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:Crosstalk between cardiac cells is critical during heart development but its role in organ maturation is still largely uncharacterised. Here, we show that endothelial cells increas the force of contraction and enhance the expression of mature sarcomeric proteins and extracellular matrix (ECM) components in human pluripotent stem cell derived cardiac organoids (hCO). Endothelial cells regulate cardiac maturation and function both directly through secretion of ECM molecules and indirectly via paracrine signaling. Laminin α5, an endothelial enriched ECM protein, was identified as a key regulator of cardiac maturation and contractility in vitro. In vivo loss-of-function studies in mice confirmed that Lama5 was required for myocardial expansion during heart development in vivo. In addition, paracrine PDGF signaling was identified as a mediator of increased ECM deposition and cardiac contractility in hCO. This study uncovers matrix regulatory functions of endothelial cells governing cardiac maturation and highlights the importance of multicellularity for organoid models.
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:Summary Single-cell RNA sequencing was performed on cardiac organoids differentiated from WTC11 hiPSCs (TTN-mEGFP) to investigate chamber-specific lineage commitment. Organoids were collected at day 7.5 of differentiation, using protocols designed to direct progenitors toward left ventricle, right ventricle, or atrium fates. This dataset enables the characterization of transcriptional programs and cellular heterogeneity underlying the early specification of the cardiac chamber. Dataset: This dataset contains single-cell RNA sequencing (scRNA-seq) data generated to compare the development of cardiac organoids differentiated from WTC11 hiPSCs (TTN-mEGFP reporter line). Organoids were harvested at day 7.5 of differentiation, a stage at which chamber-specific programs begin to emerge. Each sample corresponds to a distinct differentiation protocol designed to direct the commitment of progenitors toward a specific cardiac chamber identity: the left ventricle, the right ventricle, or the atrium. The experimental rationale, illustrated in Supplementary Figure 4 (Figure S4) of Becca et al. (2025), highlights the use of scRNA-seq to resolve the cellular heterogeneity of these chamber-directed organoids and to define transcriptional signatures associated with early cardiac chamber specification.
Project description:Human cardiac organoids were derived from hiPSCs using a regimen of small molecules and morphogens. Extracellular vesicles (cardEVs) were subsequently isolated from the conditioned medium via differential centrifugation. Finally, the proteomic cargo of cardEVs was characterized using LC-MS/MS and bioinformatically compared against EVs derived from the AC10 cardiomyocyte cell line and cadaveric human heart explants (heEVs).