Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn-Life & Brain Center and Hertie Foundation, Sigmund-Freud Straße 25, D-53105 Bonn, Germany.
Stem Cells Int. 2012;2012:549846. doi: 10.1155/2012/549846. Epub 2012 May 29.
Induced pluripotent stem (iPS) cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT) and Sox2 (Sox2-TAT) proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.
诱导多能干细胞（iPS 细胞）为细胞替代疗法以及疾病建模提供了一种有吸引力的选择，可以从中获得患者特异性的多能细胞。为了具有临床意义，安全的 iPS 细胞需要通过非病毒整合的方式产生，即避免因重编程转基因的病毒整合而导致的永久性遗传修饰。最近，已经应用了各种实验策略来实现无转基因的 iPS 细胞的衍生，包括使用非整合病毒、附加体表达或通过位点特异性重组酶或转座酶在重编程后切除转基因。一种直接诱导重编程因子的简单方法是直接递送合成的 mRNA 或生物活性蛋白。我们之前报道了细胞通透性 Oct4（Oct4-TAT）和 Sox2（Sox2-TAT）蛋白的生成，并表明 Oct4-TAT 具有重编程能力，即它可以替代编码 Oct4 的病毒。在这里，我们探索了增强 Sox2-TAT 蛋白稳定性和功能递送至体细胞的条件。我们表明富含脂质的白蛋白补充剂可以稳定细胞通透性 Sox2 蛋白，无论是在无血清补充的培养基还是在血清替代物中。采用优化的蛋白递送条件，我们证明 Sox2-TAT 蛋白能够替代病毒 Sox2。Sox2-piPS 细胞表达多能性相关标志物，并分化为所有三个胚层。
