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.     

腺病毒介导siRNA抑制全反式维甲酸诱导的骨髓间充质干细胞RARβ表达

构建携带针对大鼠维甲酸受体β(Retinoic acid receptorβ,RARβ)基因的siRNA重组腺病毒,并感染全反式维甲酸(All-trans retinoic acid,ATRA)处理的骨髓间充质干细胞(Mesenchymal stem cells,MSCs),检测其对RARβ的表达及MSCs成神经分化的影响。设计针对大鼠RARβ的4对siRNA的DNA序列,体外退火形成双链,定向克隆至含有U6/H1双启动子的腺病毒穿梭质粒pSES-HUS,随后与腺病毒骨架质粒pAd-Easy1在BJ5183细菌中同源重组,并在HEK293细胞中包装获得重组腺病毒Ad-siRARβ。腺病毒感染大鼠MSCs后经ATRA处理24 h,Real-time、Western blotting及免疫荧光检测RARβ的表达情况。改良神经诱导培养基(Modified neuronal induction medium,MNM)诱导MSCs神经分化,Real-time PCR及免疫荧光检测神经相关蛋白表达。PCR、酶切及测序鉴定均证实siRNA正确克隆至腺病毒质粒中,腺病毒感染大鼠MSCs后可观察到60%以上的细胞有红色荧光蛋白(Red fluorescent protein,RFP)表达。经ATRA处理24 h,Real-time、Westernblotting及免疫荧光检测发现RARβ表达定位于细胞核,ATRA作用后MSCs中RARβ表达增高16.5±2.34倍(P<0.05),有3组siRNA能有效抑制ATRA诱导的RARβ表达增强,抑制率分别为(66.26±9.12)%、(48.70±5.78)%、(64.09±0.53)%(P<0.05),且以pool组效果最强,抑制率为(78.09±4.24)%(P<0.01)。ATRA联合MNM诱导MSCs成神经样细胞,表达相关神经特异蛋白Nestin、NSE、MAP-2、Tau,免疫荧光结果显示神经标志蛋白Nestin、NSE、Tju1表达阳性细胞率为(50-88)%,而腺病毒介导的siRARβ能有效抑制MSCs的神经标志物表达水平及阳性细胞率(P<0.05)。成功构建了携带针对大鼠RARβ基因的siRNA重组腺病毒,能有效感染MSCs并显著抑制ATRA诱导的RARβ表达增强和MSCs的神经分化。     

Nonviral genetic modification mediates effective transgene expression and functional RNA interference in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSC) are increasingly the focus of both basic and clinical research due to their ability to strike a balance between self-renewal and commitment to mesodermal differentiation. However, the promising therapeutic utility of hMSC in regenerative medical approaches requires detailed knowledge about their molecular characteristics. Therefore, genetic modification of hMSC provides a powerful tool to understand their complex molecular regulation mechanisms. METHODS: Here we describe a proof of concept approach of separate and combined gene transfer and gene silencing by nonviral DNA transfection of enhanced green fluorescent protein (EGFP) and EGFP-targeted small interfering RNAs (siRNAs) in hMSC. For optimization of nonviral DNA and siRNA transfer different liposomal-based transfection strategies were validated. RESULTS: The highest fraction of EGFP-expressing hMSC was obtained using Lipofectamine 2000 (50%) which also mediated the highest transfection rates of siRNAs into hMSC (>or=92%). Stably EGFP-expressing hMSC maintained their proliferation capacity paired with the ability to differentiate into different mesodermal lineages (bone, cartilage, and fat) without loss of transgene expression. Based on our nonviral nucleic acid delivery technique we showed efficient, functional, and long-term RNA interference (RNAi) in hMSC by gene specific knock-down of transiently and stably expressed EGFP (88-98%). CONCLUSIONS: This is the first demonstration of efficient nonviral transfer of both nucleic acids (DNA and siRNA) into hMSC, exhibiting the potential of targeted modification of hMSC. In particular, the combination of these techniques represents a powerful gene transfer/silencing strategy, thus facilitating detailed genetic approaches to study regulatory networks in stem cell differentiation processes.     

