Cell transplantation into ischemic myocardium using mesenchymal stem cells transfected by vascular endothelial growth factor

Aims: To investigate the effects of mesenchymal stem cells (MSCs) transplantation combining with vascular endothelial growth factor (VEGF) gene therapy on myocardium rebuilding, angiogenesis, and heart function improvement in rats with myocardial infarction. Methods: SD rat MSCs were isolated, cultured in vitro, labeled with BrdU and transfected by Ad.VEGF gene. Four weeks after left anterior descending artery was ligated to create rat myocardial infarction, cardiac function was examined with echocardiography. Rats were randomly divided into four groups (n = 10 in each group): Group I: MSCs/Ad.VEGF implantation; Group II: MSCs implantation; Group III: Ad.VEGF injection; Group IV: Control. MSCs differentiation was observed 4 weeks after transplantation. Immunohistochemistry and angiogenesis were observed. Echocardiography was performed to detect the effects on heart function. Results: MSCs labeled with BrdU could be identified in host hearts in group I and II, most of them positively stained with cTnT antibody. Echocardiography indicated that the improvement of the LVEF value in group I was more significant than that in the other three groups (P < 0.01, respectively). Some cells were incorporated into the coronary capillaries in the infarcted region. The capillary density in group I was higher than that in the other three groups (P < 0.01, respectively). Conclusion: MSCs implantation combining with VEGF gene therapy can obviously repair damaged myocardium and enhance the angiogenesis in ischemic heart tissue.     

Generation of human adult mesenchymal stromal/stem cells expressing defined xenogenic vascular endothelial growth factor levels by optimized transduction and flow cytometry purification

Adult mesenchymal stromal/stem cells (MSCs) are a valuable source of multipotent progenitors for tissue engineering and regenerative medicine, but may require to be genetically modified to widen their efficacy in therapeutic applications. For example, overexpression of the angiogenic factor vascular endothelial growth factor (VEGF) at controlled levels is an attractive strategy to overcome the crucial bottleneck of graft vascularization and to avoid aberrant vascular growth. Since the regenerative potential of MSCs is rapidly lost during in vitro expansion, we sought to develop an optimized technique to achieve high-efficiency retroviral vector transduction of MSCs derived from both adipose tissue (adipose stromal cells, ASCs) or bone marrow (BMSCs) and rapidly select cells expressing desired levels of VEGF with minimal in vitro expansion. The proliferative peak of freshly isolated human ASCs and BMSCs was reached 4 and 6 days after plating, respectively. By performing retroviral vector transduction at this time point, >90% efficiency was routinely achieved before the first passage. MSCs were transduced with vectors expressing rat VEGF(164) quantitatively linked to a syngenic cell surface marker (truncated rat CD8). Retroviral transduction and VEGF expression did not affect MSC phenotype nor impair their in vitro proliferation and differentiation potential. Transgene expression was also maintained during in vitro differentiation. Furthermore, three subpopulations of transduced BMSCs homogeneously producing specific low, medium, and high VEGF doses could be prospectively isolated by flow cytometry based on the intensity of their CD8 expression already at the first passage. In conclusion, this optimized platform allowed the generation of populations of genetically modified MSCs, expressing specific levels of a therapeutic transgene, already at the first passage, thereby minimizing in vitro expansion and loss of regenerative potential.     

