Generation of Heart Organoids Modeling Early Human Cardiac Development Under Defined Conditions

Abstract

Cardiovascular-related disorders are a significant worldwide health problem. Cardiovascular disease (CVD) is the leading cause of death in developed countries, making up a third of the mortality rate in the US 1 . Congenital heart defects (CHD) affect ~1% of all live births 2 , making it the most common birth defect in humans. Current technologies provide some insight into how these disorders originate but are limited in their ability to provide a complete overview of disease pathogenesis and progression due to their lack of physiological complexity. There is a pressing need to develop more faithful organ-like platforms recapitulating complex in vivo phenotypes to study human development and disease in vitro. Here, we report the most faithful in vitro organoid model of human cardiovascular development to date using human pluripotent stem cells (hPSCs). Our protocol is highly efficient, scalable, shows high reproducibility and is compatible with high-throughput approaches. Furthermore, our hPSC-based heart organoids (hHOs) showed very high similarity to human fetal hearts, both morphologically and in cell-type complexity. hHOs were differentiated using a two-step manipulation of Wnt signaling using chemical inhibitors and growth factors in completely defined media and culture conditions. Organoids were successfully derived from multiple independent hPSCs lines with very similar efficiency. hHOs started beating at ~6 days, were mostly spherical and grew up to ~1 mm in diameter by day 15 of differentiation. hHOs developed sophisticated, interconnected internal chambers and confocal analysis for cardiac markers revealed the presence of all major cardiac lineages, including cardiomyocytes (TNNT2 + ), epicardial cells (WT1 + , TJP + ), cardiac fibroblasts (THY1 + , VIM + ), endothelial cells (PECAM1 + ), and endocardial cells (NFATC1 + ). Morphologically, hHOs developed well-defined epicardial and adjacent myocardial regions and presented a distinct vascular plexus as well as endocardial-lined microchambers. RNA-seq time-course analysis of hHOs, monolayer differentiated iPSCs and fetal human hearts revealed that hHOs recapitulate human fetal heart tissue development better than previously described differentiation protocols 3,4 . hHOs allow higher-order interaction of distinct heart tissues for the first time and display biologically relevant physical and topographical 3D cues that closely resemble the human fetal heart. Our model constitutes a powerful novel tool for discovery and translational studies in human cardiac development and disease.

Patients' perspectives on the derivation and use of organoids

“…Reflections on intestinal organoids have centered on their use in CF-related work (e.g., precision medicine, biobanking). Nonetheless, multiple other human organoid systems have been reported (Israeli et al, 2020;Kim et al, 2020). Consequently, broader inquiry is needed.…”

Optogenetic‐mediated cardiovascular differentiation and patterning of human pluripotent stem cells

“…More recently, Wnt and BMP signaling pathways were identified as critical cues for cardiac organoid selforganization and chamber formation in 3D, in which CMs and endothelial cells were both formed in the same aggregates. 19,20 With the advent of optogenetic control of BMP signaling, 23 we may be able to precisely pattern the differentiation of CMs, endothelial cells and/or epicardial cells via optogenetics to achieve spatially-organized cardiac tissues/ organoids with high consistency and reproducibility. In the future, OptoWnt could be combined with 3D cell culture, orthogonal light control of other developmental signaling pathways, and advanced optical patterning techniques to achieve more physiologically relevant hPSC organoid models with intricate geometries and orthogonally controlled cell positions for studying human development and disease.…”

“…Flow cytometry analysis demonstrated that $80% of the day 15 cells were cTnT + CMs, with a differentiation efficiency ($82.7% 17 ) comparable to our previous GiWi protocol. 15 19,20 we illuminated day 6 cardiac progenitor aggregates for 24 hours, leading to self-organized cardiac organoids with a cavity (Figure S3B). Our results highlight the potential of our OptoWnt platform for light-controllable tissue patterning in hPSCs.…”

Historical evolution of spheroids and organoids, and possibilities of use in life sciences and medicine

“…Protocols to derive 3D heart models have been tested since at least 2007, and, to date, there is a great variation of possibilities. [91] The most recent publications indicate generation of heart organoids that present different cell types such as myocytes, epicardial cells, endocardial cells, endothelial cells, and fibroblasts, [91] including models in which biological and physiological pacemakers are derived within the culture. [92] The applications include infarct modeling, drug screening, cell therapy, understanding of heart development, and even determination of function of each cardiac cell type.…”