SUMMARY Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple 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.
Abstract Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across developmental lineages. Here, we engineer barcoded induced pluripotent stem cells to generate an atlas of multilineage differentiation from pluripotency, encompassing an eight-day time course with modulation of WNT, BMP, and VEGF signalling pathways. Annotation of in vitro cell types with reference to in vivo development reveals diverse mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Interrogation of temporal and signalling-specific gene expression in this atlas, evaluated against cell type-specific gene expression in human complex trait data highlights the WNT-inhibitor gene TMEM88 as a regulator of mesendodermal lineages influencing cardiovascular and anthropometric traits. Genetic TMEM88 loss of function models show impaired differentiation of endodermal and mesodermal derivatives in vitro and dysregulated arterial blood pressure in vivo. Together, this study provides an atlas of multilineage stem cell differentiation and analysis pipelines to dissect genetic determinants of mammalian developmental physiology.
Abstract Aims The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The human lncRNA CARMEN, which is known to dictate specification toward the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced isoforms. Methods and results The CARMEN locus can be manipulated to direct human primary cardiac precursor cells (CPCs) into specific cardiovascular fates. Investigating CARMEN isoform usage in differentiating CPCs represents therefore a unique opportunity to uncover isoform-specific functions in lncRNAs. Here, we identify one CARMEN isoform, CARMEN-201, to be crucial for SMC commitment. CARMEN-201 activity is encoded within an alternatively spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1, IRX1, IRX5, and SFRP1, and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced. Conclusions These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA. They further demonstrated the crucial importance of the lncRNA isoform CARMEN-201 in SMC specification during heart development.
Abstract Methods for cell clustering and gene expression from single-cell RNA sequencing (scRNA-seq) data are essential for biological interpretation of cell processes. Here we present TRIAGE-Cluster which uses genome-wide epigenetic data from diverse bio-samples to identify genes demarcating cell diversity in scRNA-seq data. TRIAGE-Cluster integrates patterns of repressive chromatin deposited across diverse cell types with weighted density estimation to determine cell type clusters in a 2D UMAP space. We then present TRIAGE-ParseR, a machine learning method that evaluates gene expression rank lists to define gene groups governing the identity and function of cell types. We demonstrate the utility of this two-step approach using atlases of in vivo and in vitro cell diversification and organogenesis. We also provide a web accessible dashboard for analysis and download of data and software. Collectively, genome-wide epigenetic repression provides a versatile strategy to define cell diversity and study gene regulation of scRNA-seq data.
SUMMARY Determining genes orchestrating cell differentiation in development and disease remains a fundamental goal of cell biology. This study establishes a genome-wide metric based on the gene-repressive tri-methylation of histone 3 lysine 27 (H3K27me3) across hundreds of diverse cell types to identify genetic regulators of cell differentiation. We introduce a computational method, TRIAGE, that uses discordance between gene-repressive tendency and expression to identify genetic drivers of cell identity. We apply TRIAGE to millions of genome-wide single-cell transcriptomes, diverse omics platforms, and eukaryotic cells and tissue types. Using a wide range of data, we validate TRIAGE’s performance for identifying cell-type specific regulatory factors across diverse species including human, mouse, boar, bird, fish, and tunicate. Using CRISPR gene editing, we use TRIAGE to experimentally validate RNF220 as a regulator of Ciona cardiopharyngeal development and SIX3 as required for differentiation of endoderm in human pluripotent stem cells. A record of this paper’s Transparent Peer Review process is included in the Supplemental Information.
Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation in vitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation in vitro and mouse gastrulation in vivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1–3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations.
SUMMARY This study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.
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