小干扰RNA透皮递送策略研究进展

小干扰RNA (Small interfering RNA,siRNA)已被用于各种皮肤病的治疗。然而,由于siRNA具有电负性、极性强、易被核酸酶降解以及难以突破皮肤表皮屏障等缺陷,使其应用受限。因此,安全高效的siRNA递送载体是siRNA有效治疗皮肤病的前提。近年来,随着对siRNA研究的不断深入,基于脂质、聚合物、肽和纳米颗粒的递送系统的开发取得了很大进展,一些新的siRNA透皮递送载体应运而生,如类脂质体、树枝状聚合物、细胞穿透肽、球形核酸纳米颗粒等等。文中将重点介绍近年来siRNA透皮递送载体的最新研究进展。     

碳纳米管作为siRNA转运载体的研究进展

随着人们对RNA干扰分子机理的研究愈加深入,siRNA作为一种新的基因治疗药物极有可能为人类攻克癌症等难以治愈的疾病带来希望。然而,目前在RNA干扰应用中遇到的最大挑战就是如何有效地将siRNA导入靶细胞且不致引起严重的细胞毒性。碳纳米管在药物传递和基因传递等生物医学领域的潜在应用受到广泛关注;但要实现碳纳米管在基因治疗领域的应用,碳纳米管的功能化是关键,也是近几年来研究的重点。综述近年来碳纳米管作为siRNA转运载体在基因治疗领域的研究进展。     

In vitro evaluation of optimized liposomes for delivery of small interfering RNA

One of the biggest challenges for small interfering RNAs (siRNAs) as therapeutic agents is their insufficient cellular delivery efficiency. We developed long circulating and cationic liposomes to improve the cell uptake and inhibitory effectiveness of siRNA on the expression of vascular endothelial growth factor (VEGF) in cancer cells. SiRNA liposomes were obtained by polyelectrolyte complexation between negatively charged siRNA and positively charged liposome prepared by a hydration method. Gel electrophoresis was used to evaluate the loading efficiency of siRNA on the cationic liposome. The optimized siRNA liposomes were observed to be spherical in shape and had smooth surfaces with particle sizes of 167.7?±?2.0?nm and zeta potentials of 4.03?±?0.69?mV, which had no significant change when stored at 4?°C for three months. Fluorescence-activated cell sorting studies and confocal laser scanning images indicated that the cationic liposomes significantly increased the uptake of fluorescence-labeled siRNA in cancer cells. Effects of the siRNA on the inhibition of VEGF were tested by measuring concentrations of VEGF in cell culture media via an enzyme-linked immunosorbent assay and intracellular VEGF levels using a western blotting method. The liposomal siRNA was significantly effective at inhibiting the expression of VEGF in lung, liver and breast cancer cells. Optimal liposomes could effectively deliver siRNA into cancer cells and inhibit VEGF as a therapy agent.     

Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells

Mesenchymal stem cells (MSCs) have limited ability to differentiate into cardiomyocytes and the factors affect this process are not fully understood. In this study, we investigated the passage (P)-related transdifferentiation potential of MSCs into cardiomyocyte-like cells and its relationship to the proliferation ability. After 5-azacytidine treatment, only P4 but not P1 and P8 rat bone marrow MSCs (rMSCs) showed formation of myotube and expressed cardiomyocyte-associated markers. The growth property analysis showed P4 rMSCs had a growth-arrest appearance, while P1 and P8 rMSCs displayed an exponential growth pattern. When the rapid proliferation of P1 and P8 rMSCs was inhibited by 5-bromo-2-deoxyuridine, a mitosis inhibitor, only P1, not P8 rMSCs, differentiated into cardiomyocyte-like cells after 5-azacytidine treatment. These results demonstrate that the differentiation ability of rMSCs into cardiomyocytes is in proliferation ability-dependent and passage-restricted patterns. These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs.     

Polyethylenimine‐mediated gene delivery into human bone marrow mesenchymal stem cells from patients

Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for post‐infarction left ventricular (LV) dysfunction. However, age‐related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kD), one of non‐viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI‐mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analysed the influence of cell cycle on transfection and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF). hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0 μg DNA/cm². The average transfection efficiency for all tested samples, middle‐age group (<65 years), old‐age group (>65 years), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency did not show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S phase. No significant phenotypic differences were observed between these two subpopulations. Furthermore, PEI‐mediated therapeutic gene VEGF transfer could significantly enhance the expression level.     

Establishment of lentiviral-vector-mediated model of human alpha-1 antitrypsin delivery into hepatocyte-like cells differentiated from mesenchymal stem cells

Alpha-1 antitrypsin (AAT) deficiency is a lethal hereditary disorder characterized by a severe diminution in plasma levels of AAT leading to progressive liver dysfunction. Since mesenchymal stem cells can differentiate into hepatocyte-like cells they offer a potential unlimited source in autologous transplant procedures. The transfer of genetically modified hepatocyte cells derived from hMSCs into the body constitutes a novel paradigm of coupling cell therapy with gene therapy for this disease. hMSCs were isolated by density gradient centrifugation and plastic adherence. Hepatic differentiation was induced by exposing hMSC to induction medium for up to 21 days. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR and immunocytochemistry. The chimeric AAT-Jred transgene was transferred to differentiated cells using a lentiviral vector and its expression was visualized by fluorescent microscopy. Flow cytometric analysis confirmed that hMSCs were obtained. Major hepatocyte marker genes expression were confirmed by RT-PCR and immunocytochemistry. AAT gene was successfully introduced into hepatocyte-like cells differentiated from hMSCs. This established system could be suitable for generation of hMSC derived hepatocyte-like cells containing the normal AAT gene, thus offering a potential in vitro source of cells for transplantation therapy of liver diseases in AAT-deficient patients.     

