Differential Coupling of Self-Renewal Signaling Pathways in Murine Induced Pluripotent Stem Cells

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line. We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a Nanog-GFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in naïve pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors.将体细胞重编程为具有胚胎干细胞（embryonic stem cells, ESCs）样特性的诱导多能干细胞（induced pluripotent stem cells, iPSCs）的技术，已受到领域内的广泛关注，诸多研究聚焦于提升其诱导生成效率并解析重编程的分子机制。尽管该领域受到广泛关注，但目前我们对于在iPSCs中活跃、且参与调控其细胞命运的分子信号通路，尚未开展深入细致的研究。为填补这一研究空白，我们针对模式小鼠iPSC细胞系，系统表征了不同信号分子对其细胞行为的影响。我们发现该iPSC细胞系对血清存在显著响应：血清可显著促进细胞增殖，且根据所用培养体系的不同，会降低iPSCs的自我更新能力。令人意外的是，与ESCs相比，该iPSC细胞系对撤除白血病抑制因子（leukemia inhibitory factor, LIF）的敏感性更低，具体体现为Nanog-GFP报告基因的表达得以维持，且在无LIF的培养条件下自我更新能力增强。尽管抑制磷脂酰肌醇-3激酶（PI3K）信号通路会降低iPSCs的自我更新能力，但抑制糖原合成激酶3（Gsk-3）则可提升其自我更新能力，即便在无LIF的培养环境中亦是如此。该iPSC细胞系经高传代培养后，会出现特性改变，包括遗传不稳定以及自我更新能力下降。不过这一自我更新能力下降的缺陷，可通过抑制Gsk-3得到修复。综上，我们的研究数据表明，调控Gsk-3的活性在iPSC细胞命运调控中发挥关键作用。我们提出，未来应更审慎地开展不同iPSC细胞系的分子调控通路表征工作，因为若其调控模式与初始态多能ESCs中观察到的情况存在偏差，可能会使iPSCs及其分化衍生物易于出现异常甚至不良的行为表型。