Atlas of multilineage stem cell differentiation reveals TMEM88 as a developmental regulator of blood pressure

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple any developmental lineage trajectories. In this study, we engineered barcoded iPSCs to generate an atlas of multilineage differentiation from pluripotency, encompassing a time-course of WNT-induced differentiation perturbed using modulators of WNT, BMP, and VEGF signalling. Computational mapping of in vitro cell types to in vivo developmental lineages revealed a diversity of iPSC-derived cell types comprising mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Coupling this atlas of in vitro differentiation with Summary data-based Mendelian Randomisation analysis of human complex traits, we identify the WNT-inhibitor protein TMEM88 as a putative regulator of mesendodermal cell types governing development of diverse cardiovascular and anthropometric traits. Using genetic loss of function models, we show that TMEM88 is required for differentiation of diverse endoderm and mesoderm cell lineages in vitro and that TMEM88 knockout in vivo results in a significant dysregulation of arterial blood pressure. This study provides an atlas of multilineage iPSC differentiation coupled with new molecular, computational, and statistical genetic tools to dissect genetic determinants of mammalian developmental physiology. Overall design: We first used scRNA-seq to test our genomic barcode (BC)-based multiplexing method in undifferentiated human induced pluripotent stem cells (hiPSCs), pooling 18 different samples into one scRNA-seq experiment. Next, we used these 18 barcoded hiPSC lines across three scRNA-seq libraries to assess effects of nine different small molecule signalling modulation treatments (including a control) on iPSC differentiation towards diverse mesendoderm lineages across day 2, day 5, and day 9 of differentiation, with two replicates per permutation. We separately use Cell Hashing HTOs to multiplex eight time points, each 24 hours apart, from day 2 to day 9 of this mesendoderm differentiation under control conditions for a scRNA-seq time course reference. We generated two additional time course scRNA-seq datasets with Cell Hashing matching the time points from the reference. The first dataset employs a CRISPRi-mediated TMEM88 knockdown in hiPSCs to assess the effect of TMEM88 loss-of-function on mesendoderm differentiation (gain of Wnt signalling), while the hiPSCs from the second dataset are treated with Wnt inhibitor XAV-939 to observe effects of an opposite direction of Wnt signalling change.

多能干细胞为解析跨多种发育谱系轨迹的细胞分化分子调控机制提供了可规模化的研究手段。本研究构建了带条形码的诱导多能干细胞（induced pluripotent stem cells, iPSC），以生成一套源自多能状态的多谱系分化图谱，涵盖经WNT、骨形态发生蛋白（bone morphogenetic protein, BMP）和血管内皮生长因子（vascular endothelial growth factor, VEGF）信号通路调节剂扰动的WNT诱导分化时间进程。
将体外细胞类型计算映射至体内发育谱系后，研究发现诱导多能干细胞衍生的细胞类型具有多样性，包括中内胚层谱系细胞（如侧板中胚层、轴旁中胚层）、神经嵴以及原始肠组织。将该体外分化图谱与人类复杂性状的基于汇总数据的孟德尔随机化（Summary data-based Mendelian Randomisation）分析相结合，本研究鉴定出WNT抑制蛋白TMEM88作为调控中内胚层细胞类型的潜在调控因子，其可影响多种心血管及人体测量学性状的发育。
利用遗传功能丧失模型，研究证实TMEM88是体外分化多种内胚层与中胚层细胞谱系所必需的；且体内TMEM88基因敲除会导致动脉血压显著失调。本研究提供了一套多谱系诱导多能干细胞分化图谱，并配套全新的分子、计算与统计遗传学工具，用于解析哺乳动物发育生理学的遗传决定因素。
总体实验设计：首先，我们利用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）验证了基于基因组条形码（barcode, BC）的多重标记方法在未分化人类诱导多能干细胞（human induced pluripotent stem cells, hiPSC）中的应用效果，将18份不同样本混合至同一次单细胞RNA测序实验中。随后，我们依托这18株带条形码的人类诱导多能干细胞系，构建3个单细胞RNA测序文库，以评估9种不同小分子信号调节处理（含对照组）对诱导多能干细胞向多种中内胚层谱系分化的影响，分化时间点涵盖第2天、第5天与第9天，每个处理设置2次生物学重复。我们还单独采用细胞哈希（Cell Hashing）标签对对照组条件下中内胚层分化的8个时间点（间隔24小时，从第2天至第9天）进行多重标记，以构建单细胞RNA测序时间进程参考数据集。此外，我们额外生成2套采用细胞哈希标记的时间进程单细胞RNA测序数据集，其时间点与参考数据集匹配。第一套数据集利用CRISPR干扰（CRISPR interference, CRISPRi）介导的人类诱导多能干细胞中TMEM88基因敲低，以探究TMEM88功能丧失对中内胚层分化（即Wnt信号通路激活）的影响；第二套数据集则采用WNT抑制剂XAV-939处理人类诱导多能干细胞，以观察Wnt信号通路反向调控的效应。