Researchers at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) have used human pluripotent stem cells to grow sesame seed-sized heart organoids, called cardioids, that spontaneously self-organize into beating, heart chamber-like structures, without the need for experimental scaffolds. The scientists suggest their technology has allowed them to create some of the most realistic heart organoids to date,  which may revolutionize research into cardiovascular disorders and congenital heart defects.

A developing cardioid in a 7-day time course. [Video courtesy of Sasha Mendjan, IMBA]

“We want to come up with human heart models that develop more naturally and are therefore predictive of disease,” said research lead Sasha Mendjan, PhD. “Cardioids are a major milestone. Our guiding principle is that for an in vitro tissue to be fully physiological, it also needs to undergo organogenesis. We were able to achieve this, using the developmental principles of self-organization—which makes it such an exciting discovery … This way, companies will be more open to bringing more drugs into the clinical trials because they are much more certain of the outcome of the trial.”

Mendjan and team report on their development in Cell, in a paper titled, “Cardioids reveal self-organizing principles of human cardiogenesis.”

Cardiovascular diseases are the leading cause of fatalities globally, and are responsible for about 18 million deaths each year. Heart defects are also the most prevalent type of birth defect in children. However, the lack of human physiological models of the heart represents a major bottleneck to our understanding, and potential development of regenerative therapies for heart diseases and malformations.

“The human heart is the first functional organ to form in development, but is one of the most difficult organs to model in vitro,” the authors wrote. Previously, scientists have built 3D cardiac organoids via tissue engineering, an approach that generally involves assembling cells and scaffolds like building a house out of brick and mortar. “These have proven immensely useful to measure contraction force, perform compound screens, and model structural muscle and arrhythmogenic disorders,” the investigators continued. However, such, engineered organoids do not have the same physiological responses to damages as human hearts and thus often fail to serve as good disease models. “ … existing models do not recapitulate cardiac-specific self-organization to acquire in vivo-like architecture such as a CM chamber with inner endocardial cavity lining and are therefore limited as models of human cardiogenesis and heart disease.”

Mendjan explained, “Tissue engineering is very useful for many things like, for example, if you want to do measurements on contraction. But in nature, the organs aren’t built this way. In the embryo, organs develop spontaneously through a process called self-organization. During development, the cellular building blocks interact with each other, moving around and changing shape as an organ’s structure emerges and grows.

“Self-organization is how nature makes snowflake crystals or birds behave in a flock,” he continued. “This is difficult to engineer because there seems to be no plan, but still something very ordered and robust comes out. The self-organization of organs is much more dynamic, and a lot is going on that we do not understand. We think that this ‘hidden magic’ of development, the stuff we do not yet know about, is the reason why currently diseases are not modeled very well.”

The self-organizing organoid field has revolutionized biomedical research over the past decade. However, the heart has represented last major inner organ missing such a physiological model capable of recapitulating developmental and injury response processes. “Although self-organizing organoids have been reported for almost all major organs, there are currently no cardiac-specific self-organizing human cardiac organoids that autonomously pattern and morph into an in vivo-like structure.”

Mendjan and his team wanted to mimic development by enabling self-organization in a dish. During development, a heart chamber emerges from the mesoderm germ layer. For their work, the team had to establish in vivo-like mesodermal signaling conditions guiding pluripotent stem cells. They coaxed the stem cells to self-organize by activating known signaling pathways involved in embryonic heart development, in a specific order. “… we developed a differentiation approach based on temporal control of the key cardiogenic signaling pathways— activin, bone morphogenic protein (BMP), fibroblast growth factor (FGF), retinoic acid, and WNT,” they wrote.

Self-organising cardiac organoids form cavities [Image courtesy Sasha Mendjan, IMBA]

Besides a beating myocardial layer, a functional heart also contains an inner endothelial lining that later contributes to heart vasculature, and an outer epicardial layer that directs heart growth and regeneration. The cardioids recapitulate this three layered structure, shaping a heart chamber-like structure.

As the cells differentiated, they started to form the separate layers, similar to the structure of the heart wall. After one week of development, the organoids self-organized into a 3D structure that had an enclosed cavity, a similar spontaneous growth trajectory as human hearts. In addition, the team found the cardioids’ wall-like tissue contracted rhythmically to squeeze liquid around the inside the cavity.

A cardioid showing wave-like contractions [Video courtesy of Sasha Mendjan, IMBA]

“Amazingly, this led to self-organization of a heart chamber-like structure that was beating,” Mendjan said. “For the first time, we could observe something like this in a dish. It is a simple, robust and scalable model, and does not require addition of exogenous extracellular matrix like many other organoid models … It’s not that we are using something different than other researchers, but we are just using all of the signals known.”

He pointed out that not all pathways are needed to direct stem cells to become heart cells per se. “So they thought, ‘Okay, they’re not really necessary in vitro.’ But it turns out all these pathways are necessary. They are important to make the cells self-organize into an organ.”

Through their reported studies the scientists determined how signaling and transcription factors control cardioid chamber formation. For instance, the team was able to phenocopy the dramatic chamber cavity loss observed in children with hypoplastic left heart syndrome in cardioids by disrupting a transcription factor linked to this defect.

The researchers also assessed the effects on the cardioids of cryoinjury (injury by freezing), a technique mimicking myocardial infarction. They used a cold steel rod to freeze parts of the mini-hearts, which killed many cells at the site. Cell death is commonly observed after injuries such as a heart attack. Immediately, the team saw cardiac fibroblasts—a type of cell responsible for wound healing—start to migrate toward the injury sites and produce proteins to repair the damage. For the first time in a dish, the team found that this injury triggered an in vivo-like accumulation of extracellular matrix proteins in cardioids, an early hallmark of both regeneration and fibrotic heart disease.

The authors claimed, “Here, we established hPSC-derived self-organizing ‘’cardioids’’ that recapitulate chamber-like morphogenesis in the absence of non-cardiac tissues and use them to study self-organizing mechanisms of human cardiogenesis and heart disease … human cardioids represent a powerful platform to mechanistically dissect self-organization, congenital heart defects and serve as a foundation for future translational research.”

Mendjan further stated, “Cardioids bear incredible potential to unravel human congenital heart defects. As the system is physiological and scalable, this opens up huge possibilities for drug discovery and regenerative medicine.” His team has plans to grow cardiac organoids with multiple chambers that more closely recapitulate the organization of a real human heart. Many congenital heart diseases happen when other chambers start to form, so the multi-chamber model would help doctors better understand how defects develop in fetuses.

“Overall, the cardioid platform has a wide potential to explore fundamental mechanisms of self-organization and congenital defects as well as to generate future mature and complex human heart models suitable for drug discovery and regenerative medicine,” the investigators concluded.

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