Tissue transglutaminase is essential for integrin-mediated survival of bone marrow-derived mesenchymal stem cells

Autologous mesenchymal stem cell (MSC) transplantation therapy for repair of myocardial injury has inherent limitations due to the poor viability of the stem cells after cell transplantation. Adhesion is a prerequisite for cell survival and also a key factor for the differentiation of MSCs. As a novel prosurvival modification strategy, we genetically engineered MSCs to overexpress tissue transglutaminase (tTG), with intention to enhance adhesion and ultimately cell survival after implantation. tTG-transfected MSCs (tTG-MSCs) showed a 2.7-fold and greater than a twofold increase of tTG expression and surface tTG activity, respectively, leading to a 20% increased adhesion of MSCs on fibronectin (Fn). Spreading and migration of tTG-MSCs were increased 4.75% and 2.52%, respectively. Adhesion of tTG-MSCs on cardiogel, a cardiac fibroblast-derived three-dimensional matrix, showed a 33.1% increase. Downregulation of tTG by transfection of small interfering RNA specific to the tTG resulted in markedly decreased adhesion and spread of MSCs on Fn or cardiogel. tTG-MSCs on Fn significantly increased phosphorylation of focal adhesion related kinases FAK, Src, and PI3K. tTG-MSCs showed significant retention in infarcted myocardium by forming a focal adhesion complex and developed into cardiac myocyte-like cells by the expression of cardiac-specific proteins. Transplantation of 1 x 10(6) MSCs transduced with tTG into the ischemic rat myocardium restored normalized systolic and diastolic cardiac function. tTG-MSCs further restored cardiac function of infarcted myocardium as compared with MSC transplantation alone. These findings suggested that tTG may play an important role in integrin-mediated adhesion of MSCs in implanted tissues. Disclosure of potential conflicts of interest is found at the end of this article.     

通心络超微粉对猪急性心肌梗死后移植骨髓间充质干细胞的影响

目的探讨通心络超微粉能否改善猪急性心肌梗死(AMI)局部微环境,从而提高移植自体骨髓间充质干细胞(MSCs)在体存活和移植效果。方法28头中华小型猪被分为4组(n=7),分别为对照、通心络超微粉(自AMI前3 d用药直至后4 d)、MSCs移植和通心络超微粉+MSCs联合组。AMI模型由阻断冠状动脉左前降支90 min制成,再灌注后即刻经心肌内注射DAPI标记的自体骨髓MSCs(3×107cells/动物)。在移植后1周(基线)和6周(终点),以核磁共振(MRI)检测心功能。6周后处死动物,检测移植细胞在体存活和分化、梗死周边心肌凋亡和氧化应激指标。结果终点时,与对照组、MSCs组和通心络超微粉组相比,联合组纤维化和炎症细胞浸润显著减轻,并伴较多存活心肌。与MSCs移植组相比,联合组移植细胞的在体存活和成心肌分化能力均显著增强(P<0.0001)。MRI显示在基线时各心功能参数无显著差异(P>0.05);终点时,和对照组相比,仅联合组左室射血分数(LVEF)显著增加(P<0.0001),室壁运动障碍节段数、梗死室壁增厚率、梗死面积和左室质量指数均显著减小(P<0.0001)。终点时,和对照组相比,在通心络超微粉组和联合组梗死周边区的心肌凋亡均显著减轻(P<0.0001),SOD显著增加(P<0.05),MDA显著减少(P<0.05)。结论实验表明AMI再灌注后即刻心肌内移植自体骨髓MSCs生存和分化能力有限,未产生显著功能学获益。而围移植期使用通心络超微粉可显著改善梗死局部微环境,提高移植细胞存活和分化,并产生显著心功能获益。     

Repair of infarcted myocardium using mesenchymal stem cell seeded small intestinal submucosa in rabbits

