Cell Reprogramming and Stem Cells Laboratory, Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC 3168, Australia.
Stem Cells Int. 2012;2012:541014. doi: 10.1155/2012/541014. Epub 2012 Apr 26.
Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g., Thy1 in mouse). Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative) recombinant "pTAT-mcMyc" protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP(+) colony formation (i.e., reprogramming efficiency) whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK) suggested differential (and non-additive) mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1) failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming.
外源表达 Oct4、Sox2、Klf4 和 cMyc 使哺乳动物体细胞采用胚胎干细胞的分子和表型特征，首先需要抑制谱系相关基因（例如，小鼠中的 Thy1）。虽然从重编程鸡尾酒中省略 cMyc 可以最大程度地降低不受控制的增殖风险，但排除 cMyc 会导致重编程效率降低几倍。因此，评估了用（非整合的）重组“pTAT-mcMyc”蛋白递送来替代 cMyc 转基因的可行性，而不影响重编程效率或多能性表型。纯化和递送电中性/颗粒性 pTAT-mcMyc 保持了 Oct4-GFP(+)集落形成（即重编程效率），同时通过各种标准支持多能性。pTAT-mcMyc±Oct4、Sox2 和 Klf4 对 Thy1 的差异抑制表明了不同的（而非累加的）抑制机制。扩展这些发现，通过交错方法（预先抑制 Thy1）尝试提高重编程效率未能提高重编程效率。我们认为蛋白递送是一种有用的工具，可以阐明体细胞重编程的时间/分子事件。
