Project description:Self-organisation and coordinated morphogenesis of multiple cardiac lineages is essential for the development and function of the heart1-3. However, the absence of a human in vitro model that mimics the basic lineage architecture of the heart hinders research into developmental mechanisms and congenital defects4. Here, we describe the establishment of a reliable, lineage-controlled and high-throughput cardiac organoid platform. We show that cardiac mesoderm derived from human pluripotent stem cells robustly self-organises and differentiates into cardiomyocytes forming a cavity. Co-differentiation of cardiomyocytes and endothelial cells from cardiac mesoderm within these structures is required to form a separate endothelial layer. As in vivo, the epicardium engulfs these cardiac organoids, migrates into the cardiomyocyte layer and differentiates. We use this model to demonstrate that cardiac cavity formation is controlled by a mesodermal WNT-BMP signalling axis. Disruption of one of the key BMP targets in cardiac mesoderm, the transcription factor HAND1, interferes with cavity formation, which is consistent with its role in early heart tube and left chamber development5. Thus, the cardiac organoid platform represents a powerful resource for the quantitative and mechanistic analysis of early human cardiogenesis and defects that are otherwise inaccessible. Beyond understanding congenital heart disease, cardiac organoids provide a foundation for future translational research into human cardiac disorders.
Project description:Pathogenic mutations in alpha kinase 3 (ALPK3) cause cardiomyopathy and a range of musculoskeletal defects. How ALPK3 mutations result in disease remains unclear and little is known about this atypical kinase. Using a suite of engineered human pluripotent stem cells (hPSCs) we show that ALPK3 localizes to the sarcomere, specifically at the M-Band. Both sarcomeric organization and calcium kinetics were disrupted in ALPK3 deficient hPSC derived cardiomyocytes. Further, cardiac organoids derived from ALPK3 knockout hPSCs displayed reduced force generation. Phosphoproteomic profiling of wildtype and ALPK3 null hPSC derived cardiomyocytes revealed ALPK3-dependant phospho-peptides were enriched for proteins involved in sarcomere function and protein quality control. We demonstrate that ALPK3 binds to the selective autophagy receptor SQSTM1 (Sequestome 1) and is required for the sarcomeric localization of SQSTM1. We propose that ALPK3 is a myogenic kinase with an integral role in the intracellular signaling networks underlying sarcomere maintenance required for continued cardiac contractility.
Project description:Cardiac maturation is an important developmental phase where there are profound biological and functional changes after birth in mammals. Herein, we use our profiling of human heart maturation in vivo to identify key drivers of maturation in our human cardiac organoid (hCO) model. After screening of various metabolism modulating factors, we established a directed maturation (DM) protocol to induce mature cardiac expression and compared the proteomic changes to our original serum free (SF) protocol. In this dataset, we compared 4 replicates of DM to 4 replicates of SF derived cardiac organoids using global DIA-MS/MS.
Project description:The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of the adult. However, the key drivers of this process remain poorly defined. We developed as 96-well screening platform, using human pluripotent stem cell derived cardiac organoids, to determine the molecular requirements for in vitro cardiomyocyte maturation. Here, we describe gene expression changes resulting from culturing human cardiac organoids in standard cell culture conditions and under optimized maturation conditions. We assessed our maturation conditions by comparing transcriptional changes of human cardiac organoids to RNA isolated from human heart. Interesting, analysis of these data revealed that a switch to fatty acid oxidative metabolism is a key governor of cardiomyocyte maturation and mature cardiac organoids were refractory to mitogenic stimuli.
Project description:The study of cardiac physiology and disease is hindered by physiological differences between humans and small-animal models. Here, we report the generation of multi-chambered vascularized human cardiac organoids under anisotropic stress, and their applicability to study electro-metabolic coupling in cardiac tissue. The organoids are derived from human induced pluripotent stem cells, and integrate sensors for the simultaneous measurement of oxygen uptake, extracellular field potentials and cardiac contraction at resolutions higher than 10 Hz. The microphysiological system allowed us to find that 1-Hz cardiac respiratory cycles are coupled with electrical activity rather than with mechanical activity, that calcium oscillations drive a mitochondrial respiration cycle, that the pharmaceutical or genetic inhibition of electro-mitochondrial coupling results in arrhythmogenic behaviour, and that the induction of arrythmia by the chemotherapeutic mitoxantrone can be partially reversed by the co-administration of metformin. Microphysiological cardiac systems may further facilitate the study of the mitochondrial dynamics of cardiac rhythms and advance the understanding of cardiac physiology.
Project description:Stem cell organoids are powerful models for studying organ development, disease modeling, drug screening, and regenerative medicine applications. The convergence of organoid technology, tissue engineering, and artificial intelligence (AI) could potentially enhance our understanding of the design principle for organoid engineering. In this study, we utilized micropatterning techniques to create a designer library of 230 cardiac organoids with 7 geometric designs (Circle 200, Circle 600, Circle 1000, Rectangle 1:1, Rectangle 1:4, Star 1:1, and Star 1:4). We employed manifold learning techniques to analyze single organoid heterogeneity based on 10 physiological parameters. We successfully clustered and refined our cardiac organoids based on their functional similarity using unsupervised machine learning approaches, thus elucidating unique functionalities associated with geometric designs. We also highlighted the critical role of calcium rising time in distinguishing organoids based on geometric patterns and clustering results. This innovative integration of organoid engineering and machine learning enhances our understanding of structure-function relationships in cardiac organoids, paving the way for more controlled and optimized organoid design.