GDNF慢病毒载体感染食蟹猴骨髓间充质干细胞

骨髓间充质干细胞(mesenchymal stem cells,MSCs)是基因工程和细胞治疗的种子细胞之一,本研究利用含胶质源性神经营养因子(glial cell derived neurotrophic factor,GDNF)基因的慢病毒载体感染成年食蟹猴MSCs,探讨转染后GDNF在MSCs中的体外表达水平及其影响因素。首先,通过密度梯度离心法分离食蟹猴骨髓单核细胞(marrow mononuclear cells,MNCs),体外培养食蟹猴MSCs。同时构建表达GDNF的慢病毒载体,并感染食蟹猴MSCs,分别利用酶联免疫吸附(ELISA)方法和Real-time PCR方法,测定感染不同拷贝数病毒和不同转染组细胞GDNF的蛋白分泌水平和基因表达水平。实验结果显示,表达GDNF基因的慢病毒载体成功转染成年食蟹MSCs,体外培养的MSCs持续表达分泌GDNF。感染慢病毒的拷贝数可以影响GDNF分泌水平,相同条件下感染拷贝数越高,GDNF分泌量越多,其基因表达水平越高。     

巢式PCR检测食蟹猴和猕猴中SRV和SFV

目的利用巢式PCR方法检测圈养的食蟹猴（Macaca fascicularis）和猕猴（Macaca mulatta）中猴D型逆转录病毒（Simian Type D Retrovirus SRV）和猴泡沫病毒（Simian foamy virus SFV）。方法针对SRV-env和SFV-pol基因的保守区序列设计特异性外引物,然后再设计特异性内引物。将外引物扩增出的片段克隆到PJET1.2blunt载体中作为阳性对照,运用NCBI中BLAST软件比对测序结果。用巢式PCR方法分别检测食蟹猴和猕猴中SRV和SFV。结果发现食蟹猴中SFV感染率为65.2%,SRV感染率为9.5%,猕猴中SFV感染率为60.5%,SRV感染率为12.8%。结论圈养的食蟹猴和猕猴SFV的感染率均较高,SRV感染率很低。     

食蟹猴精原干细胞体外培养体系的初步建立

为了掌握食蟹猴(Macaca fascicularis)精原于细胞(spermatogonial stem cells,SSCs)体外培养生长特性,并建立其培养体系.手术法获得幼年期食蟹猴单侧睾丸,改良的两步酶消化法获得其细胞悬液,添加特定培养液进行体外培养,以碱性磷酸酶(AKP)染色鉴定培养细胞,并评价不同饲养层细胞...     

食蟹猴胚胎干细胞向间充质前体细胞诱导分化及鉴定

间充质干细胞(mesenchymal stem cells,MSCs)可以诱导分化成脂肪、软骨、骨骼和骨骼肌细胞,并可作为骨骼、软骨或肌肉移植中的再生干细胞,广泛应用于细胞治疗和组织工程。胚胎干细胞(embryonic stem cells,ESCs)具有体外培养无限增殖和多向分化的特性,能被诱导分化为机体几乎所有的细胞类型。该研究通过无血清条件下诱导食蟹猴ESCs形成类胚体(embryoid bodies,EBs),然后在血清条件下贴壁分化EBs成间充质前体细胞(mesenchymal precursor cells,MPCs),再经过长期体外培养,纯化和扩增MPCs。结果显示,纯化后的MPCs具有MSCs生物学特征,并能在体外诱导分化成脂肪细胞和骨细胞。将这些细胞皮下注射给SCID小鼠,并未发现形成肿瘤,提示食蟹猴ESCs来源的MPCs具有一定的安全性。     

猴血中SFV病毒基因PCR检测方法的建立及应用

针对猴泡沫病毒SFV(Simian FoamyVirus)多聚酶区(pol区)设计两对引物,以猴血DNA为模板进行嵌套式PCR扩增,得到500bp左右基因片段,克隆进入pUC-19载体,经测序鉴定为SFV465bp的pol区基因片段。将此段序列与SFV各型425bp的pol区基因片段进行同源性比较,它与SFV-1型的同源性最高,为92.00%。在此基础上,用这两对引物对158例猴血DNA进行检测,阳性54例,阳性率为34.2%。发现在猴群中有较高的SFV病毒的感染。     

Nested-PCR检测恒河猴泡沫病毒

目的用nested-PCR检测恒河猴泡沫病毒SFV的前病毒形式。方法从SFV-1的pol区域选择两对引物分别对原代猴肾细胞（rhesus monkey kidney,RMK）及猴外周血淋巴细胞（peripheral blood lymphocytes,PBLs）进行体外扩增,扩增产物经1.0%的琼脂糖凝胶电泳,证实其特异性。阳性对照使用具有典型泡沫样病变的RMK377细胞株的前病毒DNA,阳性对照的PCR扩增产物经测序证实,阴性对照为恒河猴的SRV-1cDNA。结果nested-PCR能快速灵敏的直接从猴外周血淋巴细胞检出SFV的前病毒形式,与RMK细胞培养结果基本相一致。结论本实验所建立的检测SFV的nested-PCR法能快速准确的检测猴群中的SFV的带毒情况,对于提高实验猴的质量具有重要意义。     

