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

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. 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), and WT V6.5 mouse ESCs were used as positive controls. ATAC-seq was measured in each time point.

将体细胞重编程为诱导多能干细胞（induced pluripotent stem cells，iPSC）的相关研究，在再生医学的临床应用领域与细胞身份调控机制解析方向均引发了广泛的研究兴趣。此前针对该过程核心分子事件的表征工作，受限于较低的重编程产率与过程本身的随机动态特性。本研究针对高效且成功的重编程过程，开展了高分辨率的分子事件全景图谱绘制，以单日为时间分辨率，覆盖转录组、表观基因组与翻译组的动态变化。实验选用携带DOX诱导型OKSM表达盒的Mbd3f/-与Gatad2a-/-小鼠胚胎成纤维细胞（mouse embryonic fibroblasts，MEFs），在5%氧分压（pO2）的胎牛血清/白血病抑制因子（FBS/LIF）培养基中加入DOX以启动iPSC重编程。在DOX诱导启动后的第3.5天，将培养基更换为添加MEK/ERK与GSK3信号通路双小分子抑制剂的LIF/KnockOut血清替代物（LIF/KSR）培养基（即2i培养基）。每24小时收集一次细胞直至第8天，随后进行文库制备与高通量测序。本研究以传代3次及以上的Mbd3f/-、Gatad2a-/- iPSC株，以及野生型（wild type，WT）V6.5小鼠胚胎干细胞（embryonic stem cells，ESCs）作为阳性对照。各时间点均开展了转座酶可及性染色质测序（ATAC-seq）检测。