Abstract
Congenital heart defects constitute the most common human birth defect, however understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro. Here we report a method to generate developmentally relevant human heart organoids by self-assembly using human pluripotent stem cells. Our procedure is fully defined, efficient, reproducible, and compatible with high-content approaches. Organoids are generated through a three-step Wnt signaling modulation strategy using chemical inhibitors and growth factors. Heart organoids are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level. They develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types, recapitulate heart field formation and atrioventricular specification, develop a complex vasculature, and exhibit robust functional activity. We also show that our organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects.
Highlights
Congenital heart defects constitute the most common human birth defect, understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro
We started by assembling Human pluripotent stem cells (hPSCs) into embryoid bodies (EBs) by centrifugation in ultra-low attachment 96-well plates followed by a 48-h incubation at 37 °C and 5% CO2 prior to induction (Fig. 1a)
Optimal cardiogenic mesoderm induction for all human embryonic stem cell and induced pluripotent stem cell lines was found at 1–4 μM CHIR99021 concentrations, rather than the typical 10–12 μM range reported for monolayer methods14,21–27. 4 μM CHIR99021 exposure resulted in the highest cardiomyocyte content with 64.9 ± 5.3% TNNT2 + cells at day 15 (Fig. 1d, Supplementary Fig. 1a)
Summary
Congenital heart defects constitute the most common human birth defect, understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro. Significant attempts have been made over the last decade to address the lack of relevant heart-on-a-chip or heart organoid models, using tissue engineering techniques[8,9,10,11,12,13,14,15] While these approaches allow for high control of the end construct, they tend to be expensive, work-intensive, and not readily scalable. Mouse embryonic stem cells (ESCs) were used to generate precardiac organoids showing distinct heart field specifications[16], cardiac crescent-like structures juxtaposed with primitive gut tube[17], and atrial and ventricular cardiomyocyte lineages[18] These studies provided us with a great deal of understanding and information of early heart development in vitro, but are faced with limitations associated with mouse models. As a proof-of-concept of the value of this system to model human cardiac disease, we utilized our organoid system to model the effects of pregestational diabetes (PGD) — clinically defined as diabetes before pregnancy and present during at least the 1st trimester of fetal development — on the developing embryonic heart
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