Promotion of reprogramming to ground state pluripotency by signal inhibition.
Promotion of reprogramming to ground state pluripotency by signal inhibition.
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通过信号抑制来促进重编程对基态多能。
DOI：
10.1371/journal.pbio.0060253
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发表时间：
2008-10-21
期刊：
影响因子：
9.8
通讯作者：
Smith, Austin
中科院分区：
文献类型：
--
作者：
Silva, Jose;Barrandon, Ornella;Nichols, Jennifer;Kawaguchi, Jitsutaro;Theunissen, Thorold W.;Smith, Austin
诱导多能干细胞（iPS）是通过基因操作从体细胞产生的。重编程需要多个转基因整合，并且在稀有细胞中明显随机发生许多天。组织干细胞可能比其他细胞受到不太严格的表观遗传限制，因此可能更适合去编程。我们报告，脑源性神经干细胞（NS）获得未分化的形态迅速和高频率后，单轮转导与重编程因子。然而，真正的多能性的关键属性，包括内源性Oct 4和Nanog的稳定表达，雌性细胞中X染色体沉默的表观遗传学擦除，以及嵌合体定殖的能力，没有达到。因此，我们应用分子定义的条件从胚胎中衍生和繁殖真正的多能干细胞。我们将丝裂原活化蛋白激酶信号传导和糖原合成酶激酶-3（GSK 3）的双重抑制（2 i）与自我更新细胞因子白血病抑制因子（LIF）相结合。2 i/LIF条件诱导Oct 4和Nanog的稳定上调、X染色体的再激活、转基因沉默以及体细胞和生殖系嵌合体的能力。使用2 i/LIF，NS细胞重编程仅需要每个转基因的1-2次整合。此外，用Sox 2和c-Myc转导是不可能的，并且Oct 4和Klf 4足以将NS细胞转化成嵌合体形成iPS细胞。这些发现表明，体细胞状态影响重编程的要求，并描绘了两个阶段的过程。捕获可以简单且高效地推进到基态多能性的前多能细胞的能力为这种显着现象的分子解剖打开了大门。生物体从胚胎到成体的发育是不可逆的，细胞逐渐分化为专门的最终表型。现在，在山中伸弥（Shinya Yamanaka）开创性地发现了诱导多能性之后，逆转发育时间已经成为可能：我们可以将成年细胞重新编程回到早期胚胎中发现的幼稚多能性状态。多能性的诱导是一种非凡的现象，但目前对其了解甚少且效率低下。我们研究了来自小鼠大脑的干细胞，发现它们比其他细胞类型更快地重新编程。然而，重新编程的脑细胞在完全多能性的边缘被阻止，并且没有获得诱导多能性的一些基本特性。在逆转发育过程的基本原理的指导下，我们探索了阻断启动胚胎多能性丧失和进入分化的信号的效果。我们使用该信号的化学抑制剂与已知促进多能性维持的第二途径的刺激相结合。这种简单的处理使得部分转化的神经干细胞有效地完成了转化，并且变得与胚胎干细胞难以区分。因此，不完全重编程的细胞，以前被认为是产生多能干细胞的无用副产品，实际上提供了获得真正的诱导多能细胞的最快，最可靠和最有效的途径。干细胞的诱导重编程通过未分化但非多能性的中间体分两个阶段进行。丝裂原活化蛋白激酶信号传导的抑制将这种中间过渡状态转化为真正的多能性。
Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency—including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras—were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1–2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon. Development of an organism proceeds irreversibly from embryo to adult, with cells differentiating progressively towards specialised final phenotypes. Now, following the pioneering discovery of induced pluripotency by Shinya Yamanaka, it has become possible to reverse developmental time: we can reprogramme an adult cell back to the naïve state of pluripotency found in the early embryo. Induction of pluripotency is an extraordinary phenomenon but is currently poorly understood and inefficient. We investigated stem cells from the mouse brain and found that they reprogrammed faster than other cell types. However, the reprogrammed brain cells arrested on the verge of full pluripotency and did not gain some essential properties of induced pluripotency. Guided by the rationale of reversing a development process, we explored the effect of blocking the signal that initiates loss of pluripotency and entry into differentiation in the embryo. We used a chemical inhibitor of this signal in combination with stimulation of a second pathway known to promote maintenance of pluripotency. This simple treatment allowed the partly converted neural stem cells to complete the transition efficiently and become indistinguishable from embryonic stem cells. Therefore, incompletely reprogrammed cells, which have previously been dismissed as useless by-products of attempts to generate pluripotent stem cells, in fact provide the fastest, most reliable, and most efficient route to obtaining authentic induced pluripotent cells. Induced reprogramming of stem cells proceeds in two phases via an intermediate that is undifferentiated but not pluripotent. Inhibition of mitogen-activated protein kinase signaling converts this intermediate transitional state to authentic pluripotency.
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影响因子：
64.8
作者：
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通讯作者：
Vallier, Ludovic
影响因子：
2.7
作者：
PALMIERI, SL;PETER, W;SCHOLER, HR
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影响因子：
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作者：
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影响因子：
64.5
作者：
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通讯作者：
SASAKI, M
影响因子：
64.8
作者：
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通讯作者：
ROGERS, D