猴血中SFV病毒基因PCR检测方法的建立及应用

针对猴泡沫病毒SFV（Simian Foany Virus）多聚酶区（pol区）设计两对引物,以猴血DNA为模板者嵌套式PCR扩增,得到500bp左右基因片段,克隆进入pUC-19载体,经测序鉴定为SFV465bp的pol区基因片段,将此段序列与SFV各型425bp的pol区基因片段进行同源性比较,它与SFV－1型的同源性最高,为92.00%。在此基础上,用这两对引物对158例猴血DNA进行检测,阳性54例,阳性率为34.2%,发现猴群中有较高的SFV病毒的感染。     

猴泡沫病毒间接免疫荧光检测方法的建立及初步应用

目的建立检测血清中猴泡沫病毒（SFV）抗体的间接免疫荧光方法,为检测实验用猴群中SFV的感染情况提供参考依据。方法用SFV-1病毒感染BHK-21细胞,待50%细胞出现病变时,用胰蛋白酶消化细胞后以2×107/mL浓度40μL的细胞滴到10孔镀膜的玻片上,丙酮固定。利用制备的抗原片通过间接免疫荧光法对34份猴血清标本进行检测。结果建立了检测SFV抗体的间接免疫荧光染色方法,SFV抗体阳性19例,15例血清检测为阴性。结论本方法具有良好的特异性,可作为SFV检测的可靠方法。     

对比不同年龄段食蟹猴骨髓间充质干细胞的生物学特征

骨髓间充质干细胞因其广泛的临床应用前景而备受关注.非人灵长类动物在基因、生理和代谢等方面与人类相似,在制作疾病模型和疾病治疗研究等方面具有无可比拟的优势.因此,来源于非人灵长类的骨髓间充质干细胞是细胞移植和组织工程研究中的重要工具.本实验对比研究了不同年龄段食蟹猴骨髓间充质干细胞的生物学特征.结果发现,与中年食蟹猴骨髓间充质干细胞比较,青少年食蟹猴骨髓间充质干细胞具有明显高的增殖和分化潜能.长期体外培养的食蟹猴骨髓间充质干细胞能发生自发转变,转变后的细胞具有明显不同于骨髓间充质干细胞的形态特征.端粒酶活性检测显示,各年龄组不同代数的骨髓间充质干细胞端粒酶活性没有明显差别,但与骨髓间充质干细胞比较,转变后的细胞端粒酶活性显著增高.另一方面,随着体外培养时间延长,染色体不稳定性发生频率相应增加.这些结果提示在使用间充质干细胞进行实验或临床研究前,必须全面考虑各种因素,包括供体的年龄等,并且完善各种检测.     

人工驯养条件下食蟹猴B病毒抗体水平变化规律研究

目的研究人工驯养条件下食蟹猴B病毒抗体水平变化规律,便于有效控制自繁食蟹猴的BV感染率。方法随机选取409只对不同月龄自繁食蟹猴,采用BVELISA法进行BV抗体监测。结果新生仔猴刚出生时均携带不同程度的BV抗体,但随着月龄的增加,BV抗体水平开始下降,至5月龄时BV抗体阳性率降至最低（12．3％）,之后BV抗体水平逐渐升高。结论人工驯养条件下食蟹猴B病毒抗体水平呈由高到低再升高的趋势,5月龄时断奶可最大限度地获得BV抗体阴性猴。     

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristics of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy.     

In vivo manipulation of gene expression in non-human primates using lentiviral vectors as delivery vehicles

Non-human primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the difficulty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this difficulty because they can infect and transduce either dividing or nondividing cells, without producing an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells, and are able to maintain long-term expression. In this article we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. Finally, we describe in detail the lentiviral vector production protocol we use and provide examples of how the vector can be employed to target peripheral tissues and the brain.     

Risk assessment: A model for predicting cross-species transmission of simian foamy virus from macaques (M. fascicularis) to humans at a monkey temple in Bali, Indonesia

Contact between humans and nonhuman primates (NHPs) frequently occurs at monkey temples (religious sites that have become associated with free-ranging populations of NHPs) in Asia, creating the potential for NHP-human disease transmission. In March 2003 a multidisciplinary panel of experts participated in a workshop designed to model the risk of NHP-human pathogen transmission. The panel developed a risk assessment model to describe the likelihood of cross-species transmission of simian foamy virus (SFV) from temple macaques (Macaca fascicularis) to visitors at monkey temples. SFV is an enzootic simian retrovirus that has been shown to be transmitted from NHPs to humans. In operationalizing the model field data, laboratory data and expert opinions were used to estimate the likelihood of SFV transmission within this context. This model sets the stage for a discussion about modeling as a risk assessment tool and the kinds of data that are required to accurately predict transmission.     

