Zaragosi Laure-Emmanuelle, Billon Nathalie, Ailhaud Gérard, Dani Christian
Institut de Recherche, Signalisation, Biologie du Développement et Cancer, CNRS UMR6543, Centre de Biochimie, Faculté des Sciences, Université Nice Sophia-Antipolis, 06108 Nice Cedex 2, France.
Stem Cells. 2007 Mar;25(3):790-7. doi: 10.1634/stemcells.2006-0235. Epub 2006 Dec 7.
Adipose tissue-derived stem cells are a powerful tool for in vitro study of adult stem cell biology. So far, they have not been extensively used for gain or loss of function studies since they are resistant to most common transfection methods. Herein, we tested several classic transfection methods on human multipotent adipose tissue-derived stem (hMADS) cells. Our results showed that lipofectants and calcium phosphate were poorly efficient for transgene delivery in hMADS cells. In contrast, nucleofection, an electroporation-based method that is assumed to target plasmid DNA directly to the cell nucleus, led to a significant transient transgene expression in hMADS cells (up to 76% enhanced green fluorescent protein [EGFP]-positive cells were detected). Furthermore, after selection of hMADS cells that were nucleofected with a selectable plasmid coding for EGFP, stable EGFP expressing clones could be propagated in culture and efficiently induced to differentiate into EGFP-positive adipocytes and osteoblasts. Finally, we verified that nucleofected hMADS cells could produce a functional, transgene-encoded, secreted protein. To this aim, hMADS cells were nucleofected with a plasmid coding for leukemia inhibitory factor (LIF). This protein was detected at high concentrations in supernatants from pCAG-LIF transfected hMADS cells. Moreover, supernatants were able to maintain mouse embryonic stem cells' undifferentiated phenotype, indicating that hMADS cells could secrete a functional LIF protein. Taken together, our data demonstrate that nucleofection allows both transient and stable gene expression in adipose tissue-derived stem cells, without impairing their differentiation potential.
脂肪组织来源的干细胞是体外研究成体干细胞生物学的有力工具。到目前为止，由于它们对大多数常见的转染方法具有抗性，因此尚未广泛用于功能获得或缺失研究。在此，我们在人多能脂肪组织来源的干细胞（hMADS细胞）上测试了几种经典的转染方法。我们的结果表明，脂质体转染试剂和磷酸钙在hMADS细胞中的转基因递送效率很低。相比之下，核转染是一种基于电穿孔的方法，假定可将质粒DNA直接靶向细胞核，可导致hMADS细胞中显著的瞬时转基因表达（检测到高达76%的增强型绿色荧光蛋白[EGFP]阳性细胞）。此外，在用编码EGFP的可选择质粒进行核转染的hMADS细胞经过筛选后，稳定表达EGFP的克隆可在培养中传代，并能有效诱导分化为EGFP阳性的脂肪细胞和成骨细胞。最后，我们验证了经核转染的hMADS细胞能够产生一种功能性的、转基因编码的分泌蛋白。为此，用编码白血病抑制因子（LIF）的质粒对hMADS细胞进行核转染。在pCAG-LIF转染的hMADS细胞的上清液中检测到高浓度的这种蛋白。此外，上清液能够维持小鼠胚胎干细胞的未分化表型，表明hMADS细胞能够分泌功能性的LIF蛋白。综上所述，我们的数据表明，核转染能够在脂肪组织来源的干细胞中实现瞬时和稳定的基因表达，而不损害其分化潜能。