Bcl-2 engineered MSCs inhibited apoptosis and improved heart function

Engraftment of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for postinfarction left ventricular dysfunction. However, limited cell viability after transplantation into the myocardium has restricted its regenerative capacity. In this study, we genetically modified MSCs with an antiapoptotic Bcl-2 gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a rat left anterior descending ligation model via intracardiac injection. Rat MSCs were manipulated to overexpress the Bcl-2 gene. In vitro, the antiapoptotic and paracrine effects were assessed under hypoxic conditions. In vivo, the Bcl-2 gene-modified MSCs (Bcl-2-MSCs) were injected after myocardial infarction. The surviving cells were tracked after transplantation. Capillary density was quantified after 3 weeks. The left ventricular function was evaluated by pressure-volume loops. The Bcl-2 gene protected MSCs against apoptosis. In vitro, Bcl-2 overexpression reduced MSC apoptosis by 32% and enhanced vascular endothelial growth factor secretion by more than 60% under hypoxic conditions. Transplantation with Bcl-2-MSCs increased 2.2-fold, 1.9-fold, and 1.2-fold of the cellular survival at 4 days, 3 weeks, and 6 weeks, respectively, compared with the vector-MSC group. Capillary density in the infarct border zone was 15% higher in Bcl-2-MSC transplanted animals than in vector-MSC treated animals. Furthermore, Bcl-2-MSC transplanted animals had 17% smaller infarct size than vector-MSC treated animals and exhibited functional recovery remarkably. Our current findings support the premise that transplantation of antiapoptotic gene-modified MSCs may have values for mediating substantial functional recovery after acute myocardial infarction.     

骨髓间充质干细胞和心脏Sca1阳性细胞移植治疗心肌梗死的效果比较及其机制研究

目的探讨骨髓来源的间充质干细胞(MSCs)和心脏来源的Sca1阳性干细胞移植对小鼠急性心肌梗死后的心功能恢复的影响。方法分别从成年C57BL/C小鼠的骨髓分离获取骨髓间充质干细胞,从心脏利用流式分选获取Sca1阳性干细胞,并在体外进行培养、扩增。C57BL/C小鼠雄性小鼠24只,按照以下方式分组,每组6只,假手术组,生理盐水对照组,MSCs治疗组和Sca1阳性细胞治疗组。小鼠麻醉后开胸,利用结扎心脏冠状动脉的左前降支建立急性心肌梗死模型,建模成功后分别在梗死区周围心肌内注射上述细胞或者生理盐水,假手术组小鼠不结扎冠状动脉。4周后,利用心脏超声观察小鼠左心室射血分数(LVEF)以及左心室舒张末期容积,以此评估心功能改变。细胞培养至第4代,采用荧光半定量PCR的方式分析细胞表达血管内皮生长因子(VEGFa和VEGFb)的情况。结果分离培养的骨髓MSCs贴壁生长,呈梭形;Sca1阳性干细胞在心脏组织的阳性率约为18%,培养后细胞贴壁生长,成短梭形,形态较MSCs更饱满。在细胞移植治疗后,与Sca1阳性干细胞治疗相比,MSCs治疗可以增加心梗后小鼠的存活率。细胞移植治疗4周后,骨髓来源MSCs治疗组的LVEF明显高于心脏来源Sca1阳性干细胞,且更有利于减少心梗后左心室舒张末期容积的扩大,改善心肌重塑。骨髓来源的MSCs表达VEGFa和VEGFb明显高于Sca1阳性细胞。结论心脏来源的Sca1阳性干细胞和骨髓来源的MSCs均可提高LVEF,促进心功能的恢复,且后者优于前者,这可能与MSCs高表达VEGF有关。     

Hypoxia-inducible factor-1α is essential for hypoxia-induced mesenchymal stem cell mobilization into the peripheral blood

Mobilization of mesenchymal stem cells (MSCs) is a promising strategy for tissue repair and regenerative medicine. The establishment of an appropriate animal model and clarification of the underlying mechanisms are beneficial to develop the mobilization regimens for therapeutic use. In this study, we therefore established a rat MSC mobilization model and investigated the related mechanisms, using continuous hypoxia as the mobilizing stimulus. We found that MSCs could be mobilized into peripheral blood of rats exposed to short-term hypoxia (2 days) and the mobilization efficiency increased in a time-dependent manner (2-14 days). Hypoxia-inducible factor-1α (HIF-1α) was upregulated during hypoxic exposure and was expressed continuously in bone marrow. Inhibition of HIF-1α expression by YC-1 remarkably reduced the number of mobilized MSCs, suggesting that HIF-1α is essential for hypoxia-induced MSC mobilization. Further, we investigated the potential role of HIF-1α target genes, vascular endothelial growth factor (VEGF), and stromal cell-derived factor-1α (SDF-1α). VEGF expression was elevated from day 2 to day 7 of hypoxia, stimulating an increase in bone marrow sinusoidal vessels and possibly facilitating the egress of MSCs. SDF-1α protein levels were increased in the peripheral blood of rats during MSC mobilization and promoted the migration of MSCs under hypoxic conditions in vitro. These results suggest that HIF-1α plays a pivotal role in hypoxia-induced MSC mobilization, possibly acting via its downstream genes VEGF and SDF-1α. These data provide a novel insight into the mechanisms responsible for MSC mobilization and may help in the development of clinically useful therapeutic agents.     