Derivation,characterization and gene modification of cynomolgus monkey mesenchymal stem cells

Mesenchymal stem cells (MSCs) have received considerable attention in recent years. Particularly exciting is the prospect that MSCs could be differentiated into specialized cells of interest, which could then be used for cell therapy and tissue engineering. MSCs derived from nonhuman primates could be a powerful tool for investigating the differentiation potential in vitro and in vivo for preclinical research. The purpose of this study was to isolate cynomolgus mesenchymal stem cells (cMSCs) from adult bone marrow and characterize their growth properties and multipotency. Mononuclear cells were isolated from cynomolgus monkey bone marrow by density-gradient centrifugation, and adherent fibroblast-like cells grew well in the complete growth medium with 10 μM Tenofovir. cMSCs expressed mesenchymal markers, such as CD29, CD105, CD166 and were negative for hematopoietic markers such as CD34, CD45. Furthermore, the cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages under certain conditions, maintaining normal karyotype throughout extended culture. We also compared different methods (lipofection, nucleofection and lentivirus) for genetic modification of cMSCs and found lentivirus proved to be the most effective method with transduction efficiency of up to 44.6% and lowest level of cell death. The cells after transduction stably expressed green fluorescence protein (GFP) and maintained the abilities to differentiate down osteogenic and adipogenic lineages. In conclusion, these data showed that cMSCs isolated from cynomolgus bone marrow shared similar characteristics with human MSCs and might provide an attractive cell type for cell-based therapy in higher-order mammalian species disorder models.     

Relative Antiviral Resistance Induced in Homologous and Heterologous Cells by Cross-Reacting Interferons

Human cells incubated with human interferon become more resistant to vesicular stomatitis virus (VSV) than to Semliki Forest virus (SFV); monkey cells treated with monkey interferon become more resistant to SFV than to VSV. However, monkey cells incubated with human interferon developed relative antiviral activity identical to that induced by homologous interferon, and human cells developed characteristic human interferon-induced relative antiviral activity when exposed to monkey interferon. Therefore, cross-reacting interferons induce the relative antiviral activity characteristic of the interferon-treated cell rather than the cell of the interferon's origin. This relationship supports the hypothesis that interferon is not itself antiviral but rather induces cells to develop their own antiviral activity.     

Replication in a superficial epithelial cell niche explains the lack of pathogenicity of primate foamy virus infections

Foamy viruses (FVs) are ancient retroviruses that are ubiquitous in nonhuman primates (NHPs). While FVs share many features with pathogenic retroviruses, such as human immunodeficiency virus, FV infections of their primate hosts have no apparent pathological consequences. Paradoxically, FV infections of many cell types in vitro are rapidly cytopathic. Previous work has shown that low levels of proviral DNA are found in most tissues of naturally infected rhesus macaques, but these proviruses are primarily latent. In contrast, viral RNA, indicative of viral replication, is restricted to tissues of the oral mucosa, where it is abundant. Here, we perform in situ hybridization on tissues from rhesus macaques naturally infected with simian FV (SFV). We show that superficial differentiated epithelial cells of the oral mucosa, many of which appear to be shedding from the tissue, are the major cell type in which SFV replicates. Thus, the innocuous nature of SFV infection can be explained by replication that is limited to differentiated superficial cells that are short-lived and shed into saliva. This finding can also explain the highly efficient transmission of FVs among NHPs.     

Improving the Safety of Mesenchymal Stem Cell-Based Ex Vivo Therapy Using Herpes Simplex Virus Thymidine Kinase

Human mesenchymal stem cells (MSCs) are multipotent stem cells that have been intensively studied as therapeutic tools for a variety of disorders. To enhance the efficacy of MSCs, therapeutic genes are introduced using retroviral and lentiviral vectors. However, serious adverse events (SAEs) such as tumorigenesis can be induced by insertional mutagenesis. We generated lentiviral vectors encoding the wild-type herpes simplex virus thymidine kinase (HSV-TK) gene and a gene containing a point mutation that results in an alanine to histidine substitution at residue 168 (TK(A168H)) and transduced expression in MSCs (MSC-TK and MSC-TK(A168H)). Transduction of lentiviral vectors encoding the TK(A168H) mutant did not alter the proliferation capacity, mesodermal differentiation potential, or surface antigenicity of MSCs. The MSC-TK(A168H) cells were genetically stable, as shown by karyotyping. MSC-TK(A168H) responded to ganciclovir (GCV) with an half maximal inhibitory concentration (IC₅₀) value 10-fold less than that of MSC-TK. Because MSC-TK(A168H) cells were found to be non-tumorigenic, a U87-TK(A168H) subcutaneous tumor was used as a SAE-like condition and we evaluated the effect of valganciclovir (vGCV), an oral prodrug for GCV. U87-TK(A168H) tumors were more efficiently ablated by 200 mg/kg vGCV than U87-TK tumors. These results indicate that MSC-TK(A168H) cells appear to be pre-clinically safe for therapeutic use. We propose that genetic modification with HSV-TK(A168H) makes allogeneic MSC-based ex vivo therapy safer by eliminating transplanted cells during SAEs such as uncontrolled cell proliferation.     

Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.     

Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.