Bosch P, Fouletier-Dilling C, Olmsted-Davis E A, Davis A R, Stice S L
Department of Animal and Dairy Science, The University of Georgia, Athens, Georgia 30602, USA.
Mol Reprod Dev. 2006 Nov;73(11):1393-403. doi: 10.1002/mrd.20593.
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.
间充质干细胞（MSC）介导的基因治疗研究主要在啮齿动物身上进行。合适的大型动物模型可能会在人体临床试验之前提供额外的安全性和有效性信息。本研究的目的是：（a）使用基于多胺的商业转染试剂（GeneJammer，Stratagene，拉霍亚，加利福尼亚州）优化猪骨髓间充质干细胞的腺病毒转导效率，以及（b）确定转导后的间充质干细胞是否保留分化为中胚层谱系的能力。在不同条件下用携带绿色荧光蛋白（GFP）基因的复制缺陷型腺病毒载体感染猪间充质干细胞（pMSCs），并分析GFP表达情况。将转导后的细胞在体外诱导分化为脂肪生成、软骨生成和成骨谱系。当携带35型腺病毒纤维的5型腺病毒（Ad5F35eGFP）与GeneJammer联合使用时，我们观察到表达GFP的pMSCs百分比增加了5.5倍。在GeneJammer存在的情况下，以10.3 - 13.8 MOI（1,500 - 2,000 vp/细胞）转导pMSCs产生了最高百分比的表达GFP的细胞（约90%），且不影响细胞活力。当pMSCs用Ad5eGFP载体感染时也检测到了类似的积极效果。腺病毒转导过程中胎牛血清（FBS）的存在增强了GeneJammer处理组和对照组中载体编码的转基因表达。在GeneJammer存在的情况下用腺病毒载体转导的pMSCs经历了脂肪生成、软骨生成和成骨分化。在pMSCs的腺病毒感染过程中添加GeneJammer可以扭转pMSCs转导效率低下的问题，同时保留其多能分化能力。GeneJammer增强的转导将有助于在MSC介导的基因治疗模型和疗法中使用腺病毒载体。