血管内皮生长因子基因转染骨髓间充质干细胞同种异体移植治疗大鼠急性心肌梗死

目的探讨同种异体血管内皮生长因子(VEGF)基因转染的骨髓间充质干细胞(MSCs)在大鼠梗死心脏局部存活、分化及对心功能的影响;明确同种异体干细胞及VEGF基因转染干细胞移植治疗急性心肌梗死(AMI)的可行性及效果。方法雄性SD大鼠30只,随机分为单纯注射培养基对照组、MSCs治疗组及VEGF基因转染MSCs治疗组。分离纯化雄性Wistar大鼠骨髓间充质干细胞(rMSCs),于左冠状动脉前降支结扎1h后植入到SD大鼠心组织,移植4周后检测心功能并取心脏行组织染色检查。结果异体大鼠MSCs可在梗死心组织定居、生存;免疫组化检测MSCs转化为心肌细胞及血管内皮细胞;与对照组比较VEGF基因转染异体细胞移植组左室射血分数升高(P<0.05),梗死边缘区心肌面毛细血管数目明显增加(P<0.05)。结论同种异体VEGF基因转染MSCs移植治疗AMI可行、有效。     

Polymeric vector-mediated gene transfection of MSCs for dual bioluminescent and MRI tracking in vivo

MSC's transplantation is a promising cell-based therapy for injuries in regenerative medicine, and in vivo visualization of transplanted MSCs with noninvasive technique is essential for the tracking of cell infusion and homing. A new cationic polymer, poly(ethylene glycol)-block-poly(l-aspartic acid)-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (PAI/SPION), was constructed as a magnetic resonance imaging (MRI)-visible non-viral vector for the delivery of plasmids DNA (pDNA) encoding for luciferase and red fluorescence protein (RFP) as reporter genes into MSCs. As a result, the MSCs were labeled with SPION and reporter genes. The PAI/SPION complexes exhibited high transfection efficiency in transferring pDNA into MSCs, which resulted in efficient luciferase and RFP co-expression. Furthermore, the complexes did not significantly affect the viability and multilineage differentiation capacity of MSCs. After the labeled MSCs were transplanted into the rats with acute liver injury via the superior mesenteric vein (SMV) injection, the migration behavior and organ-specific accumulation of the cells could be effectively monitored using the in vivo imaging system (IVIS) and MRI, respectively. The immunohistochemical analysis further confirmed that the transplanted MSCs were predominantly distributed in the liver parenchyma. Our results indicate that the PAI/SPION is a MRI-visible gene delivery agent which can effectively label MSCs to provide the basis for bimodal bioluminescence and MRI tracking in vivo.     

