Biosafety assessment of human mesenchymal stem cells engineered by hybrid baculovirus vectors

Mesenchymal stem cells (MSCs) hold promise for cell therapy, and implantation of MSCs engineered with a baculovirus transiently expressing the growth factor can augment the bone repair. To prolong the baculovirus-mediated transgene expression, we developed hybrid baculovirus vectors exploiting the FLP/Frt-mediated recombination for circular episome formation. Transduction of human MSCs with the hybrid baculovirus vectors harboring the osteoinductive bmp2 gene substantially extended the BMP2 expression and improved the cellular osteogenic differentiation. To confirm the potential in the clinical setting, the present study evaluated the biosafety profile of human MSCs engineered by the hybrid vectors. We unraveled that transduction of MSCs with the hybrid baculovirus vectors slightly impeded the cell proliferation after transduction, probably due to the perturbation of cellular gene expression and induction of innate responses. Nonetheless, the hybrid baculovirus vectors did not compromise the cell viability and cellular differentiation. No transgene integration into the host chromosome and disruption of the karyotype of the MSCs were observed. Additionally, no upregulation of proto-oncogenes or downregulation of tumor suppressor genes occurred in the MSCs transduced with the hybrid baculovirus vectors. Neither did the transduced MSCs induce tumor formation in nude mice. This study not only supported the safety of MSCs for cell therapy but also implicated the potential of the human MSCs engineered by the hybrid baculovirus vectors for their applications in clinical scenarios necessitating sustained transgene expression.     

Rat adipose-derived stromal cells expressing BMP4 induce ectopic bone formation in vitro and in vivo

AIM: Bone morphogenetic protein 4 (BMP4) is one of the main local contributing factors in callus formation in the early phase of fracture healing. Adipose-derived stromal cells (ADSC) are multipotent cells. The present study was conducted to investigate the osteogenic potential of ADSC when exposed to adenovirus containing BMP4 cDNA (Ad-BMP4). METHODS: ADSC were harvested from Sprague-Dawley rats. After exposure to Ad-BMP4, ADSC were assessed by alkaline phosphatase activity (ALP) assay, RT-PCR and von Kossa staining. BMP4 expression was assessed by RT-PCR, immunofluorescence and Western blot analysis. ADSC transduced with Ad-BMP4 were directly injected into the hind limb muscles of athymic mice. ADSC Ad-EGFP(enhanced green fluorescence protein) served as controls. All animals were examined by X-ray film and histological analysis. RESULTS: The expression of BMP4 was confirmed at both mRNA and protein levels. The expression of the osteoblastic gene, ALP activity and von Kossa staining confirmed that ADSC transduced with Ad-BMP4 underwent rapid and marked osteoblast differentiation, whereas ADSC transduced with Ad-EGFP and cells left alone displayed no osteogenic differentiation. X-ray and histological examination confirmed new bone formation in athymic mice transplanted with ADSC transduced with Ad-BMP4. CONCLUSION: Our data demonstrated successful osteogenic differentiation of ADSC transduced with Ad-BMP4 in vitro and in vivo. ADSC may be an ideal source of mesenchyme lineage stem cells for gene therapy and tissue engineering.     

