Aluigi Michela, Fogli Miriam, Curti Antonio, Isidori Alessandro, Gruppioni Elisa, Chiodoni Claudia, Colombo Mario P, Versura Piera, D'Errico-Grigioni Antonia, Ferri Elisa, Baccarani Michele, Lemoli Roberto M
Institute of Hematology and Medical Oncology L. e A. Seràgnoli, Via Massarenti, 9, 40137 Bologna, Italy.
Stem Cells. 2006 Feb;24(2):454-61. doi: 10.1634/stemcells.2005-0198. Epub 2005 Aug 11.
Viral-based techniques are the most efficient systems to deliver DNA into stem cells because they show high gene transduction and transgene expression in many cellular models. However, the use of viral vectors has several disadvantages mainly involving safety risks. Conversely, nonviral methods are rather inefficient for most primary cells. The Nucleofector technology, a new nonviral electroporation-based gene transfer technique, has proved to be an efficient tool for transfecting hard-to-transfect cell lines and primary cells. However, little is known about the capacity of this technique to transfect adult stem cells. In this study, we applied the Nucleofector technology to engineer human bone marrow- derived mesenchymal stem cells (hMSCs). Using a green fluorescent protein reporter vector, we demonstrated a high transgene expression level using U-23 and C-17 pulsing programs: 73.7%+/-2.9% and 42.5%+/-3.4%, respectively. Cell recoveries and viabilities were 38.7%+/-2.9%, 44.5%+/-3.9% and 91.4%+/-1.3%, 94.31%+/-0.9% for U-23 and C-17, respectively. Overall, the transfection efficiencies were 27.4%+/-2.9% (U-23) and 16.6%+/-1.4% (C-17) compared with 3.6%+/-2.4% and 5.4%+/-3.4% of other nonviral transfection systems, such as FUGENE6 and DOTAP, respectively (p<.005 for all comparisons). Nucleofection did not affect the immunophenotype of hM-SCs, their normal differentiation potential, or ability to inhibit T-cell alloreactivity. Moreover, the interleukin-12 gene could be successfully transfected into hMSCs, and the immunomodulatory cytokine was produced in great amount for at least 3 weeks without impairment of its biological activity. In conclusion, nucleofection is an efficient nonviral transfection technique for hMSCs, which then may be used as cellular vehicles for the delivery of biological agents.
基于病毒的技术是将DNA导入干细胞最有效的系统，因为它们在许多细胞模型中显示出高基因转导和转基因表达。然而，使用病毒载体有几个缺点，主要涉及安全风险。相反，非病毒方法对大多数原代细胞来说效率相当低。Nucleofector技术是一种基于电穿孔的新型非病毒基因转移技术，已被证明是转染难转染细胞系和原代细胞的有效工具。然而，关于该技术转染成体干细胞的能力知之甚少。在本研究中，我们应用Nucleofector技术对人骨髓来源的间充质干细胞（hMSCs）进行基因工程改造。使用绿色荧光蛋白报告载体，我们通过U-23和C-17脉冲程序证明了高转基因表达水平，分别为73.7%±2.9%和42.5%±3.4%。U-23和C-17的细胞回收率和活力分别为38.7%±2.9%、44.5%±3.9%和91.4%±1.3%、94.31%±0.9%。总体而言，与其他非病毒转染系统（如FUGENE6和DOTAP）分别为3.6%±2.4%和5.4%±3.4%相比，转染效率分别为27.4%±2.9%（U-23）和16.6%±1.4%（C-17）（所有比较p<0.005）。核转染不影响hMSCs的免疫表型、其正常分化潜能或抑制T细胞同种异体反应性的能力。此外，白细胞介素-12基因可以成功转染到hMSCs中，并且至少3周内大量产生免疫调节细胞因子且其生物活性不受损害。总之，核转染是一种用于hMSCs的高效非病毒转染技术，hMSCs随后可作为生物制剂递送的细胞载体。