pEGFP/hVEGF121融合蛋白真核表达载体的构建及在骨髓间质干细胞中的表达

目的：构建携带人血管内皮细胞生长因子121及绿色荧光蛋白报告基因的融合蛋白真核表达质粒并检测其在骨髓间质干细胞（MSC）中的表达。 方法： 采用PCR技术，以pCD/hVEGF121质粒为模板扩增VEGF121基因全长，采用PCR产物的粘端克隆法，将VEGF121定向克隆入pEGFP-C1的多克隆位点，构建pEGFP/hVEGF121重组质粒，酶切、PCR及序列分析鉴定，脂质体介导转染体外培养的MSC，荧光显微镜及免疫细胞化学染色检测EGFP/VEGF融合蛋白的表达。 结果： PCR、酶切及测序证实目的基因VEGF121正确连接至pEGFP-C1的多克隆位点，pEGFP/hVEGF121重组质粒转染MSC后，荧光显微镜及免疫细胞化学检测EGFP/VEGF蛋白在MSC中存在表达。 结论： 成功构建了携带人VEGF121及EGFP报告基因的融合蛋白真核表达质粒，并在MSC中获得表达，为进一步研究VEGF基因治疗缺血性心血管疾病及MSC的分化奠定了实验基础。     

骨髓间充质干细胞膜微粒促进大鼠心肌梗死后血管新生

目的:观察骨髓间充质干细胞膜微粒(MSC-MPs)对大鼠心肌梗死后血管新生以及心功能的影响。方法:提取Sprague-Dawley大鼠MSCs并培养,在低氧低营养条件下培养72 h,以诱导细胞凋亡释放MSC-MPs。将培养上清液超速离心获取MSC-MPs,透射电镜下观察其大小及形态,并用流式细胞术分析其表型。建立SD大鼠心肌梗死模型,心肌梗死边缘区注射膜微粒及对照试剂。超声心动图检测心功能,Masson染色检测心梗面积,免疫组织化学染色技术检测梗死边缘区血管α-平滑肌肌动蛋白和von Willebrand因子以确定血管新生情况,real-time PCR检测心梗组织中血管内皮生长因子(VEGF)表达变化。结果:MSCs凋亡后可以释放膜微粒,MSC-MPs来自MSCs,直径为100~1 000 nm。心肌梗死大鼠心肌内注射MSC-MPs后,第7天和第28天时心功能明显改善,第28 d时心梗面积比对照组减小,新生血管密度明显增加,第7天时心梗组织VEGF的表达增加。结论:MSC-MPs可以促进大鼠心肌梗死后的血管新生,改善心功能。     

Gene Delivery Approaches for Mesenchymal Stem Cell Therapy: Strategies to Increase Efficiency and Specificity

A significant number of clinical trials have been undertaken to explore the use of mesenchymal stem cells (MSCs) for the treatment of several diseases such as Crohn’s disease, diabetes, bone defects, myocardial infarction, stroke etc., Due to their efficiency in homing to the tissue injury sites, their differentiation potential, the capability to secrete a large amount of trophic factors and their immunomodulatory effects, MSCs are becoming increasingly popular and expected to be one of the promising therapeutic approaches. However, challenges associated with the isolation of pure MSC populations, their culture and expansion, specific phenotypic characterization, multi-potential differentiation and challenges of efficient transplantation limit their usage. The current strategies of cell-based therapies emphasize introducing beneficial genes, which will improve the therapeutic ability of MSCs and have better homing efficiency. The continuous improvement in gene transfer technologies has broad implications in stem cell biology. Although viral vectors are efficient vehicles for gene delivery, construction of viral vectors with desired genes, their safety and immunogenicity limit their use in clinical applications. We review current gene delivery approaches, including viral and plasmid vectors, for transfecting MSC with beneficial genes. The review also discusses the use of a few emerging technologies that could be used to improve the transfer/induction of desirable genes for cell therapy.     