Myocardial infarction (MI) remains a common and deadly disease. Using tissue-engineered cardiac grafts to repair infarcted myocrdium is considered to be a therapeutic approach. This study tested the feasibility of using MSCs-seeded SIS to repair chronic myocardial infarction in a rabbit model. MI in rabbits was created by ligation of the left anterior descending artery. BrdU-labeled mesenchymal stem cells (MSCs) were seeded on the small intestinal submucosa and cultured for 5–7 days prior to implantation. Four weeks after myocardial infarction, cardiac grafts were implanted onto the epicardial surface of infarcted myocardium. Four weeks after implantation of the membranes, a serial of tests including echocardiography, hemodynamics, histology and immunohistochemistry were undertaken to evaluate the effect of the implanted grafts on recovery of the infarcted myocardium. It was shown that left ventricular contractile function and dimension, the capillary density of the infarcted region, and myocardial pathological changes were significantly improved in rabbits implanted either SIS or MSCs-seeded SIS. But the MSCs-seeded SIS was more effective. Immunofluorescence staining demonstrated the migration of Brdu-labeled MSCs from the membrane into the infarcted area and their differentiation to cardiomyocytes and smooth muscle cells. Taken together, these results suggest that MSCs-seeded SIS can be used to repair chronic myocardial infarction, which enhances myocardial regeneration.     

Cellular cardiomyoplasty: improvement of left ventricular function correlates with the release of cardioactive cytokines

A growing number of studies are reporting beneficial effects of the transplantation of alleged cardiac stem cells into diseased hearts after myocardial infarction. However, the mechanisms by which transplanted cells might help to promote repair of cardiac tissue are not understood and might involve processes different from the differentiation of transplanted cells into cardiomyocytes. We have compared the effects exerted by skeletal myoblasts (which are not able to form new cardiomyocytes) and ESC-derived cardiomyocytes after implantation into infarcted mouse hearts by echocardiographic follow-up and histological analysis and related these effects to the release of cardioactive cytokines. We found that both cell types led to a long-lasting improvement of left ventricle function and to an improvement of tissue architecture. Since no relevant amounts of myoblast-derived cells were present in infarcted hearts 28 days after transplantation, we investigated the release of cytokines from implanted cells both before and after transplantation into infarcted hearts. ESC-derived cardiomyocytes and myoblasts secreted substantial amounts of interleukin (IL)-1alpha, IL-6, tumor necrosis factor-beta, and oncostatin M, which strongly supported survival and protein synthesis of cultured cardiomyocytes. We postulate that the beneficial effects of the transplantation of myoblasts and cardiomyocytes on heart function and morphology only partially (if at all) depend on the integration of transplanted cells into the myocardium but do depend on the release of a complex blend of cardioactive cytokines.     

Impact of myocardial infarct proteins and oscillating pressure on the differentiation of mesenchymal stem cells: effect of acute myocardial infarction on stem cell differentiation

Stem cell transplantation in acute myocardial infarction (AMI) has emerged as a promising therapeutic option. We evaluated the impact of AMI on mesenchymal stem cell (MSC) differentiation into cardiomyocyte lineage. Cord blood-derived human MSCs were exposed to in vitro conditions simulating in vivo environments of the beating heart with acute ischemia, as follows: (a) myocardial proteins or serum obtained from sham-operated rats, and (b) myocardial proteins or serum from AMI rats, with or without application of oscillating pressure. Expression of cardiac-specific markers on MSCs was greatly induced by the infarcted myocardial proteins, compared with the normal proteins. It was also induced by application of oscillating pressure to MSCs. Treatment of MSCs with infarcted myocardial proteins and oscillating pressure greatly augmented expression of cardiac-specific genes. Such expression was blocked by inhibitor of transforming growth factor beta(1) (TGF-beta(1)) or bone morphogenetic protein-2 (BMP-2). In vitro cellular and electrophysiologic experiments showed that these differentiated MSCs expressing cardiomyocyte-specific markers were able to make a coupling with cardiomyocytes but not to selfbeat. The pathophysiologic significance of in vitro results was confirmed using the rat AMI model. The protein amount of TGF-beta(1) and BMP-2 in myocardium of AMI was significantly higher than that in normal myocardium. When MSCs were transplanted to the heart and analyzed 8 weeks later, they expressed cardiomyocyte-specific markers, leading to improved cardiac function. These in vitro and in vivo results suggest that infarct-related biological and physical factors in AMI induce commitment of MSCs to cardiomyocyte-like cells through TGF-beta/BMP-2 pathways.     

