High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [WGBS]. High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [WGBS]

Reprogramming of somatic cells to induced pluripotent stem cells provoke immense interest both for clinical applications in regenerative medicine, and in understanding the regulation of cell identity. Previous characterization of the molecular events that underlie this process were limited by the low yield and by the stochastic dynamics of this process. Here we present an in-depth mapping of the molecular events that underlie an efficient and successful reprogramming, covering transcriptional, epigenetic and translational changes in a single day resolution. Overall design: Mbd3f/- and Gatad2a-/- MEFs that carry DOX-inducible OKSM cassette were used, and iPSC reprogramming was initiated by addition of DOX in FBS/LIF medium in 5% pO2 conditions. On day 3.5 after DOX initiation, medium was changed to LIF/KSR-based with the addition of two small molecule inhibitors for MEK/ERK and GSK3 signaling (2i). Cells were harvested every 24 hours until day 8, and processed for library preparation followed by high-throughput sequencing. Mbd3f/- and Gatad2a-/- established iPSC line (after 3 passages or more) WGBS was measured in each time point. A wildtype cell line, WT-2, isogenic to Gatad2a-/- was used as a control.

体细胞向诱导多能干细胞（induced pluripotent stem cells，iPSC）的重编程，无论在再生医学的临床应用领域，还是解析细胞身份调控机制方面，均受到广泛关注。此前针对该过程核心分子事件的表征研究，受限于该过程的诱导效率低下与随机动态特性，进展有限。本研究针对高效且成功的体细胞重编程过程，开展了深入的分子事件图谱绘制，实现了单日分辨率下转录组、表观基因组与翻译组变化的全覆盖分析。

实验设计概述：本研究采用携带多西环素（doxycycline，DOX）诱导型OKSM重编程元件的Mbd3f/-与Gatad2a-/-小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEF），在5%氧气分压的胎牛血清（fetal bovine serum, FBS）/白血病抑制因子（leukemia inhibitory factor, LIF）培养基中加入DOX以启动iPSC重编程。DOX诱导3.5天后，将培养基更换为添加MEK/ERK与GSK3信号通路小分子抑制剂的LIF/KnockOut血清替代物（KnockOut Serum Replacement, KSR）培养基（即2i培养体系）。自DOX诱导启动后，每24小时收集细胞一次，直至第8天，收集的细胞用于文库制备及高通量测序。此外，在各时间点均对传代3次及以上的Mbd3f/-与Gatad2a-/-已建立iPSC株开展全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing, WGBS）。本研究同时采用与Gatad2a-/-同基因背景的野生型细胞系WT-2作为对照。