血红素氧合酶-1修饰的骨髓间充质干细胞心肌移植对梗死后心肌细胞凋亡及左心室功能的影响

目的： 探讨血红素氧合酶-1(HO-1)修饰的骨髓间充质干细胞(MSCs)对心肌梗死后心肌细胞凋亡及左心室功能的影响。方法: 取大鼠骨髓,体外分离扩增培养MSCs,HO-1腺病毒转染。结扎左前降支1 h后,分别将HO-1-MSCs、MSCs多点注射到大鼠心脏梗死区周边,对照组注射等量PBS。移植后第4 d,Western blotting检测梗死区周边HO-1蛋白、促凋亡蛋白Bax的表达;ELISA检测梗死区周边组织细胞因子血管内皮生长因子(VEGF)、b-成纤维生长因子(bFGF)的表达,第7 d超声心动图检测各组大鼠心功能变化。4周后,取梗死区周边心肌行Masson染色和免疫组化CD34因子染色。结果: Adv-HO-1转染MSCs后获稳定表达;HO-1蛋白在HO-1-MSCs组的表达明显高于MSCs组和对照组(P<0.01);促凋亡蛋白Bax的表达明显低于其它2组(P<0.01);细胞因子VEGF、bFGF的表达不同程度地高于其它2组(P<0.01)。HO-1-MSCs组左室收缩功能各项指标明显优于其它2组(P<0.01)。移植4周后,HO-1-MSCs组梗死区周边毛细血管密度明显高于MSCs组和对照组(P<0.01),HO-1-MSCs组心室壁变厚,心室腔明显缩小,胶原蛋白沉积减少。结论: HO-1修饰的MSCs分泌细胞因子协同HO-1蛋白抑制心肌细胞凋亡,促血管新生,抑制心室重构,改善心功能。     

移植转染ADM或HGF基因的骨髓间充质干细胞治疗大鼠心肌梗死

目的探讨移植转染两种不同基因的骨髓间充质干细胞移植治疗大鼠心肌梗死的作用。方法分离、扩增培养大鼠骨髓间充质干细胞;ELISA检测细胞上清液ADM或HGF的含量;制备大鼠心肌梗死模型,心肌局部注射移植经DAPI标记的MSCs;多普勒超声检测大鼠心功能;荧光显微镜观察细胞存活及分布;免疫组化检测新生血管密度及移植细胞在心梗区的分化。结果转染Ad-ADM或Ad-HGF后,MSCs可有效表达ADM或HGF;与单纯MSCs组相比,两基因组细胞均表达TNI,均有connexin 43的表达增加(P<0.05),心梗区新生血管密度增高(P<0.01),左室射血分数(EF)增加(P<0.01);两组间各指标无差别。结论不同基因修饰的MSCs移植均可增强单纯MSCs对心肌梗死大鼠的治疗作用。     

Advances in individualized and regenerative medicine

Molecular and cell biology have resulted in major advances in our understanding of disease pathogenesis as well as in novel strategies for the diagnosis, therapy and prevention of human diseases. Based on modern molecular, genetic and biochemical methodologies it is on the one hand possible to identify for example disease-related point mutations and single nucleotide polymorphisms. On the other hand, using high throughput array and other technologies, it is for example possible to simultaneously analyze thousands of genes or gene products (RNA and proteins), resulting in an individual gene or gene expression profile (‘signature’). Such data increasingly allow to define the individual disposition for a given disease and to predict disease prognosis as well as the efficacy of therapeutic strategies in the individual patient (‘individualized medicine’). At the same time, the basic discoveries in cell biology, including embryonic and adult stem cells, induced pluripotent stem cells, genetically modified cells and others, have moved regenerative medicine into the center of biomedical research worldwide with a major translational impact on tissue engineering as well as transplantation medicine. All these aspects have greatly contributed to the recent advances in regenerative medicine and the development novel concepts for the treatment of many human diseases, including liver diseases.     

Transplantation of genetically engineered mesenchymal stem cells improves cardiac function in rats with myocardial infarction: benefit of a novel nonviral vector, cationized dextran

It is expected that mesenchymal stem cells (MSCs) will be a cell source for cardiac reconstruction because of their differentiation potential and ability to supply growth factors. However, poor viability at the transplanted site often hinders the therapeutic potential of MSCs. Here, in a trial designed to address this problem, a non-viral carrier of cationized polysaccharide is introduced for genetic engineering of MSCs. Spermine-introduced dextran of cationized polysaccharide (spermine-dextran) was internalized into MSCs by way of a sugar-recognizable receptor to enhance the expression level of plasmid deoxyribonucleic acid (DNA). When genetically engineered by the spermine-dextran complex with plasmid DNA of adrenomedullin (AM), MSCs secreted a large amount of AM, an anti-apoptotic and angiogenic peptide. Transplantation of AM gene-engineered MSCs improved cardiac function after myocardial infarction significantly more than MSCs alone. Thus, this genetic engineering technology using the non-viral spermine-dextran is a promising strategy to improve MSC therapy for ischemic heart disease.     