抑制组蛋白乙酰化酶活性对心肌微环境中间充质干细胞向心肌细胞分化的影响

目的 通过检测移植经干扰乙酰化作用基因Gen5表达重组质粒ZJ3转染间充质干细胞(mesenchymal stem cells,MSCs)的Wistar大鼠心肌组织内乙酰化酶活性及乙酰化作用基因Gen5、心肌发育基因GATA4的表达量,探讨体内心肌微环境下乙酰化修饰在干细胞特化心肌样细胞转录调控中的作用.方法 提取干扰组蛋白乙酰化酶Gcn5表达重组质粒ZJ3,经脂质体包载转染MSCs24h后移植至Wistar大鼠心肌组织内,2w后检测移植区域心肌组织内乙酰化酶活性及乙酰化作用基因Gcn5、心肌发育基因GATA4的表达量.结果 实验组心肌组织乙酰化酶活性较正常对照组、阴性对照组及试剂对照组均有显著性降低;实验组心肌组织内Gen5及GATA4表达量较正常对照组、阴性对照组及试剂对照组均有明显减弱.结论 相关特异性基因的检测结果显示封阻干细胞染色质组蛋白乙酰化修饰后,在体内心肌微环境诱导下亦可以抑制干细胞特化心肌细胞转录过程,这为进一步研究组蛋白末端乙酰化修饰与干细胞分化调节机制奠定了实验室基础.     

外周血间充质干细胞移植心肌梗死兔新生血管及心功能的变化

背景：药物治疗和支架置入治疗尚不能修复心肌梗死后已坏死的心肌。 目的：观察外周血间充质干细胞移植治疗对心肌梗死兔新生血管及心功能的影响。 方法：随机抽签法将36只大白兔分为假手术组,间充质干细胞移植组和对照组,结扎兔冠状动脉左室支建立心肌梗死模型。 结果与结论：移植后4周,流式细胞仪分析显示绝大部分间充质干细胞表达CD44,极少量细胞表达CD34和CD45。间充质干细胞移植组梗死心肌组织有移植的间充质干细胞存活,超声心动仪示间充质干细胞移植组左心室射血分数及短轴缩短率明显高于对照组(P < 0.01);左心室收缩末内径和舒张末内径明显小于对照组(P < 0.01)。间充质干细胞移植组心肌纤维化程度、心肌梗死面积均明显小于对照组(P < 0.01)。免疫组织化学染色显示间充质干细胞移植组新生毛细血管密度明显高于对照组(P < 0.01)。提示外周血间充质干细胞移植增加了梗死心肌新生血管密度,改善心脏的功能。     

