Genome stability in different mouse embryonic stem cells [exome-seq]. Genome stability in different mouse embryonic stem cells [exome-seq]

Naïve mouse ESCs exhibit full term developmental competence thus hold great potential in regenerative medicine. Maintaining genome stability is essential for potential application of ESCs in stem cell therapy. While ESC potency fluctuate with retrovirus activity, it is unclear whether long-term cultures affect retrotransposition and genomic stability. We compared retrotransposons in naïve ESCs using various approaches. We show that feeder-serum based culture conditions and small molecule LCDM maintain appropriate expression of retrovirus activity following long-term cultures, whereas cultures under 2iLif and a2i result in aberrant upregulation of retrotransposons. This leads to high frequent retrotransposition. Moreover, naïve ESCs on feeder-serum based culture conditions and small molecule LCDM still generate complete ESC pups by TEC assay, functional test of pluripotency, following long-term cultures, despite that all culture conditions maintain high expression levels of pluripotent genes such as Oct4, Nanog, Klf4 and Sox2. Meanwhile, long telomeres are maintained in ESCs under cultures in Feeder-serum and LCDM conditions but telomeres shortened in other conditions with increasing passages. These data provide insights into retrotransposition, genomic stability and pluripotency of naïve ESCs. Overall design: We used 5 different ESC culture conditions to do Exome-seq for analyzing the mutations in different ESCs; and we also do ATAC-seq for analyzing mutations in turmors derived from ESC-derived mice (by tetraploid complementary assay)

初始态小鼠胚胎干细胞（naïve mouse ESCs）具备全程发育潜能，在再生医学领域具有巨大应用潜力。维持基因组稳定性是胚胎干细胞（ESCs）应用于干细胞治疗的核心前提。尽管胚胎干细胞的干性状态随逆转录病毒活性发生波动，但目前尚不明确长期体外培养是否会影响逆转录转座过程与基因组稳定性。本研究采用多种方法对初始态小鼠胚胎干细胞中的逆转录转座子开展比较分析。结果显示，经长期体外培养后，饲养层-血清培养体系与小分子LCDM培养体系可维持逆转录病毒活性的正常表达水平；而2iLif与a2i培养体系则会导致逆转录转座子出现异常上调，进而引发高频次的逆转录转座事件。此外，尽管所有培养体系均能维持Oct4、Nanog、Klf4及Sox2等多能基因的高表达水平，但经长期体外培养后，采用饲养层-血清与小分子LCDM培养的初始态小鼠胚胎干细胞仍可通过四倍体互补试验（Tetraploid Embryo Complementation assay, TEC）获得完整的胚胎干细胞来源仔鼠。与此同时，采用饲养层-血清与LCDM培养的胚胎干细胞可维持较长端粒长度，而其余培养体系中的端粒长度则会随传代次数增加逐渐缩短。本研究数据为揭示初始态小鼠胚胎干细胞的逆转录转座调控、基因组稳定性维持及多能性维持机制提供了重要研究视角。整体实验设计：本研究采用5种不同的胚胎干细胞培养体系，通过外显子组测序（Exome-seq）分析不同胚胎干细胞中的突变情况；同时还通过转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）分析经四倍体互补试验获得的胚胎干细胞来源小鼠所罹患肿瘤中的突变特征。