Non-viral gene delivery to mesenchymal stem cells: methods, strategies and application in bone tissue engineering and regeneration

Mesenchymal stem cells (MSCs) can be isolated from several tissues in the body, have the ability to self-renewal, show immune suppressive properties and are multipotent, being able to generate various cell types. At present, due to their intrinsic characteristics, MSCs are considered very promising in the area of tissue engineering and regenerative medicine. In this context, genetic modification can be a powerful tool to control the behavior and fate of these cells and be used in the design of new cellular therapies. Viral systems are very effective in the introduction of exogenous genes inside MSCs. However, the risks associated with their use are leading to an increasing search for non-viral approaches to attain the same purpose, even if MSCs have been shown to be more difficult to transfect in this way. In the past few years, progress was made in the development of chemical and physical methods for non-viral gene delivery. Herein, an overview of the application of those methods specifically to MSCs is given and their use in tissue engineering and regenerative medicine therapeutic strategies highlighted using the example of bone tissue. Key issues and future directions in non-viral gene delivery to MSCs are also critically addressed.     

Mesenchymal stem cells derived from bone marrow favor tumor cell growth in vivo

Mesenchymal stem cells (MSCs) have generated a great deal of interest in clinical application because of their potential use in regenerative medicine and tissue engineering. However, the therapeutic application of MSCs still remain limited unless the favorable effect of MSCs for tumor growth in vivo and the long-term safety of the clinical applications of MSCs are better understood. In this study, MSCs derived from fetal bone marrow (FMSCs) and adult MSCs (AMSCs) alone or FMSCs and AMSCs with tumor cell line (F6 or SW480) together were transplanted subcutaneously into BALB/c-nu/nu mice to observe the outgrowth of tumor, and the characteristics of tumor cells were investigated by pathological and immunohistochemical methods, flow cytometry and real-time quantitative PCR. The results showed that both FMSCs and AMSCs could favor tumor growth in vivo. The pathologic examination revealed that tumor tissues had rich vessel distribution, extensive necrosis and invasion surrounding normal tissues, such as muscular tissue and subcutaneous tissue. In the immunohistochemical examination, tumor cells mixed with MSCs transplanted subcutaneously exhibited elevated capability of proliferation, rich angiogenesis in tumor tissues and highly metastatic ability. To understand whether MSCs affected the general properties of the tumor cells in vivo, the expression of some surface antigens and Bmi-1 gene of tumor tissue cells was detected in this study. The results indicated that these parameters were not affected after the interaction of MSCs with tumor cells in vivo. These findings suggested that MSCs could favor tumor growth in vivo. It is necessary to carry out a study for assurance of the long-term safety before MSCs were used as a therapy tools in regenerative medicine and tissue engineering.     

Mesenchymal stem cell-based tumor-targeted gene therapy in gastrointestinal cancer

Mesenchymal stem (or stromal) cells (MSCs) are nonhematopoietic progenitor cells that can be obtained from bone marrow aspirates or adipose tissue, expanded and genetically modified in vitro, and then used for cancer therapeutic strategies in vivo. Here, we review available data regarding the application of MSC-based tumor-targeted therapy in gastrointestinal cancer, provide an overview of the general history of MSC-based gene therapy in cancer research, and discuss potential problems associated with the utility of MSC-based therapy such as biosafety, immunoprivilege, transfection methods, and distribution in the host.     