梗死心肌组织裂解液对鼠骨髓间充质干细胞诱导分化作用的体外研究

 目的 探讨梗死心肌组织裂解液对骨髓间充质干细胞（MSC）向心肌细胞分化的诱导作用。 方法 取 SD 大鼠梗死区心肌组织及其周围区域正常心肌组织分别制备梗死心肌组织裂解液和正常心肌组织裂解液；用 Balb/c 小鼠骨髓细胞进行 MSC 培养，取生长较好的第 2 代 MSC 制备 MSC 贴片，分别用 DMEM 培养基（空白对照组）、加入正常心肌组织裂解液的 DMEM 培养基（正常心肌组织裂解液组）和加入梗死心肌组织裂解液的 DMEM 培养基（梗死心肌组织裂解液组）培养。取各组培养 14 d 的 MSC，透射电镜下观察细胞超微结构；并进行免疫细胞化学染色，观察 α-肌动蛋白（actin）、心肌特异性转录因子 4（GATA-4）、心肌特异性肌球蛋白重链（MHC）、心肌特异性肌钙蛋白 T（cTnT）、心肌特异性肌钙蛋白 I（cTnI）、心肌增强因子 2（MEF-2）、连接蛋白（Cx）43 和 Cx45 的表达情况。 结果 超微结构观察显示空白对照组细胞器结构正常；正常心肌组织裂解液组细胞内未见明显的肌丝样结构；而梗死心肌组织裂解液组部分细胞内可见细小的肌丝样结构。免疫细胞化学染色分析显示，空白对照组 MSC 不表达心肌细胞特异性蛋白；正常心肌组织裂解液组 MSC 仅表达 α-actin；而梗死心肌组织裂解液组 MSC 表达 α-actin、GATA-4、MHC、cTnT、cTnI 和 MEF-2 等心肌特异性标志蛋白，但不表达 Cx43 和 Cx45。 结论 梗死心肌组织裂解液可以诱导骨髓间充质干细胞向心肌样细胞分化。     

Intravenously Injected Mesenchymal Stem Cells Home to Infarcted Myocardium Without Altering Cardiac Function

Background： Systemic delivery of mesenchymal stem cells （MSCs） to the infarcted myocardium is an attractive noninvasive strategy, but therapeutic effect of this strategy remain highly controversial. Methods： Myocardial infarction was induced in female Sprague-Dawley rats by transient ligation of the left anterior descending coronary artery for 60 min. Either 2.5 × 106 DiI-labeled MSCs or equivalent saline was injected into the tail vein at 24 h after infarction. Results： Three days later, MSCs localized predominantly in the infarct region of heart rather than in the remote region. MSCs were also observed in spleen, lung and liver. At 4 weeks after infarction, echocardiographic parameters, including ejection fraction, fractional shortening, left ventricular end-diastolic and end-systolic diameters, were not significantly different between MSCs and saline groups. Hemodynamic examination showed that ± dp/dtmax were similar between MSCs and saline-treated animals. Histological evaluation revealed that infarct size and vessel density were not significantly changed by MSCs infusion. Conclusion： Intravenously injected MSCs can home to infarcted myocardium, but plays a limited role in cardiac repair following myocardial infarction.     

The use of green fluorescence gene (GFP)-modified rabbit mesenchymal stem cells (rMSCs) co-cultured with chondrocytes in hydrogel constructs to reveal the chondrogenesis of MSCs

This study was conducted to reveal the chondrogenesis of mesenchymal stem cells that had been genetically modified with the green fluorescence protein (GFP) gene and then co-cultured with chondrocytes in vitro and in vivo. Subsequent mixing of chondrocytes in the hydrogel constructs induced increased chondrogenic differentiation of the transfected hMSCs. The proliferation and differentiation of MSCs that were transfected with the GFP gene and co-cultured with chondrocytes (1:1 and 1:3) or chondrocytes alone were evaluated by a live/dead assay, MTT assay, GAG & DNA assay, RT-PCR, real time-PCR, and histological and immunochemical analysis in vitro and in vivo. Real-time PCR revealed that the expression of aggrecan and COMP by genetically modified hMSCs co-cultured with chondrocytes was 2 or 3 times greater than that of genetically modified MSCs alone. Moreover, the expression of collagen type II was more than 3.5 times greater than that of genetically modified MSCs alone. 3-D hydrogel constructs co-cultured with chondrocytes and genetically modified MSCs showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study, regardless of whether they were co-cultured in the presence of chondrocytes. These findings indicate that co-culture with chondrocytes and genetically modified MSCs can be used to engineer well designed implants for the formation of neocartilage by transplanted genetically modified MSCs.     

Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium

Neovascularization may improve cardiac function and prevent further scar tissue formation in infarcted myocardium. A number of studies have demonstrated that bone marrow-derived cells have the potential to induce neovascularization in ischemic tissues. In this study, we hypothesized that implantation of bone marrow mononuclear cells (BMMNCs) using injectable fibrin matrix further enhances neovascularization in infarcted myocardium compared to BMMNC implantation without matrix. To test this hypothesis, infarction was induced in rat myocardium by cryoinjury. Three weeks later, rat BMMNCs were mixed with fibrin matrix and injected into the infarcted myocardium. Injection of either BMMNCs or medium alone into infarcted myocardium served as controls. Eight weeks after the treatments, histological analyses indicated that implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration in the infarcted myocardium compared to BMMNC implantation without matrix. Examination with fluorescence microscopy revealed that cells labeled with a fluorescent dye prior to implantation survived in the infarcted myocardium at 8 weeks of implantation. Importantly, implantation of BMMNCs using fibrin matrix resulted in much more extensive neovascularization in infarcted myocardium than BMMNC implantation without matrix. The microvessel density in infarcted myocardium was significantly higher (p < 0.05) when BMMNCs were implanted using fibrin matrix (350 +/- 22 microvessels/mm2) compared to BMMNC implantation without matrix (262 +/- 13 microvessels/mm2) and medium injection (76 +/- 9 microvessels/mm2). In addition, average internal diameter of microvessels was significantly larger (p < 0.05) in BMMNC implantation with fibrin matrix group (14.6 +/- 1.2 microm) than BMMNC implantation without matrix group (10.2 +/- 0.7 microm) and medium injection group (7.3 +/- 0.5 microm). These results suggest that fibrin matrix could serve as a cell implantation matrix that enhances neovascularization efficacy for myocardial infarction treatment.     

The stimulation of the cardiac differentiation of mesenchymal stem cells in tissue constructs that mimic myocardium structure and biomechanics

We investigated whether tissue constructs resembling structural and mechanical properties of the myocardium would induce mesenchymal stem cells (MSCs) to differentiate into a cardiac lineage, and whether further mimicking the 3-D cell alignment of myocardium would enhance cardiac differentiation. The tissue constructs were generated by integrating MSCs with elastic polyurethane nanofibers in an electrical field. Control of processing parameters resulted in tissue constructs recapitulating the fibrous and anisotropic structure, and typical stress-strain response of native porcine myocardium. MSCs proliferated in the tissue constructs when cultured dynamically, but retained a round morphology. mRNA expression demonstrated that cardiac differentiation was significantly stimulated. Enhanced cardiac differentiation was achieved by 3-D alignment of MSCs within the tissue constructs. Cell alignment was attained by statically stretching tissue constructs during culture. Increasing stretching strain from 25% to 75% increased the degree of 3-D cell alignment. Real time RT-PCR results showed that when cells assuming a high degree of alignment (with application of 75% strain), their expression of cardiac markers (GATA4, Nkx2.5 and MEF2C) remarkably increased. The differentiated cells also developed calcium channels, which are required to have electrophysiological properties. This report to some extent explains the outcome of many in?vivo studies, where only a limited amount of the injected MSCs differentiated into cardiomyocytes. It is possible that the strain of the heartbeat (～20%) cannot allow the MSCs to have an alignment high enough for a remarkable cardiac differentiation. This work suggests that pre-differentiation of MSCs into cardiomyocytes prior to injection may result in a greater degree of cardiac regeneration than simply injecting un-differentiated MSCs into heart.     

Collagen remodeling after myocardial infarction in the rat heart.

