Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

This publication details a standardized, scalable protocol for producing complex, self-assembling human heart organoids (hHOs) directly from human pluripotent stem cells (hPSCs). Congenital heart defects affect approximately 1% of live births globally, yet modeling human heart development in vitro has historically been limited by the lack of complex 3D human tissue systems. The protocol overcomes this challenge by combining embryoid body generation in 96-well ultra-low attachment plates with a three-step Wnt pathway modulation strategy in fully defined media. The differentiation process begins on Day 0 with mesoderm induction using the Wnt activator CHIR99021 combined with BMP4 and Activin A. On Day 2, cardiac mesoderm specification is triggered using the Wnt inhibitor Wnt-C59. Finally, on Day 7, a brief secondary Wnt activation step with CHIR99021 directs proepicardial organ tissue formation. Over 15 days, the organoids expand from 200 µm to up to 1 mm in diameter and display robust, spontaneous beating starting around Day 6. Characterization through immunofluorescence and RNA sequencing confirms that hHOs recapitulate early cardiogenesis milestones, including first and second heart field specification (marked by HAND1 and HAND2, respectively). By Day 15, hHOs feature a cellular makeup analogous to the fetal human heart, comprising ~58% cardiomyocytes, ~15% epicardial cells, ~13% endocardial cells, ~12% cardiac fibroblasts, and ~1% endothelial cells. The organoids exhibit internal microchambers lined by endocardial tissue, an outer epicardial layer, and a spontaneous, interconnected vascular network. Functional assays using live Fluo-4 calcium imaging confirm regular action potential kinetics and synchronized calcium fluxes in organoid cardiomyocytes. Additionally, the protocol outlines optimized techniques for handling and mounting large 3D organoids using microbeads to maintain structural integrity during confocal imaging. Overall, this high-throughput organoid platform provides a robust tool for investigating human heart development, disease etiology, and pharmaceutical screening.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs. The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and at day 15, highly complex tissues inside hHOs, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

The discussion section highlights that this protocol offers an accessible, highly reproducible, and cost-effective methodology to generate complex human heart organoids from human pluripotent stem cells using precise sequential Wnt signaling modulation. The organoids closely resemble the cell type composition and transcriptomic profile of the developing human fetal heart, consisting of ~58% cardiomyocytes alongside non-myocyte populations including epicardial cells, endocardial cells, cardiac fibroblasts, and endothelial cells. A distinct feature of this protocol is the spontaneous development of an internal endothelial vascular network without requiring external growth factor additions. Furthermore, functional characterization using Fluo-4 live calcium imaging demonstrates normal action potential dynamics across individual cardiomyocytes. Future directions involve adapting the protocol to promote organoid maturation toward adult-like phenotypes, as current constructs represent early fetal development and are intended for developmental studies, disease modeling, and drug toxicity screening rather than therapeutic tissue transplantation.