细胞色素P450基因修饰的脐带间充质干细胞联合环磷酰胺对急性B淋巴细胞白血病的治疗作用

目的 研究细胞色素P450（Cyt P450）基因修饰的脐带间充质干细胞（UC-MSCs）联合环磷酰胺（CPA）对急性B淋巴细胞白血病的治疗作用。方法 通过基因工程方法设计Cyt P450 2B6（CYP2B6）基因序列，采用慢病毒载体基因转染UC-MSCs，得到可高表达CYP2B6蛋白的CYP2B6-MSC种子细胞。采用实时荧光定量PCR（qRT-PCR）检测CYP2B6基因的表达评估转染效果；采用流式细胞术检测CYP2B6细胞表面标志物表达；采用体外诱导分化检测CYP2B6细胞分化能力。将CYP2B6-MSC与Nalm-6共培养，CCK8法和Annexin-V FITC/PI试剂盒检测UC-MSCs/CYP2B6-MSC联合CPA对Nalm-6细胞的增殖和凋亡的影响。结果 qRT-PCR检测结果显示，CYP2B6-MSC可高表达CYP2B6蛋白，而hUC-MSC（对照组细胞）不表达CYP2B6蛋白；流式细胞术及诱导分化检测发现，CYP2B6基因修饰后的MSC流式表型及分化能力均未有显著变化；肿瘤杀伤实验发现，CYP2B6-MSC与Nalm-6细胞共培养，同时加入CPA后，与UC-MSC及未加CPA组比较，CYP2B6-MSC+CPA对Nalm-6细胞具有显著的增殖抑制及促凋亡作用（P<0.01）。结论 CYP2B6基因修饰后的MSC联合CPA对Nalm-6具有显著的生长抑制和促凋亡作用。     

Adenovirus vector-mediated gene transfer into stem cells

Stem cells, including embryonic stem (ES) cells, mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs), are defined by their capacity for self-renewal and multilineage differentiation. Efficient gene transfer into stem cells is essential for the basic research in developmental biology and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (Ad) vectors, based on Ad type 5, can efficiently and transiently introduce the exogenous gene into many cell types via the primary receptor, coxsackievirus, and adenovirus receptor (CAR). However, some kinds of stem cells, such as MSCs and HSCs, cannot be efficiently transduced with conventional Ad vectors based on Ad serotype 5 (Ad5), because of the lack of CAR expression. To overcome this problem, fiber-modified Ad vectors and an Ad vector based on another serotype of Ad have been developed. Here, we review the advances in the development of Ad vectors suitable for stem cells and discuss their application in basic biology and clinical medicine.     

基因修饰间充质干细胞治疗肿瘤研究进展

间充质干细胞（MSCs）是来源于中胚层的成体干细胞,体内分布广泛,可从骨髓、脂肪、牙髓、脐带/胎盘等组织中分离获取,具有高度的可增殖和分化潜能,较低的免疫原性,同时具有向炎症损伤部位微环境的趋向性,在疾病治疗方面可作为基因药物的载体实现精准治疗。癌症被认为是永远无法愈合的伤口,其组织微环境在损伤与修复中呈无休止的动态变化,MSCs在其中扮演了重要角色。利用MSCs具有的向肿瘤组织中归巢及定位的特点,对其进行抗肿瘤药物的基因工程改造可能在癌症的治疗上是一种新的策略。概述MSCs在癌症发生发展中的作用以及基因修饰MSCs治疗癌症的研究进展,旨在为临床使用基因修饰MSCs治疗癌症提供策略和见解。     

In vitro and in vivo induction of bone formation based on adeno-associated virus-mediated BMP-7 gene therapy using human adipose-derived mesenchymal stem cells

Aim： To determine whether adeno-associated virus （AAV）-2-mediated, bone morphogenetic protein （BMP）-7-expressing human adipose-derived mesenchymal stem cells （ADMS） cells would induce bone formation in vitro and in vivo. Methods： ADMS cells were harvested from patients undergoing selective suction-assisted lipectomy and transduced with AAV carrying the human BMP-7 gene. Non-trans- duced cells and cells transduced with AAV serotype 2 carrying the enhanced green fluorescence protein gene served as controls. ADMS cells were qualitatively assessed for the production of BMP-7 and osteocalcin, and subjected to alkaline phosphatase （ALP） and Chinalizarin staining. A total of 2.5×10^6 cells mixed with type I collagen were implanted into the hind limb of severe combined immune-deficient （SCID） mice and subjected to a histological analysis 3 weeks post implantation. Results： Transfection of the ADMS cells achieved an efficiency of 99% at d 7. Transduction with AAV2-BMP-7 induced the expression of BMP-7 until d 56, which was markedly increased by d 7. The cells were positively stained for ALP. Osteocalcin production and matrix mineralization further con- firmed that these cells differentiated into osteoblasts and induced bone formation in vitro. A histological examination demonstrated that implantation of BMP-7- expressing ADMS cells could induce new bone formation in vivo. Conclusion： The present in vitro and in vivo study demonstrated that human ADMS cells would be a promising source of autologous mesenchymal stem cells for BMP gene therapy and tissue engineering.     

