Klf4 reverts developmentally programmed restriction of ground state pluripotency
Klf4 reverts developmentally programmed restriction of ground state pluripotency
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DOI：
10.1242/dev.030957
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发表时间：
2009-04-01
期刊：
影响因子：
4.6
通讯作者：
Smith, Austin
中科院分区：
文献类型：
--
作者：
Guo, Ge;Yang, Jian;Smith, Austin
源自多能的早期epiblast衍生的小鼠胚胎干（ES）细胞对嵌合体的所有胎儿谱系有功能分化的后代。相比之下，植入后上皮化的上皮细胞的认知细胞（Episc）线无法在胚胎中定居，即使它们表达了核心多能基因Oct4，Sox2和Nanog。我们检查了这两种细胞类型之间的互连。 ES细胞可以响应生长因子提示而容易成为EPISC。相比之下，EPISC不会变成ES细胞。我们利用PiggyBac换位将单个重编程因子KLF4引入Episcs。在Episc培养条件下没有明显的作用，但是在基态ES细胞条件下，形成未分化的菌落的细胞的一部分。这些EPISC衍生的诱导多能茎（Epi-IPS）细胞激活了ES细胞特异性转录本，包括内源性KLF4，以及谱系规范的下调标记。 X在Epi-IPS细胞中删除了雌性细胞中的X染色体沉默，这是Episc状态的特征。他们产生了高量贡献的嵌合体，产生了种系传播。这些特性在CRE介导的KLF4转基因的缺失后保持了，并正式表明了发育表型的完整和稳定的重编程。因此，在适当的环境中重新表达KLF4可以从Episcs中再生中殿的基础状态。重编程取决于抑制外在生长因子刺激，并在不到1％的细胞中完成。这证明了Episc在发育，表观遗传和功能上与ES细胞区分开的论点。但是，由于单个转基因是达到基态的最低要求，因此EPISC为重新编程过程的未知组件提供了一个有吸引力的机会。
Mouse embryonic stem (ES) cells derived from pluripotent early epiblast contribute functionally differentiated progeny to all foetal lineages of chimaeras. By contrast, epistem cell (EpiSC) lines from post-implantation epithelialised epiblast are unable to colonise the embryo even though they express the core pluripotency genes Oct4, Sox2 and Nanog. We examined interconversion between these two cell types. ES cells can readily become EpiSCs in response to growth factor cues. By contrast, EpiSCs do not change into ES cells. We exploited PiggyBac transposition to introduce a single reprogramming factor, Klf4, into EpiSCs. No effect was apparent in EpiSC culture conditions, but in ground state ES cell conditions a fraction of cells formed undifferentiated colonies. These EpiSC-derived induced pluripotent stem (Epi-iPS) cells activated expression of ES cell-specific transcripts including endogenous Klf4, and downregulated markers of lineage specification. X chromosome silencing in female cells, a feature of the EpiSC state, was erased in Epi-iPS cells. They produced high-contribution chimaeras that yielded germline transmission. These properties were maintained after Cre-mediated deletion of the Klf4 transgene, formally demonstrating complete and stable reprogramming of developmental phenotype. Thus, re-expression of Klf4 in an appropriate environment can regenerate the nave ground state from EpiSCs. Reprogramming is dependent on suppression of extrinsic growth factor stimuli and proceeds to completion in less than 1% of cells. This substantiates the argument that EpiSCs are developmentally, epigenetically and functionally differentiated from ES cells. However, because a single transgene is the minimum requirement to attain the ground state, EpiSCs offer an attractive opportunity for screening for unknown components of the reprogramming process.