A comparative study of endoderm differentiation in humans and chimpanzees

There is substantial interest in the genetic regulatory framework that is established in early human development, and in the evolutionary forces that shaped early developmental processes in humans. Progress in these areas has been slow because it is difficult to obtain relevant biological samples. Recent technological developments in the generation and differentiation of inducible pluripotent stem cells (iPSCs) provide the ability to develop in vitro models of early human and non-human primates developmental stages. We have previously established matched iPSC panels from humans and chimpanzees. Using these panels, we comparatively characterized gene regulatory changes through a four-day timecourse differentiation of iPSCs (day 1) into primary streak (day 2), endoderm progenitors (day 3), and definitive endoderm (day 4). As might be expected, we found that differentiation stage (in effect, cell type) is the major driver of variation in gene expression levels in our study, followed by species. We then identified thousands of differentially expressed genes between humans and chimpanzees in each differentiation stage. Yet, when we considered gene-specific dynamic regulatory trajectories throughout the timecourse, we found that 75% of genes, including nearly all known endoderm developmental markers, have conserved trajectories in the two species. Interestingly, we observed a marked reduction of both intra- and inter-species variation in gene expression levels in primitive streak samples compared to the iPSCs, with a recovery of variation in endoderm progenitors. The reduction in variation in gene expression levels at a specific developmental stage, paired with the high degree of conservation of temporal expression across species, is consistent with the dynamics of developmental canalization. Overall, we conclude that endoderm development in iPSC-based models are highly conserved and canalized between humans and our closest evolutionary relative. Overall design: We used a panel of 6 human and 4 chimpanzee iPSC lines previously derived and characterized in the Gilad lab. We differentiated the iPSCs into definitive endoderm, a process that was completed over 3 days, and included replicates of cell lines over the differentiation process. We harvested RNA from iPSCs (day 0) prior to differentiation and subsequently every 24 hours to capture intermediate populations corresponding to primitive streak (day 1), endoderm progenitors (day 2), and definitive endoderm (day 3). Overall, we collected a total of 32 human samples and 32 chimpanzee samples. We assessed used RNA sequencing to estimate gene expression levels in all samples. There are a total of 64 samples (6 human cell lines + 2 technical replicates at 4 time points; 4 chimpanzee cell lines x 2 technical replicates at 4 time points.)学界对人类早期发育过程中建立的基因调控框架，以及塑造人类早期发育进程的进化动力，均抱有浓厚研究兴趣。然而该领域的研究进展始终缓慢，核心难点在于难以获取相关的生物样本。近年来，诱导多能干细胞（inducible pluripotent stem cells, iPSCs）的构建与分化技术取得重要进展，使得我们能够建立人类及非人灵长类早期发育阶段的体外模型。此前我们已构建了匹配的人类与黑猩猩iPSC细胞系面板。依托该细胞面板，我们对iPSC为期4天的分化过程进行了比较分析，以刻画基因调控变化：以iPSC为起始（对应第1天），经分化依次于第2天形成原条（primitive streak）、第3天形成内胚层前体细胞（endoderm progenitors）、第4天形成定型内胚层（definitive endoderm）。正如预期，本研究发现，分化阶段（本质上即为细胞类型）是基因表达水平差异的最主要驱动因素，物种差异次之。随后我们在每个分化阶段中，均鉴定出数千个人与黑猩猩之间的差异表达基因。但当我们分析全时间进程中基因特异性的动态调控轨迹时，发现75%的基因（包括几乎所有已报道的内胚层发育标记基因）在两个物种间的调控轨迹均保守。值得注意的是，与初始iPSC相比，原条阶段样本的基因表达水平在种内与种间变异均显著降低，而在内胚层前体细胞阶段变异水平得以恢复。特定发育阶段的基因表达变异降低，结合跨物种的时序表达高度保守性，这与发育渠化（developmental canalization）的动态特征相符。综上，我们认为在基于iPSC的模型中，内胚层发育过程在人类及其亲缘关系最近的演化近亲黑猩猩之间，具有高度保守性与发育渠化特征。 研究设计：我们使用了此前由Gilad实验室构建并表征的细胞面板，包含6株人类iPSC与4株黑猩猩iPSC。我们将iPSC诱导分化为定型内胚层，整个分化过程耗时3天，并在分化进程中设置了细胞系生物学重复。我们在分化前的iPSC（第0天）收集样本，随后每24小时收集一次，以获取对应阶段的中间细胞群：原条（第1天）、内胚层前体细胞（第2天）与定型内胚层（第3天）。最终我们共收集到32份人类样本与32份黑猩猩样本。我们采用RNA测序技术对所有样本的基因表达水平进行定量。本次实验共包含64个样本：6株人类细胞系 × 4个时间点 × 2个技术重复；4株黑猩猩细胞系 × 4个时间点 × 2个技术重复。