5-氟胞嘧啶在体外对转导胞嘧啶脱氨酶基因的肝癌细胞的细胞毒作用

As to investigate the cytotoxic effects of 5-fluorocytosine on hepatocarcinoma cells genetically modified with cytosine deaminase gene, the gene was transduced into the tumour cells with retroviral method. The cytotoxicity and the bystanding killing effects of 5-fluorocytosine on the tumour cells were measured with MTT assay. It was found that the prodrug 5-fluorocytosine had significant cytotoxic effects on human hepatocarcinoma cells which were transduced with cytosine deaminase in vitro. The IC₅₀ value of 5-
fluorocytosine on transgenic and non-transgenic hepatocarcinoma cells were 110 and 1 249 mg·L^-1, respectively. Moreover, the result showed that cytosine deaminase gene/5-fluorocytosine system possessed bystander killing effects on hepatocarcinoma. The experiment demonstrates the potential value of the cytosine deaminase gene/5-fluorocytosine system in the treatment of human hepatocarcinoma.     

Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation

Periosteum has been demonstrated to contain mesenchymal progenitor cells differentiating to osteoblasts, and both bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) may play important roles in cell-based approaches to bone regeneration. The purpose of this study was to evaluate the feasibility and efficacy of BMP-2 and/or VEGF on periosteal cell differentiation to osteoblasts in vitro and ectopic bone formation in vivo. Human periosteum-derived cells were transfected with BMP-2, VEGF, BMP-2 + VEGF, or vehicle as a control by non-viral gene transfer and then cultured and implanted to nude mice intramuscularly. Real-time polymerase chain reaction analysis of the culture revealed that transgenes for BMP-2 and BMP-2 + VEGF induced more mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than VEGF and vehicle treatments; additionally, alizarin red S staining, alkaline phosphatase staining, and alkaline phosphatase activity were significantly higher in the BMP-2 + VEGF transgene than in the other versions. After implantation, ectopic bone was observed at 4 weeks and greatly increased at 8 weeks in all groups. In particular, the combination of BMP-2 and VEGF formed significantly more bone at 4 weeks, and VEGF transfection resulted in more blood vessels relative to the conditions without VEGF. Thus, VEGF might enhance BMP2-induced bone formation through modulation of angiogenesis.     

Genetically modified mesenchymal stem cells for improved islet transplantation

The use of adult stem cells for therapeutic purposes has met with great success in recent years. Among several types of adult stem cells, mesenchymal stem cells (MSCs) derived from bone marrow (BM) and other sources have gained popularity for basic research and clinical applications because of their therapeutic potential in treating a variety of diseases. Because of their tissue regeneration potential and immune modulation effect, MSCs were recently used as cell-based therapy to promote revascularization, increase pancreatic β-cell proliferation, and avoid allograft rejection in islet transplantation. Taking advantage of the recent progress in gene therapy, genetically modified MSCs can further enhance and expand the therapeutic benefit of primary MSCs while retaining their stem-cell-like properties. This review aims to gain a thorough understanding of the current obstacles to successful islet transplantation and discusses the potential role of primary MSCs before or after genetic modification in islet transplantation.     

