Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120, USA.
Stem Cells. 2011 Nov;29(11):1696-704. doi: 10.1002/stem.730.
Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.
诱导多能干细胞（iPSCs）彻底改变了干细胞领域。iPSCs 通常是通过使用逆转录病毒来产生的。然而，由此产生的细胞可能不适合临床应用。已经开发了许多制造 iPSCs 的替代策略，但非整合策略往往效率低下，而整合策略则涉及随机整合。在这里，我们报告了一种使用质粒 DNA 和序列特异性重组酶进行单次转染来创建小鼠 iPSCs 的简便策略。PhiC31 整合酶用于将重编程盒插入基因组，产生 iPSCs。然后使用 Cre 重组酶切除重编程基因。在切除重编程盒之前和之后，通过体外和体内标准证明 iPSCs 具有多能性。该策略与逆转录病毒方法的效率相当，但对使用者没有危险，操作简单，并且导致重编程盒的非随机整合，可轻松删除。我们在两种易于获得的细胞类型（成纤维细胞和脂肪干细胞）中证明了这种重编程和切除策略的效率。这种简单的策略产生了具有在临床环境中应用潜力的多能干细胞。
