DNA methylation abnormal accumulation during cloned TSCs maintaining [RRBS]

Trophoblast stem cells (TSCs) are derived from the trophoectoderm of blastocysts and maintain ability to self-renewal and differentiation. TSC is a good model to research placenta development in vitro. It will contribute to understanding and improving cloned placentomegaly to compare the transcription and methylation between cloned and natural fertilized embryos throughout TSC derivation process. In the present study, we used SCNT (NT) and SCNT with HDACi treatment (SNT) as cloned groups and natural fertilized (NF) embryos to derive TSCs and chosed 5-time points to peform RNA-seq and RRBS. We found only NT got a barriar in TSC maintenace and both cloned groups exhited abnormal accumulating DNA methylation and it might be resiponsible for some malformations of cloned placentas. We generated embryos from 3 different sources natural fertilization (NF), somatic nuclear transfer (NT), and somatic nuclear transfer with HDACi Scriptaid treatment (SNT) and chosed 5-time points during the TSC maintaining, E3.5 blastocyst trophectoderm (TE3.5), E4.5 blastocyst trophectoderm (TE4.5), outgrowths at half-time progression (outgrowth), passage 1 TSCs (TS_P1), and passage 3-4 TSCs (TS_Pn) to perform RNA-seq and RRBS. For each sample, we performed several independent replicates.

滋养层干细胞（Trophoblast stem cells, TSCs）源自囊胚的滋养外胚层，具备自我更新与分化能力。TSCs是体外研究胎盘发育的理想模型。通过在TSCs建系过程中对比克隆胚胎与自然受精胚胎的转录组与甲基化组差异，有助于解析并改善克隆性胎盘肥大问题。本研究以体细胞核移植（Somatic cell nuclear transfer, SCNT，简称NT）组、组蛋白去乙酰化酶抑制剂（Histone deacetylase inhibitors, HDACi）处理的体细胞核移植（SNT）组作为克隆组，以自然受精（NF）胚胎诱导获得TSCs，并选取5个时间点进行RNA测序（RNA-seq）与简化重亚硫酸盐测序（Reduced Representation Bisulfite Sequencing, RRBS）。研究发现仅NT组在TSCs维持阶段存在增殖障碍，且两组克隆组均出现DNA甲基化异常积累现象，该现象可能与克隆胎盘的部分畸形发生相关。本研究构建了3种不同来源的胚胎：自然受精（NF）胚胎、体细胞核移植（NT）胚胎以及经组蛋白去乙酰化酶抑制剂Scriptaid处理的体细胞核移植（SNT）胚胎，并在TSCs维持阶段选取5个样本时点，分别为E3.5囊胚滋养外胚层（TE3.5）、E4.5囊胚滋养外胚层（TE4.5）、增殖半程时的出芽结构（outgrowth）、第1代TSCs（TS_P1）及第3-4代TSCs（TS_Pn），对上述样本进行RNA-seq与RRBS检测。每个样本均设置了独立生物学重复。