Optimization of an effective directed differentiation medium for differentiating mouse bone marrow mesenchymal stem cells into hepatocytes in vitro

We have used a uniform design to explore the most effective directed differentiation medium (MEDDM) for differentiating mouse bone marrow mesenchymal stem cells (mMSCs) into hepatocytes. Our methods involved arranging eight differentiation medium groups following uniform design. Flow cytometry was used to evaluate the percentage of ALB+ and CK18+ cells in each group. Factors and their concentrations in the MEDDMs were then identified. The MEDDMs were evaluated by their ability to differentiate mMSCs into hepatocytes by RNA and protein expressions and synthesis functions. FGF at 35 ng/ml and OSM at 30 ng/ml in the medium yielded the highest percentage of ALB+ and CK18+ cells. During directed differentiation using MEDDMs, ALB, CK18, TTR, AFP mRNAs were expressed. ALB and CK18 proteins were detected in the cells. The differentiated cells produced albumin and urea in a time dependent manner. Uniform design was adequate for choosing the MEDDM of mMSCs. MEDDM containing 35 ng/ml FGF and 30 ng/ml OSM was effective in differentiating mMSCs into hepatocytes.     

Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.     

Functionalization of carbon nanotubes enables non-covalent binding and intracellular delivery of small interfering RNA for efficient knock-down of genes

The lipophilic nature of biological membranes restricts the direct intracellular delivery of potential drugs and molecular probes and makes intracellular transport one of the key problems in gene therapy. Because of their ability to cross cell membranes, single walled carbon nanotubes (SWNTs) are of interest as carriers of biologically active molecules, such as small interfering RNAs (siRNAs). We developed a strategy for chemical functionalization of SWNTs with hexamethylenediamine (HMDA) and poly(diallyldimethylammonium)chloride (PDDA) to obtain a material that was able to bind negatively charged siRNA by electrostatic interactions. PDDA-HMDA-SWNTs exhibited negligible cytotoxic effects on isolated rat heart cells at concentrations up to 10 mg/l. PDDA-HMDA-SWNTs loaded with extracellular signal-regulated kinase (ERK) siRNA were able to cross the cell membrane and to suppress expression of the ERK target proteins in primary cardiomyocytes by about 75%. PDDA-functionalized SWNTs thus present an effective carrier system for applications in siRNA-mediated gene silencing.     

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.     

miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have self-renewal and differentiation capabilities but the regulatory mechanisms of MSC fate determination remain poorly understood. Here, we aimed to identify microRNAs enriched in hMSCs that modulate differentiation commitments. Microarray analysis revealed that miR-140-5p is commonly enriched in undifferentiated hMSCs from various tissue sources. Moreover, bioinformatic analysis and luciferase reporter assay validated that miR-140-5p directly represses bone morphogenic protein 2 (BMP2). Furthermore, blocking miR-140-5p in hMSCs increased the expression of BMP signaling components and critical regulators of osteogenic differentiation. We propose that miR-140-5p functionally inhibits osteogenic lineage commitment in undifferentiated hMSCs.     

Banking of perinatal mesenchymal stem/stromal cells for stem cell-based personalized medicine over lifetime: Matters arising

Mesenchymal stromal/stem cells (MSCs) are currently applied in regenerative medicine and tissue engineering. Numerous clinical studies have indicated that MSCs from different tissue sources can provide therapeutic benefits for patients. MSCs derived from either human adult or perinatal tissues have their own unique advantages in their medical practices. Usually, clinical studies are conducted by using of cultured MSCs after thawing or short-term cryopreserved-then-thawed MSCs prior to administration for the treatment of a wide range of diseases and medical disorders. Currently, cryogenically banking perinatal MSCs for potential personalized medicine for later use in lifetime has raised growing interest in China as well as in many other countries. Meanwhile, this has led to questions regarding the availability, stability, consistency, multipotency, and therapeutic efficiency of the potential perinatal MSC-derived therapeutic products after long-term cryostorage. This opinion review does not minimize any therapeutic benefit of perinatal MSCs in many diseases after short-term cryopreservation. This article mainly describes what is known about banking perinatal MSCs in China and, importantly, it is to recognize the limitation and uncertainty of the perinatal MSCs stored in cryobanks for stem cell medical treatments in whole life. This article also provides several recommendations for banking of perinatal MSCs for potentially future personalized medicine, albeit it is impossible to anticipate whether the donor will benefit from banked MSCs during her/his lifetime.