Mesenchymal stem cells for the treatment of neurodegenerative disease

Mesenchymal stem cells/marrow stromal cells (MSCs) present a promising tool for cell therapy, and are currently being tested in US FDA-approved clinical trials for myocardial infarction, stroke, meniscus injury, limb ischemia, graft-versus-host disease and autoimmune disorders. They have been extensively tested and proven effective in preclinical studies for these and many other disorders. There is currently a great deal of interest in the use of MSCs to treat neurodegenerative diseases, in particular for those that are fatal and difficult to treat, such as Huntington's disease and amyotrophic lateral sclerosis. Proposed regenerative approaches to neurological diseases using MSCs include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation into the brain, MSCs promote endogenous neuronal growth, decrease apoptosis, reduce levels of free radicals, encourage synaptic connection from damaged neurons and regulate inflammation, primarily through paracrine actions. MSCs transplanted into the brain have been demonstrated to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons. Therapies will capitalize on the innate trophic support from MSCs or on augmented growth factor support, such as delivering brain-derived neurotrophic factor or glial-derived neurotrophic factor into the brain to support injured neurons, using genetically engineered MSCs as the delivery vehicles. Clinical trials for MSC injection into the CNS to treat traumatic brain injury and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of neurodegenerative disorders are discussed.     

Angiomyogenesis using liposome based vascular endothelial growth factor-165 transfection with skeletal myoblast for cardiac repair

We aim to investigate the feasibility and efficacy of cholesterol (Chol)+DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (hVEGF(165)) gene transfer into human skeletal myoblasts (hSkM) for cardiac repair. The feasibility and efficacy of CD liposome for gene transfer with hSkM was characterized using plasmid carrying enhanced green fluorescent protein (pEGFP). Based on the optimized transfection procedure, hSkM were transfected with CD lipoplexes carrying plasmid-hVEGF(165) (CD-phVEGF(165)). The genetically modified hSkM were transplanted into rat heart model of acute myocardial infarction. Flow cytometry revealed that about 7.99% hSkM could be transfected with pEGFP. Based on the optimized transfection condition, transfected hSkM expressed hVEGF(165) up to day-18 (1.7+/-0.1ng/ml) with peak at day-2 (13.1+/-0.52ng/ml) with >85% cell viability. Animal studies revealed that reduced apoptosis, improved angiogenesis with blood flow in group-3 animal's heart were achieved as compared to group-1 and 2. Ejection fraction was best recovered in group-3 animals. The study demonstrates that though gene transfection efficiency using CD liposome mediated hVEGF(165) gene transfer with hSkM was low; hVEGF(165) gene expression efficiency was sufficient to induce neovascularization, improve blood flow and injured heart function.     

In situ forming hydrogels with long-lasting miR-21 enhances the therapeutic potential of MSC by sustaining stimulation of target gene

Regulating stem cells by microRNA (miRNA) is promising in regenerative medicine. However, non-viral transfection is usually transient while stably lentiviral transfection is accompanied with oncogenic risk. In the study, we explored the feasibility to retain the microRNAs within biopolymer hydrogels for their long-lasting working and sustaining stimulation of target gene. miRNA-21 (MiR-21), a reported microRNA enhancing the therapeutic potential of mesenchymal stem cells (MSCs) was used. We demonstrated that miR-21 could be efficiently retained within collagen hydrogel after forming complex with cationic polymer polyethylenimine (PEI). Due to the electronic interaction with positively charged PEI, the release of miR-21 was largely prevented during 2 week incubation (<20%), while free miR-21 encapsulated in hydrogels was largely released (>50%). When MSCs were cultivated in the PEI/miR-21-incorporated hydrogels, the sustained activation of targeted gene HIF-1α was observed, resulting in the sustaining up-regulation of several downstream therapeutic cytokines. Then, the hydrogels encapsulating miR-21/PEI were coated onto tissue plate for MSC cultivation, which further confirmed the long-lasting retention and efficacy of miR-21 on the plate surface. In addition, under H₂O₂-simulated stress condition, we also demonstrated that the anti-apoptotic capacity of MSCs was significantly improved when growing on miR-21-retained hydrogels. Our study provided a safe and promising method for long-lasting stem cell regulation with miRNAs.