Smad6信号干扰对MSCs骨向分化中Smad5及Smurf1基因表达的影响

目的研究在骨形态发生蛋白2(BMP-2)诱导的骨髓间充质干细胞(MSCs)骨向分化中Smad6信号干扰对Smad5、Smurf1基因表达的影响。方法培养小鼠骨髓MSCs,并分为3组:A组为对照细胞;B组细胞用携带绿色荧光蛋白(GFP)的空白载体病毒转染+BMP-2骨向诱导;C组细胞用Smad6重组RNA干扰载体转染+BMP-2诱导。结果病毒转染后GFP在MSCs中有效表达,病毒转染效率接近100%。与A组比较,BMP-2诱导24h显著提高了B组Smurf1 mRNA水平(P<0.01);而Smad6信号干扰在两个时间点则进一步提高了C组Smurf1 mRNA水平,并显著高于B组(P<0.01)。C组磷酸化的Smad5(pSmad5)和Smurf1水平在两个时间点也显著高于B组(P<0.01),而Smad5蛋白水平则未受影响。结论在BMP-2诱导的MSCs骨向分化中,Smad6 RNA干扰可促进Smad5磷酸化,并引起Smurf1基因表达代偿性增高。     

IL-37b gene transfer enhances the therapeutic efficacy of mesenchumal stromal cells in DSS-induced colitis mice

Aim:

To investigate whether the transfer of the IL-37b gene, a newly identified inhibitor of both innate and adaptive immunity, could improve the therapeutic efficacy of mesenchumal stromal cells (MSCs) in inflammatory bowel disease (IBD).

Methods:

The expression of IL-37 in biopsied specimens of the patients with active ulcerative colitis (UC) was detected using RT-PCR and immunohistochemistry. Mice were treated with 3% dextran sulfate sodium (DSS) for 8 days to induce colitis. Before DSS treatment, the mice were injected with MSCs, MSC-eGFP or MSC-IL37b. Their body weight was measured each day, and the colons and spleens were harvested on d 10 for pathological and biochemical analyses.

Results:

In biopsied specimens of the patients with active UC, the expression of IL-37 was dramatically elevated in inflamed mucosa, mainly in epithelial cells and infiltrating immune cells. Compared to MSC-eGFP or MSCs, MSC-IL37b administration significantly attenuated the body weight and colon length reduction, and decreased the histological score in DSS-induced colitis mice. Furthermore, MSC-IL37b administration increased the percentage of myeloid-derived suppressor cells (MDSCs) among total splenic mononuclear cells as well as the percentage of regulatory T cells (Tregs) among splenic CD4+ T cells in the mice. Moreover, MSC-IL37b administration increased the IL-2+ cells and decreased the IFN-γ+ cells among splenic CD4+ T cells.

Conclusion:

IL-37 is involved in the pathophysiology of UC. IL-37b gene transfer enhances the therapeutic efficacy of MSCs in DSS-induced colitis mice by inducing Tregs and MDSCs and regulating cytokine production.     

Cyclic RGD-poly(ethylene glycol)-polyethyleneimine is more suitable for glioblastoma targeting gene transfer in vivo

Arginine-glycine-aspartic acid (RGD) is a widely chosen ligand to improve the specific gene targeting transfection efficiency of polyethyleneimine (PEI) in vivo. However, the optimal RGD conjugating mode, RGD-poly(ethylene glycol)-PEI (RGD-PEG-PEI) or RGD-PEI-methoxyl poly(ethylene glycol) (RGD-PEI-mPEG) still remains controversial. In this study, RGD-PEG-PEI and RGD-PEI-mPEG were synthesized and compared with respects to their glioblastoma cell-binding capability and tumor-targeting ability of their complexes with plasmid DNA. These results demonstrated that RGD-PEG-PEI/plasmid enhanced green fluorescent protein (pEGFP)-N2 complexes had higher binding affinities with U87 cells than RGD-PEI-mPEG/pEGFP-N2 complexes. The gene transfection was also performed on U87 cells in vitro and in vivo. In vitro, both of the RGD-modified PEI derivatives enhanced the gene transfection efficiency to some extent. However, all of the complexes (with or without RGD modification) had high transfection efficiency. The biodistribution of RGD-PEG-PEI/pEGFP-N2 complexes in mice bearing subcutaneous glioblastomas were significantly greater than that of RGD-PEI-mPEG/pEGFP-N2 complexes, suggesting a more efficient gene transfection in vivo. In the RGD-PEG-PEI, the use of a PEG spacer was particularly important. These results indicated that RGD-PEG-PEI was more suitable for targeted gene transfer in vivo.     

