Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells
Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells
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DOI：
10.1095/biolreprod.105.045138
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发表时间：
2006-01-01
影响因子：
3.6
通讯作者：
Stice, SL
中科院分区：
文献类型：
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作者：
Bosch, P;Pratt, SL;Stice, SL
骨髓间充质干细胞（MSCs）是一种成体多能细胞，被认为是人类细胞治疗的重要资源。了解间充质干细胞的临床潜力可能需要将其用于临床前大型动物模型，如猪。本研究的目的是：1)建立猪间充质干细胞（pMSC）培养；2)优化pMSC体外培养条件，3)研究pMSC是否适合基因操作，4)利用体细胞核移植（SCNT）确定pMSC重编程潜力。从骨髓中分离的pMSCs生长，附着在具有成纤维细胞样形态的塑料上，表达间充质表面标记物THY1，但不表达造血标记物ITGAM。此外，当暴露于特定的诱导条件下，pMSCs经历了脂肪生成、软骨生成和成骨分化。pMSCs在多种培养基中生长良好，在低氧环境下培养可提高其增殖能力。用携带绿色荧光蛋白（GFP; Ad5-F35eGFP）基因的人腺病毒瞬时转导pMSCs和等基因皮肤成纤维细胞（SFs），与SFs相比，pMSCs表达GFP更多。用含有GFP基因的质粒转染pMSCs，获得了稳定的遗传修饰和转基因扩展表达的细胞系。腺相关病毒感染pMSC和SF细胞系可产生约12%的转基因细胞，这些细胞在单细胞繁殖后形成转基因无性系。pMSCs可以在体外扩增并用作核供体以产生SCNT胚胎。因此，pMSCs是大型动物自体和同种异体细胞治疗模型和SCNT转化的一种有吸引力的细胞类型。
Bone marrow mesenchymal stem cells (MSCs) are adult pluripotent cells that are considered to be an important resource for human cell-based therapies. Understanding the clinical potential of MSCs may require their use in preclinical large-animal models, such as pigs. The objectives of the present study were 1) to establish porcine MSC (pMSC) cultures; 2) to optimize in vitro pMSC culture conditions, 3) to investigate whether pMSCs are amenable to genetic manipulation, and 4) to determine pMSC reprogramming potential using somatic cell nuclear transfer (SCNT). The pMSCs isolated from bone marrow grew, attached to plastic with a fibroblast-like morphology, and expressed the mesenchymal surface marker THY1 but not the hematopoietic marker ITGAM. Furthermore, pMSCs underwent lipogenic, chondrogenic, and osteogenic differentiation when exposed to specific inducing conditions. The pMSCs grew well in a variety of media, and proliferative capacity was enhanced by culture under low oxygen atmosphere. Transient transduction of pMSCs and isogenic skin fibroblasts (SFs) with a human adenovirus carrying the gene for green fluorescent protein (GFP; Ad5-F35eGFP) resulted in more pMSCs expressing GFP compared with SFs. Cell lines with stable genetic modifications and extended expression of transgene were obtained when pMSCs were transfected with a plasmid containing the GFP gene. Infection of pMSC and SF cell lines by an adeno-associated virus resulted in approximately 12% transgenic cells, which formed transgenic clonal lines after propagation as single cells. The pMSCs can be expanded in vitro and used as nuclear donors to produce SCNT embryos. Thus, pMSCs are an attractive cell type for large-animal autologous and allogenic cell therapy models and for SCNT transgenesis.