Reinforced STAT3 activity sustains self-renewal of human embryonic stem cells in a naive-like state. Homo sapiens

Only rodent embryonic stem (ES) cells can self-renew in the pristine state of pluripotency called the naive or ground state. Human ES (hES) cells self-renew in the so-called primed state of pluripotency, which is an obstacle to research, hindering cost-effective cultivation in media devoid of animal-derived products, genetic stability, and genome engineering. Here we show that forced expression of a hormone-dependent STAT3-ERT2, in combination with LIF and inhibitors of MEK and GSK3beta, allows hES cells to escape from the primed state, and enter a new state designated as TL2i, characterized by the activation of STAT3 target genes, regular passaging by single cell dissociation, and the expression of naive state-specific transcription factors. We used microarrays to analyse the gene expression changes occuring during the adaptation to the naive culture conditions. We then compared our dataset to previously published dataset of mouse ESc and EpiSc, human primed and naive ESc, and human embryos. Overall design: Human ES cells were genetically modified to overexpress the STAT3-ERT2 transcription factor. The modified cells were then cultured, on MEFs, in different conditions : (i) F = bFGF, (ii) TL = LIF + Tamoxifen, (iii) TL2i = LIF + Tamoxifen + MEK inhibitor +GSK3b inhibitor. We overexpressed STAT3-ERT2 in two different human ES cell lines : the H9 cell line and a cell line newly derived in our lab, designated Oscar. We analysed two clones H9-STAT3T-ERT2 (cl2 and cl14) and one clone Oscar-STAT3-ERT2. The TLOscar wer reverted to bFGF culture condition. We included three biological replicates per condition.

仅啮齿类胚胎干细胞（rodent embryonic stem, ES cells）可在被称为初始态（naive state）或原始态（ground state）的纯净多能性状态下实现自我更新。人类ES（hES）细胞则在所谓的预激活态（primed state）多能性状态下自我更新，该状态已成为相关研究的阻碍因素，不仅限制了无动物源成分培养基中的低成本培养，还对遗传稳定性维持与基因组编辑研究造成了干扰。
本研究证实，激素依赖型STAT3-ERT2与白血病抑制因子（LIF）、丝裂原活化蛋白激酶激酶（MEK）抑制剂及糖原合成激酶3β（GSK3β）抑制剂联合作用时，可使hES细胞脱离预激活态，进入一种被命名为TL2i的全新多能状态。该状态具有以下特征：STAT3靶基因激活、可通过单细胞解离实现常规传代，以及初始态特异性转录因子的表达。
我们利用基因芯片（microarrays）分析了细胞适应初始态培养条件过程中发生的基因表达变化，并将本数据集与已发表的小鼠ES细胞、小鼠外胚层干细胞（EpiSc）、人类预激活态与初始态ES细胞以及人类胚胎的相关数据集进行了比对。
实验整体设计：将人类ES细胞进行基因修饰以过表达STAT3-ERT2转录因子。随后将修饰后的细胞在小鼠胚胎成纤维细胞（MEFs）饲养层上，于三种不同培养条件下培养：(i) F组：添加碱性成纤维细胞生长因子（bFGF）；(ii) TL组：添加LIF与他莫昔芬（Tamoxifen）；(iii) TL2i组：添加LIF、他莫昔芬、MEK抑制剂及GSK3β抑制剂。
我们在两种不同的人类ES细胞系中过表达STAT3-ERT2：H9细胞系以及本实验室新近构建并命名为Oscar的细胞系。本次分析共包含两个H9-STAT3T-ERT2克隆（cl2与cl14）以及一个Oscar-STAT3-ERT2克隆。将TLOscar细胞逆转回bFGF培养条件。每个培养条件均设置三个生物学重复。