of intracoronary stem cell therapy affects the therapeutic response in patients with myocardial infarction.The natural course of healing the infarction and the presence of putative homing signals within the damaged myocardium appear to favor cell engraftment during the transendothelial passage in the early days after reperfusion.However,the adverse inflammatory environment,with its high oxidative stress,might be deleterious if cells are administered too early after reperfusion.Here we highlight several aspects of the timing of intracoronary stem cell therapy.Our results showed that transplantation of bone marrow mesenchymal stem cells at 2 4 weeks after myocardial infarction is more favorable for reduction of the scar area,inhibition of left ventricular remodeling,and recovery of heart function.Coronary injection of autologous bone marrow mesenchymal stem cells at 2 4 weeks after acute myocardial infarction is safe and does not increase the incidence of complications.     

Efficient adenoviral-mediated gene delivery into porcine mesenchymal stem cells

Mesenchymal stem cell (MSC) mediated gene therapy research has been conducted predominantly on rodents. Appropriate large animal models may provide additional safety and efficacy information prior to human clinical trials. The objectives of this study were: (a) to optimize adenoviral transduction efficiency of porcine bone marrow MSCs using a commercial polyamine-based transfection reagent (GeneJammer, Stratagene, La Jolla, CA), and (b) to determine whether transduced MSCs retain the ability to differentiate into mesodermal lineages. Porcine MSCs (pMSCs) were infected under varying conditions, with replication-defective adenoviral vectors carrying the GFP gene and GFP expression analyzed. Transduced cells were induced to differentiate in vitro into adipogenic, chondrogenic, and osteogenic lineages. We observed a 5.5-fold increase in the percentage of GFP-expressing pMSCs when adenovirus type 5 carrying the adenovirus type 35 fiber (Ad5F35eGFP) was used in conjunction with GeneJammer. Transduction of pMSCs at 10.3-13.8 MOI (1,500-2,000 vp/cell) in the presence of Gene Jammer yielded the highest percentage of GFP-expressing cells ( approximately 90%) without affecting cell viability. A similar positive effect was detected when pMSCs were infected with an Ad5eGFP vector. Presence of fetal bovine serum (FBS) during adenoviral transduction enhanced vector-encoded transgene expression in both GeneJammer-treated and control groups. pMSCs transduced with adenovirus vector in the presence of GeneJammer underwent lipogenic, chondrogenic, and osteogenic differentiation. Addition of GeneJammer during adenoviral infection of pMSCs can revert the poor transduction efficiency of pMSCs while retaining their pluripotent differentiation capacity. GeneJammer-enhanced transduction will facilitate the use of adenoviral vectors in MSC-mediated gene therapy models and therapies.