Synthesis, structure and tumour necrosis factor-alpha production-enhancing properties of novel adamantylamino heterocyclic derivatives

The synthesis of several adamantylated aminoheterocycles is reported. The attack of the adamantyl cation formed from 1-adamantanol in refluxing trifluoroacetic acid or induced by microwave irradiation provides adamantylamino-derivatives of respective heterocycles. Adamantylated heterocycles enhance the induction of tumour necrosis factor alpha (TNF-alpha) in genetically modified murine melanoma cells transduced with the gene for human TNF-alpha. Of the studied collection of adamantylated compounds, the most biologically active are 2-adamantylamino-6-methylpyridine and 2-adamantylamino4-methylpyrimidine. The crystal structure of 2-adamantylamino-6-methylpyridine is reported.     

运用血清药理学方法探讨滋肾降糖丸对小鼠MSC成骨分化的影响及其机制

目的：运用血清药理学方法研究滋肾降糖丸对小鼠骨髓基质干细胞（MSC）模型成骨分化的作用及其机制。方法：首先空腹6h后的小鼠口服滋肾降糖溶液,剂量为1.5g·kg^-1,随后在0、0.5、1、2和4h不同时间段收集含药血清;随后用茜素红染色和碱性磷酸酶活性检测的方法调查上述含药血清对MSC成骨诱导分化的影响;最后用实时荧光定量PCR的方法进一步调查含药血清对MSC成骨诱导过程中相关关键基因骨形态发生蛋白（BMP2）及成骨特异性转录因子（RunX2）的表达。结果：含药血清可显著促进MSC的成骨分化,以2h后收集的血清效果最强。Q-RT-PCR实验结果显示,2h含药血清显著促进诱导后第3、6dMSC细胞BMP2及RunX2基因的表达。结论：滋肾降糖丸抗骨质疏松的作用可能与上调MSC上BMP2及RunX2的基因表达,从而促进MSC的成骨分化有关。     

Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice

Mesenchymal stem cells (MSCs) were found to provide an effective therapeutic role in inflammatory diseases by modulating inflammatory responses and tissue regeneration by their differentiation ability. The present work sought to demonstrate the potential therapeutic use of MSCs in treating chronic inflammatory bowel disease (IBD) in mice. A new model to induce chronic IBD based on alternative administration periods of Dextran Sodium Sulfate (DSS) was established. Mice were divided into 2 groups; one was treated with MSCs and the other was treated with phosphate-buffered saline (PBS). Assessment of therapeutic efficacy of MSCs was by measuring weight, stool scoring, histopathological examination, and measuring the gene expression of inflammatory markers: Interleukin-23 (IL-23), Tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), and Intercellular adhesion molecule-1 (ICAM-1). The results showed that DSS administration causes bloody and watery stool, weight loss, and altered histopathologic picture. MSC treated mice showed a significant improvement in stool condition, weight gain, and normal histopathologic picture compared to the PBS treated mice. Moreover, gene expressions of inflammatory markers in the intestines of the MSC treated mice were also significantly lower than those of the PBS treated mice. In conclusion, the data here showed that MSCs have a clear potential efficacy in the treatment for IBD, as their immune modulation effects include inhibition in the expression of key inflammatory markers that each plays an important role in the pathogenesis of IBD.     

