Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada.
Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada.
Proc Natl Acad Sci U S A. 2022 Dec 6;119(49):e2207824119. doi: 10.1073/pnas.2207824119. Epub 2022 Dec 1.
Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.
揭示与重编程不同体细胞类型为多能性相关的分子事件对于理解诱导多能干细胞（iPSC）治疗衍生物的特征至关重要。可诱导重编程因子转基因细胞或动物——被指定为二级（2°）重编程系统——不仅为这些研究提供了极好的实验工具，而且还提供了一种策略来研究由于不同的体外和体内环境导致的细胞重编程结果的差异。为了使这些研究不那么繁琐，理想情况下是有各种高效的可重编程小鼠系统来诱导体细胞类型的成功大规模重编程。在这里，我们报告了两种转基因小鼠系的开发，通过这些小鼠系，2°细胞以前所未有的效率进行重编程。这些系统是通过使含有可诱导的多能性因子表达的原代重编程细胞暴露于瞬时的转基因表达中断，从而选择具有强大转基因反应的亚克隆而衍生出来的。这些系统还包括报告基因，能够方便地读取内源性激活（GFP）的读数，指示多能性，以及重编程转基因的表达（mCherry）。值得注意的是，这些 2°小鼠系的各种胎儿和成年组织来源的体细胞产生了高效且快速的重编程，在诱导后 1 周内就出现了具有独立转基因的 iPSC 集落。这些小鼠系是探索重编程中的变异性和高效重编程系统的机制基础的有力工具。
