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

This study introduces a robust, high-throughput protocol for generating human heart organoids (hHOs) using human pluripotent stem cells. By utilizing a controlled, three-step Wnt signaling modulation strategy in a 96-well format, the researchers successfully generate complex organoids that self-organize to represent key fetal cardiac structures, including distinct atrial and ventricular chambers, endocardial linings, and epicardial tissues. The resulting organoids demonstrate significant structural complexity, including an intricate vascular network and a diverse cellular composition that mirrors the fetal heart. The researchers confirm these findings using immunofluorescence to identify key cardiac lineages and RNA-sequencing to track transcriptomic development. Functional analysis through live-cell calcium imaging validates the organoids' physiological relevance, showing regular, robust calcium transients that suggest functional cardiomyocyte activity. This method offers a scalable and cost-effective tool for researchers to investigate early cardiac development, test pharmacological responses, and model congenital heart disease in vitro. By enabling the creation of consistent human heart models, this protocol facilitates research into the underlying mechanisms of heart defects that are difficult to study in standard animal models.

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 authors discuss the reproducibility and scalability of their hHO protocol, noting that it effectively integrates techniques for cardiomyocyte and epicardial cell differentiation with Wnt signaling modulation. They highlight that the organoids accurately model developmental milestones, such as heart field specification and internal chamber formation, making them useful for studying heart development and disease etiology. Future directions include building on this model to promote further maturation toward adult-like features, while acknowledging that the current model is primarily designed for research applications rather than clinical transplantation.