Peister A, Mellad J A, Wang M, Tucker H A, Prockop D J
Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA 70112, USA.
Gene Ther. 2004 Jan;11(2):224-8. doi: 10.1038/sj.gt.3302163.
Human marrow stromal cells (hMSCs) are an attractive source of adult stem cells for autologous cell and gene therapy. To transfect hMSCs without the use of viruses, we developed improved conditions for stable transfection of the cells by electroporation. hMSCs were isolated by adherence to plastic, and were electroporated at 600 V and 100 micros in a 2-mm gap cuvette with a plasmid containing enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase gene (neo(r)). After electroporation of 10(6) cells with 10 microg of the linearized plasmid DNA, hMSCs with stable DNA integration were selected by culturing with 200 microg/ml G418. The transfected hMSCs were expanded another 300-fold in 14 days to obtain 89 million cells, of which 98% expressed EGFP. Chloroquine increased the number of hMSCs transiently expressing EGFP from 12% to over 50%, but decreased stable integration. Stable integration of plasmid DNA into rat MSCs by electroporation was also successful. The transfected MSCs retained their capacity to differentiate into both adipocytes and osteoblasts. Thus, MSCs were stably transfected with plasmid DNA and retained their differentiation capacity after expansion.
人骨髓基质细胞（hMSCs）是用于自体细胞和基因治疗的成人干细胞的一个有吸引力的来源。为了在不使用病毒的情况下转染hMSCs，我们开发了通过电穿孔稳定转染这些细胞的改进条件。hMSCs通过贴壁于塑料培养瓶进行分离，并在2毫米间隙的比色皿中以600伏和100微秒进行电穿孔，所用质粒含有增强型绿色荧光蛋白（EGFP）和新霉素磷酸转移酶基因（neo(r)）。在用10微克线性化质粒DNA对10^6个细胞进行电穿孔后，通过用200微克/毫升G418培养来选择具有稳定DNA整合的hMSCs。转染的hMSCs在14天内再扩增300倍，获得8900万个细胞，其中98%表达EGFP。氯喹将瞬时表达EGFP的hMSCs数量从12%增加到50%以上，但降低了稳定整合率。通过电穿孔将质粒DNA稳定整合到大鼠MSCs中也获得成功。转染的MSCs保留了分化为脂肪细胞和成骨细胞的能力。因此，MSCs被质粒DNA稳定转染，并在扩增后保留了它们的分化能力。
