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. 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.