Smad6信号干扰对MSCs骨向分化的影响

目的研究Smad6信号干扰对骨形态发生蛋白2（BMP-2）诱导的骨髓间充质干细胞（MSCs）骨向分化的促进效应。方法培养小鼠MSCs,用BMP-2诱导骨向分化。细胞分为3组：A组细胞用携带绿色荧光蛋白（GFP）的Smad6重组RNA干扰载体转染;B组细胞用空白载体转染;C组细胞作为对照。结果病毒转染后GFP在MSCs中有效表达,病毒转染效率达98．5％。与B组比较,Smad6RNA干扰显著提高了A组细胞AIP活性和骨钙素水平（P〈0．01）,而C组ALP活性和骨钙素水平均显著低于其他2组（P〈0．01）。茜素红染色显示,A组矿化结节数目显著多于B组（P〈0．05）,而c组无矿化结节形成。结论Smad6RNA干扰可有效促进BMP-2诱导的MSCs骨向分化,该研究为骨组织工程中骨缺损修复提供了一个极具价值的手段。     

Graphene oxide enhances alginate encapsulated cells viability and functionality while not affecting the foreign body response

The combination of protein-coated graphene oxide (GO) and microencapsulation technology has moved a step forward in the challenge of improving long-term alginate encapsulated cell survival and sustainable therapeutic protein release, bringing closer its translation from bench to the clinic. Although this new approach in cell microencapsulation represents a great promise for long-term drug delivery, previous studies have been performed only with encapsulated murine C₂C₁₂ myoblasts genetically engineered to secrete murine erythropoietin (C₂C₁₂-EPO) within 160?µm diameter hybrid alginate protein-coated GO microcapsules implanted into syngeneic mice. Here, we show that encapsulated C₂C₁₂-EPO myoblasts survive longer and release more therapeutic protein by doubling the micron diameter of hybrid alginate-protein-coated GO microcapsules to 380?µm range. Encapsulated mesenchymal stem cells (MSC) genetically modified to secrete erythropoietin (D1-MSCs-EPO) within 380?µm-diameter hybrid alginate-protein-coated GO microcapsules confirmed this improvement in survival and sustained protein release in vitro. This improved behavior is reflected in the hematocrit increase of allogeneic mice implanted with both encapsulated cell types within 380?µm diameter hybrid alginate-protein-coated GO microcapsules, showing lower immune response with encapsulated MSCs. These results provide a new relevant step for the future clinical application of protein-coated GO on cell microencapsulation.     

基于转录组数据探讨不同组织来源的间充质干细胞的生物学异质性

目的 基于转录组数据探讨脂肪组织（AD）、胎盘绒毛膜（HC）、胎盘羊膜（HA）和脐带（UC）来源的间充质干细胞 （MSCs） 的生物学异质性。方法 从人 AD、HC、HA 和 UC 中分离 MSCs,流式细胞术检测细胞表面阳性标志物（CD73、CD90、CD105）和阴性标志物（CD14、CD34、CD45、CD79a、HLA-DR）的表达,改良版茜素红染色、油红O 染色、阿利辛蓝染色检测细胞的三系分化能力;Trizol 法提取细胞总 RNA,用于转录组测序,应用 GFOLD（1.1.4）进行差异基因表达分析;使用 DAVID 数据库对差异表达的基因进行基因本体（GO）功能富集分析。结果 P2 代的不同来源的 MSCs,CD73、CD90、CD105 均为阳性表达,CD14、CD34、CD45、CD79a、HLA-DR 均为阴性表达,培养的 MSCs 均具有三系分化能力。新生儿来源的 MSCs（HA、HC 和 UC）相关性大于成人来源的 MSCs（AD）,在功能富集分析中,与来源于 AD 的MSCs 相比,HA 和 UC 来源的 MSCs 表现出更优异的增殖能力。来源于 AD 的 MSCs 有更好的分化潜力以及促进血管生成能力,而来源于 UC 的 MSCs 支持神经元的发育并分泌可以调节免疫环境的趋化因子和抗炎因子。结论 不同来源的 MSCs 具有不同的生物学特征,提示不同来源的 MSCs 可能具有不同临床应用的最佳选择。