In this study changes in the amount and distribution of types I and III collagen mRNA and protein were investigated in the rat heart after induction of a left ventricular myocardial infarction (MI). Sham operated rats served as controls. The animals were sacrificed at different time intervals after operation. Northern blotting of cardiac RNA and hybridization with cDNA probes for types I and III procollagen revealed a 5- to 15-fold increase in the infarcted left ventricle. Type III procollagen mRNA levels were already increased at day 2 after MI, whereas type I procollagen mRNA followed this response at day 4 after MI. This increase was sustained for at least 21 days in the infarcted left ventricle for type III procollagen mRNA, whereas type 1 procollagen mRNA levels were still elevated at 90 days after MI. In the noninfarcted right ventricle a 5- to 7-fold increase was observed for both type I and type III procollagen mRNA levels, but only at day 4 after MI. In the non-infarcted septum a transient increase was observed for type I procollagen mRNA from day 7-21 (4- to 5-fold increase) and a decline to sham levels thereafter. In the septum type III procollagen mRNA levels were only elevated at 7 days after MI (4- to 5-fold increase) compared with sham operated controls. In situ hybridization with the same types I and III procollagen probes showed procollagen mRNA-producing cells in the infarcted area around necrotic cardiomyocytes, and in the interstitial cells in the non-infarcted part of the myocardium. No labeling was detected above cardiomyocytes. Combined in situ hybridization and immunohistochemistry showed that the collagen mRNA producing cells have a myofibroblast-like phenotype in the infarcted myocardium and are fibroblasts in the noninfarcted septum and right ventricle. The increase in types I and III procollagen mRNA in both infarcted and non-infarcted myocardium was followed by an increased collagen deposition, measured by computerized morphometry on sirius red-stained tissue sections as well as by the hydroxyproline assay. In the non-infarcted septum and right ventricle the collagen-positive area was maximal at day 14 (3- to 5-fold increase compared with sham operated controls) and slightly declined at day 21. In the infarcted myocardium the collagen-positive area was 57 +/- 10% at day 14 after MI. Hydroxyproline contents were significantly increased in the noninfarcted septum.(ABSTRACT TRUNCATED AT 400 WORDS)     

低氧增强骨髓间充质干细胞对缺氧诱导心肌细胞凋亡的可能性

背景：骨髓间充质干细胞移植入缺血心肌后存活率低,而低氧有可能增强骨髓间充质干细胞的增殖,促进其存活。 目的：体外模拟心肌细胞缺血微环境,探索低氧预处理后,骨髓间充质干细胞对持续缺氧诱导的心肌细胞凋亡的保护作用。 方法：取第4代SD大鼠骨髓间充质干细胞用于制备条件培养液。取胚胎大鼠心肌细胞株,随机分成4组：对照组：心肌细胞正常培养组;模型组：心肌细胞单纯缺氧;骨髓间充质干细胞组：心肌细胞与骨髓间充质干细胞条件培养液共缺氧;低氧组：心肌细胞与骨髓间充质干细胞低氧条件培养液共缺氧。MTT检测各组细胞活力变化,Annexin V-FITC双染标记心肌细胞凋亡,免疫组化检测各组Bax和Bcl-2蛋白的表达。 结果与结论：免疫组化显示,低氧组的Bcl-2表达较其他各组增强,而Bax的表达比模型组和骨髓间充质干细胞组减弱,Bcl-2/Bax比值最大。与对照组和骨髓间充质干细胞组相比,低氧组的细胞活力高(P < 0.05),凋亡率降低(P < 0.05)。提示低氧可能是通过增强旁分泌机制,从而对Bax和Bcl-2进行调节,对心肌细胞凋亡有保护效应。     

Lysophosphatidic acid protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis

Bone marrow-derived mesenchymal stem cells (MSCs) have shown great promise for cardiac repair. However, poor viability of transplanted MSCs within the ischemic heart has limited their therapeutic potential. Our previous studies have documented that hypoxia and serum deprivation (hypoxia/SD), induced MSCs apoptosis through the mitochondrial apoptotic pathway. Since serum lysophosphatidic acid (LPA) levels are known to be significantly elevated after acute myocardial infarction and that LPA enhanced survival of other cell systems, we embarked on determining whether LPA protects MSCs against hypoxia/SD-induced apoptosis. We have also investigated the potential mechanism(s) that may mediate such actions of LPA. All experiments were carried out on rat bone marrow MSCs. Apoptosis was induced by exposure of cells to hypoxia/SD in a sealed GENbox hypoxic chamber. Effects of LPA were investigated in the absence and presence of inhibitors that target either G(i)proteins, the mitogen activated protein kinases ERK1/2, or phosphoinositide 3-kinase (PI3K). The data obtained showed that hypoxia/SD-induced apoptosis was significantly attenuated by LPA through Gi-coupled LPA(1) receptors linked to the downstream ERK1/2 and PI3K/Akt signaling pathways that function in parallel. Additional studies have demonstrated that hypoxia/SD-induced activation of mitochondrial dysfunction was virtually abolished by LPA treatment and that inhibition of the LPA(1) receptor, Gi proteins, the PI3K/Akt pathway, or ERKs effectively reversed this protective action of LPA. Taken together, our findings indicate that LPA is a novel, potent survival factor for MSCs and this may prove to be of considerable therapeutic significance in terms of exploiting MSC-based therapy in the infracted myocardium.     

The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction

Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction.     

Stem cell-based delivery of Hypoxamir-210 to the infarcted heart: implications on stem cell survival and preservation of infarcted heart function

This study seeks to test our hypothesis that transgenic induction of miR-210 in mesenchymal stem cells (MSC) simulates the pro-survival effects of ischemic preconditioning (IPC) and that engraftment of (PC)MSC helps in the functional recovery of ischemic heart by miR-210 transfer to host cardiomyocytes through gap junctions. miR-210 expression in MSC was achieved by IPC or nanoparticle-based transfection of miR-210 plasmid ((miR)MSC) and functional recovery of the infarcted heart of rat transplanted with (PC)MSC or (miR)MSC was evaluated. Both (PC)MSC and (miR)MSC showed higher survival under lethal anoxia as compared to (non-PC)MSC and scramble-transfected MSC ((Sc)MSC) controls with concomitantly lower CASP8AP2 expression. Similarly, both (PC)MSC and (miR)MSC survived better and accelerated functional recovery of ischemic heart post-transplantation. To validate our hypothesis that MSC deliver miR-210 to host cardiomyocytes, in vitro co-culture between cardiomyocytes and (PC)MSC or (miR)MSC (using (non-PC)MSC or (Sc)MSC as controls) showed co-localization of miR-210 with gap-junctional connexin-43. miR-210 transfer to cardiomyocytes was blocked by heptanol pretreatment. Moreover, higher survival of cardiomyocytes co-cultured with (PC)MSC was observed with concomitant expression of CASP8AP2 as compared to cardiomyocytes co-cultured with (non-PC)MSC thus suggesting that miR-210 was translocated from MSC to protect host cardiomyocytes. Induction of miR-210 in MSC promoted their survival post-engraftment in the infarcted heart. Moreover, direct transfer of pro-survival miR-210 from (miR)MSC to host cardiomyocytes led to functional recovery of the ischemic heart.     

