After 13 years, the mini heart finally got a coat

Produced by: Science Popularization China

Author: Li Yuhuan (Jilin University)

Producer: China Science Expo

When you rush to catch the morning rush hour in the subway, or when you stay up late to work overtime in the office, who silently bears these burdens? It is the heart, the engine of life, which continuously supplies the source of life for us every moment. However, the heart is not an iron machine, and it also needs our care.

Heart disease is the number one killer that threatens human life and health, and heart health is also an eternal proposition in the field of life sciences. Let us learn about the story of how scientists spent thirteen years to finally dress the "mini heart".

Small organs have big effects

The human body is so delicate and complex that scientists have created "organoids" in culture dishes to better study the development and physiological and pathological states of human organs. They are three-dimensional cell cultures that have similar spatial organization to corresponding organs and can reproduce some of the functions of corresponding organs, providing a highly physiologically relevant system.

Figure 2 Organoids are a powerful tool for studying human organ development and physiology and pathology

(Image source: Genome Institute of Singapore)

Due to the complexity of the human heart microenvironment, many results of animal studies cannot be fully transferred to humans. Cardiac organoids, or "mini hearts," are of great significance for understanding heart development and studying the treatment of congenital heart disease.

The history of cardiac organoids

Professor Alessandra Moretti's team at the University of Munich in Germany is the first group of researchers in the world to successfully create organoids containing cardiomyocytes and the outer layer of heart wall cells (epicardium). As early as 2010, Professor Moretti described how stem cells can be used to create human myocardial tissue; in 2015, new discoveries were made on how to use stem cells to generate self-organized exocardium; in 2018, single-cell transcriptome technology was used to explore the molecular mechanism of stem cell generation of cardiac tissue; in 2021, gene editing technology was used to correct genetic mutations in heart disease.

In the field of cardiac organoids, there has always been an unresolved issue, that is, pluripotent stem cell-derived cardiac organoids cannot spontaneously form a true epicardium (which has a key function in cardiac development and repair). In a paper published in Nature Biotechnology on April 3, 2023, Professor Moretti's team solved this problem, creating another important milestone in cardiac development research.

Figure 3 Alessandra Moretti's research group published an article in Nature Biotechnology, showing "epicardial organoids"

(Image source: Nature magazine official website)

So, how does this mini heart coat come into being and work? This requires us to discuss it from the following aspects.

1. How are epicardial organoids generated?

In order to build a heart organoid that is closer to the real thing, Professor Moretti's team added retinoic acid, which can promote the development of the epicardium, to the formula for forming spheres using human pluripotent stem cells, and then embedded the spheres in gel for 3D culture. It was eventually found that in addition to forming a core of cardiomyocytes, the spheres treated with retinoic acid also formed a thick encapsulation layer (Figure 4). The researchers were pleasantly surprised to find that this encapsulation layer contained a large number of cells expressing epicardial markers, which is exactly the "outer coat" of the heart organoids that everyone has been looking for for 13 years!

Figure 4 Schematic diagram of the process of generating epicardial organoids using human pluripotent stem cells

(Image source: adapted from reference [1])

Through further optimized culture, the researchers finally obtained cardiac organoids that could show functional ventricular myocardium and epicardial self-organization, and named them "epicardioids."

2. How similar are epicardial organoids to the human heart?

After obtaining epicardial organoids, the researchers analyzed their cellular components through single-cell sequencing and found that they had the same cell types as human fetal epicardium, including mesothelial epicardial cells, epicardium-derived mesenchymal cells, and proliferating cells. Moreover, this small organoid perfectly reproduced the human ventricular pattern: the action potential repolarization time of the dense outer myocardium was significantly shorter than that of the inner myocardium (Figure 5).

Figure 5 The outer myocardium (OM) and inner myocardium (IM) of epicardial organoids

(Image source: Reference [1])

It can be seen that epicardial organoids extremely well simulate the structure, function and cellular complexity of the human heart.

3. How do different heart cells interact with each other?

Further analysis of the single-cell sequencing data revealed a large number of interactions between epicardial cells and other cell types. The researchers focused on the ligand-receptor interaction between insulin-like growth factor 2 (IGF2) in epicardial cells and insulin-like growth factor 1 receptor (IGF1R) in cardiomyocytes because this is a major driver of myocardial compaction in rodents but has not been studied in the human system.

The results showed that treatment with IGF1R inhibitors significantly reduced the division of cardiomyocytes in epicardial organoids (Figure 6); when cardiac organoids without epicardium were treated with IGF2, the cardiomyocyte density increased with increasing IGF2 concentration, indicating that IGF2 is sufficient to induce myocardial compaction even in the absence of epicardium.

Figure 6 The interaction between epicardial cell IGF2 and cardiomyocyte IGF1R promotes myocardial compaction.

(Image source: adapted from reference [1])

4. Who are the “ancestors” of epicardial cells?

Where do such important epicardial cells come from? Previously, the ontogeny of epicardial progenitor cells was unclear, and even less was known about their human counterparts.

In this study, Professor Moretti used single-cell sequencing combined with computational biology methods to track cell trajectories on a timeline and ultimately found that the precursor cells of the "pre-JCF" are the main source of epicardial cells. Moreover, the "pre-JCF" precursor cells are "dual-capable" in humans, and can generate both cardiomyocytes and epicardial cells (Figure 7).

Figure 7 Lineage tracing shows that “pre-JCF” progenitor cells differentiate into epicardial cells and cardiomyocytes

(Image source: Reference [1])

5. What can epicardial organoids do?

One of the main functions of organoids is to simulate diseases. After obtaining epicardial organoids, researchers are eager to use this model to solve problems that two-dimensional in vitro models cannot solve, such as the key role of fibrosis in the progression of heart failure. The researchers first treated epicardial organoids with vasoconstrictors, which resulted in hypertrophy of cardiomyocytes and also caused them to exhibit the recognized characteristics of failing hearts: frequent arrhythmias and reduced amplitude of calcium transients. (Figure 8).

Figure 8 Treatment of epicardial organoids with the vasoconstrictor ET150 increased arrhythmia frequency and reduced calcium transient amplitude.

(Image source: adapted from reference [1])

In addition, the researchers tested the ability of epicardial organoids to mimic congenital myocardial fibrosis. They generated patient-specific epicardial organoids using induced pluripotent stem cells from patients with Noonan syndrome (who present with severe left ventricular hypertrophy and myocardial fibrosis at birth) and found in culture that the cellular environment of the epicardium does allow for fibrotic changes associated with developmental defects.

Based on these findings, epicardial organoids can be used in preclinical testing to determine the effects of drugs for myocardial fibrosis, thus avoiding the risk of harm caused by direct exposure to human subjects.

Conclusion

To some extent, it can be said that organoids are a powerful tool for scientists to unravel the mysteries of life. In the field of heart research, epicardial organoids provide a unique platform to solve basic problems in developmental biology, cardiovascular medicine, and drug discovery. It is foreseeable that this mini heart in a coat will bring more discoveries. But before that, let us exercise and protect our hearts!

References:

Meier AB, et al., Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease. Nat Biotechnol. 2023 Apr 3.