Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Abstract

Congenital heart defects (CHD) constitute the most common birth defect in humans, affecting approximately 1% of all live births. Our ability to understand how these disorders originate is hindered by our limited ability to model the complexity of the human heart in vitro. 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 a novel method to generate human heart organoids by self-assembly using pluripotent stem cells. Our method is fully defined, highly efficient, scalable, shows high reproducibility and is compatible with screening and high-throughput approaches. Human heart organoids (hHOs) are generated using a two-step canonical Wnt signaling modulation strategy using a combination of chemical inhibitors and growth factors in completely defined culture conditions. hHOs faithfully recapitulate human cardiac development and are similar to age-matched fetal cardiac tissues at the transcriptomic, structural and cellular level. hHOs develop sophisticated internal chambers with well-organized multi-lineage cell-type regional identities reminiscent of the heart fields and the atrial and ventricular chambers, as well as the epicardium, endocardium, and coronary vasculature, and exhibit functional activity. We also show that hHOs can recreate complex metabolic disorders associated with CHD by establishing the first in vitro human model of diabetes during pregnancy (DDP) to study embryonic CHD. morphological and metabolically effects of increased glucose and insulin, showing the capability of modeling the effects of diabetes during pregnancy (DDP). Our heart organoid model constitutes a powerful novel tool for translational studies in human cardiac development and disease.

Heart Organoids and Engineered Heart Tissues: Novel Tools for Modeling Human Cardiac Biology and Disease

“…Formation of PSC aggregates can be performed by either high-density cell seeding, allowing 3D aggregates to form and transferring them to ultra-low-attachment wells [ 63 ], or by seeding the cells into round bottom ultra-low-attachment plates to allow 3D aggregate formation by undisturbed incubation [ 44 , 64 ] or centrifugation of the plate [ 45 , 46 , 47 ]. For aggregates prepared in 96-well plates, the number of seeded cells ranges from as little as 300–700 cells per well [ 64 ] to up to 10,000 cells per well [ 46 ]. Most protocols initiate differentiation of the PSC aggregates 2 days after seeding [ 45 , 46 , 63 , 64 ], except for Hofbauer et al, which starts after 1 day [ 47 ], and Lee et al, which starts after 4 days [ 44 ].…”

“…For aggregates prepared in 96-well plates, the number of seeded cells ranges from as little as 300–700 cells per well [ 64 ] to up to 10,000 cells per well [ 46 ]. Most protocols initiate differentiation of the PSC aggregates 2 days after seeding [ 45 , 46 , 63 , 64 ], except for Hofbauer et al, which starts after 1 day [ 47 ], and Lee et al, which starts after 4 days [ 44 ]. These protocols yield a range of cardiac organoids mimicking various aspects of cardiogenesis.…”

“…While these organoids demonstrate some overlap in the resulting features, there are key differences between them when it comes to cell lineages, morphological structure, functional capabilities, and demonstration of translational applicability. Several of the reported protocols used mouse ESCs (mESCs) [ 44 , 63 , 64 ], while the most recent ones used human iPSCs or ESCs [ 45 , 46 , 47 ], with the added benefit of pre-clinical and translational applicability to humans.…”

“…Two groups recently reported heart organoids with more complex morphological features than those described above, including chamber formation and self-vascularization [ 46 , 47 ]. Lewis-Israeli et al [ 46 ] generated self-assembling human heart organoids (hHOs) by plating ~10,000 human PSCs per well in 96-well plates, the most cells per aggregate among the reported articles.…”

“…The established cardiac stem cell lineages produced release their own local morphogens and trigger higher levels of structure and organization quickly. Organoids generated via this method can develop very high levels of complexity, with most or all of the cell types found in the heart, and spontaneously acquire relevant rudimentary anatomical morphology, such as chamber organization and atrioventricular specification [ 44 , 45 , 46 , 47 ]. The cost of this approach is lower control over the final result in comparison to organoids fabricated via direct assembly.…”