兔骨髓间充质干细胞来源的心肌(样)细胞的诱导分化研究

目的体外诱导骨髓间充质干细胞(Mesenchymal stem cells,MSCs)向肌源性细胞分化,探索诱导后的MSCs移植于心肌梗死区的存活和分化情况。方法提取、分离、培养兔的MSCs。经5-氮胞苷诱导后,进行免疫组化,电镜观察。4',6二乙酞基-2-苯基吲哚(DAPI)标记MSCs,建立兔心肌梗死模型。实验动物随机分两组:实验组(n=10)在心梗区域注入经诱导后的MSCs;对照组(n=10)在心梗区域注入不含MSCs的培养液。移植4周后,进行病理标本观察和免疫组化检测。结果5-氮胞苷诱导MSCs4周,部分细胞表达肌钙蛋白T(troponin T),电镜观察到肌丝形成。MSCs在体外用DAPI标记,用荧光显微镜观察细胞发蓝色荧光。移植4周后,在实验组中用荧光显微镜观察可见梗死区组织标本中可见DAPI标记带蓝色荧光的供体细胞核,移植细胞表达troponin T。结论MSCs经5-氮胞苷诱导后可向心肌细胞转化。移植细胞可在心肌存活,并向心肌细胞(样)转化。     

Surface coating of bone marrow cells with N-acetylglucosamine for bone marrow implantation therapy

Bone marrow implantation (BMI) has been performed clinically for the treatment of ischemic cardiovascular diseases. To achieve BMI effectively, accumulation of many bone marrow cells (BMCs) in an infarcted area of the myocardium is important. Previously, we reported that cardiomyocytes show strong interaction with N-acetylglucosamine (GlcNAc) and they can take up GlcNAc-conjugated liposomes. Thus, we examined whether GlcNAc-coated BMCs exhibit strong interaction with cardiomyocytes. The cell surface of BMCs was coated with GlcNAc without causing cell injury by GlcNAc-lipophilic polymers. It was found that the GlcNAc-coated BMCs exhibited strong interaction with cardiomyocytes. At 7 days of coculturing the GlcNAc-coated BMCs with cardiomyocytes, BMC-derived cardiomyocytes were generated. The number of BMC-derived cardiomyocytes was higher following coculture with GlcNAc-coated BMCs than following coculture with uncoated and maltose (MA)-coated BMCs. In this study, we demonstrated that the surface coating of BMCs with GlcNAc can be performed easily by using GlcNAc-lipophilic polymers and that GlcNAc-coated BMCs exhibited strong interaction with cardiomyocytes. Therefore, we think that cell surface coating with GlcNAc would help promote accumulation of BMCs in the infarcted area of the myocardium and that this accumulation would be helpful in the treatment of ischemic cardiovascular diseases with BMI.     

Priming of mesenchymal stem cells with oxytocin enhances the cardiac repair in ischemia/reperfusion injury

Oxytocin stimulates the cardiomyogenesis of embryonic stem cells and adult cardiac stem cells. We previously reported that oxytocin has a promigratory effect on umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). In this study, UCB-MSCs were cultured with oxytocin and examined for their therapeutic effect in an infarcted heart. UCB-MSCs were pretreated with 100 nM oxytocin and cardiac markers were assessed by immunofluorescence staining. Next, oxytocin-supplemented USC-MSCs (OT-USCs) were cocultured with hypoxia/reoxygenated neonatal rat cardiomyocytes and cardiac markers and dye transfer were then examined. For the in vivo study, ischemia/reperfusion was induced in rats, and phosphate-buffered saline (group 1), 1-day OT-USCs (group 2), or 7-day OT-USCs (group 3) were injected into the infarcted myocardium. Two weeks after injection, histological changes and cardiac function were examined. UCB-MSCs expressed connexin 43 (Cnx43), cardiac troponin I (cTnI), and α-sarcomeric actin (α-SA) after oxytocin supplementation and coculture with cardiomyocytes. Functional gap junction formation was greater in group 3 than in groups 1 and 2. Cardiac fibrosis and macrophage infiltration were lower in group 3 than in group 2. Restoration of Cnx43 expression was greater in group 3 than in group 2. Cnx43- and cTnI-positive OT-USCs in the peri-infarct zone were observed in group 2 and more frequently in group 3. The ejection fraction (EF) was increased in groups 2 and 3 in 2 weeks. The improved EF was sustained for 4 weeks only in group 3. Our findings suggest that the supplementation of UCB-MSCs with oxytocin can contribute to the cardiogenic potential for cardiac repair.