Induction of Human Naïve Pluripotency Using 5i/L/A Medium.
Induction of Human Naïve Pluripotency Using 5i/L/A Medium.
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DOI：
10.1007/978-1-0716-1908-7_2
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发表时间：
2022
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在植入之前，哺乳动物上胚层中的细胞构成幼稚多能状态，其特征在于不存在谱系引发、不受表观遗传限制和表达一组独特的转录因子。然而，在常规条件下衍生的人胚胎干细胞（hESC）已经退出这种幼稚状态，并获得了对应于植入后外胚层的更高级的“致敏”多能状态。我们已经开发了一种包含五种激酶抑制剂和两种生长因子（5i/L/A）的混合物，其能够诱导引发的hESC中幼稚多能性的定义特征。这些条件也可以应用于诱导患者特异性诱导多能干细胞（iPSC）中的幼稚多能性。在这里，我们提供了一个详细的方案，诱导幼稚多能性在致敏的hESC和iPSC和方法的幼稚身份的常规验证。我们还概述了使用两种荧光报告系统来跟踪活细胞中幼稚身份的获得：（i）与内源性OCT 4等位基因连接的GFP报告基因，其中引发的特异性近端增强子已被删除（ii）靶向X连锁基因的两个等位基因的双色报告系统，其报告雌性细胞中X染色体的状态（MECP 2-GFP/tdTomato）。本文所述的条件已经使人多能干细胞（hPSC）的各个方面得到了深入了解，包括其独特的转座子转录谱、X染色体状态和胚外潜能。
Prior to implantation, the cells in the mammalian epiblast constitute a naïve pluripotent state, which is distinguished by absence of lineage priming, freedom from epigenetic restriction, and expression of a unique set of transcription factors. However, human embryonic stem cells (hESCs) derived under conventional conditions have exited this naïve state and acquired a more advanced “primed” pluripotent state that corresponds to the post-implantation epiblast. We have developed a cocktail comprising five kinase inhibitors and two growth factors (5i/L/A) that enables induction of defining features of naïve pluripotency in primed hESCs. These conditions can also be applied to induce naïve pluripotency in patient-specific induced pluripotent stem cells (iPSCs). Here we provide a detailed protocol for inducing naïve pluripotency in primed hESCs and iPSCs and methods for the routine validation of naïve identity. We also outline the use of two fluorescent reporter systems to track acquisition of naïve identity in live cells: (i) a GFP reporter linked to an endogenous OCT4 allele in which the primed-specific proximal enhancer has been deleted (OCT4-ΔPE-GFP); and (ii) a dual color reporter system targeted to both alleles of an X-linked gene that reports on the status of the X chromosome in female cells (MECP2-GFP/tdTomato). The conditions described herein have given insight into various aspects of naïve human pluripotent stem cells (hPSCs), including their unique transposon transcription profile, X chromosome status, and extraembryonic potential.