Image Courtesy of Kiera Suh.
Imagine a world where any organ could be grown in the lab. A sample from a cheek swab could become a functioning heart in just a matter of weeks. New hearts would be grown from patients’ stem cells whenever they were needed, and organ transplant lists and waiting times would virtually disappear.
While this world is still far in the future, researchers at the lab of Stuart Campbell, Yale Associate Professor of Biomedical Engineering & Cellular and Molecular Physiology, have taken major strides in accelerating the maturation of stem-cell derived cardiomyocytes (iPSC-CMs) to grow engineered heart tissues (EHTs) in the lab that can even contract in response to electrical stimuli.
Growing Fetal Heart Cells
The primary function of EHTs is to provide a model of the human heart to study its features and responses to stimuli without having to access a human heart directly. These models are created by differentiating induced pluripotent stem cells into cardiomyocytes–specifically, stem-cell derived cardiomyocytes (iPSC-CMs). This process involves first washing a thin slice of a pig heart in a detergent to clear away pig cells. The extracellular matrix is then used as a template for seeding a mixture of human heart cells, including iPSC-CMs and human cardiac fibroblasts.
However, since stem cells can differentiate into any type of cell, iPSC-CMs are still relatively immature, representing fetal cardiomyocytes rather than mature ones. This limits their functionality as studies on them may not represent the characteristics of a fully grown human heart. Thus, to accurately represent mature heart tissue, iPSC-CMs need to undergo a maturation protocol that can currently take anywhere from forty days to six months.
One proposed technique for speeding up the maturation of iPSC-CMs is progressive electrical ramp pacing, which involves exposing the cells to an increasing rate of electrical current pulses over time. Previous studies have shown that this leads to heart cells that are more mature as they have more advanced electrophysiology and better calcium handling.
Calcium is also known to play a large role in cardiac physiology, as it is essential in inducing the contraction of the heart. When a membrane potential reaches the cardiac muscle, calcium channels open, allowing calcium to flow in and bind to troponin, which triggers the heart muscle cell contraction. Greater calcium levels increase contractile force. However, calcium’s role in the maturation of EHTs has not been previously considered. Most EHTs were grown in solutions containing only a fraction of the calcium concentration normally found in the heart.
“When our group realized how underutilized and underrated those calcium mediums have been across the field, we thought it might be interesting just to try it out,” said Shi Shen, the primary researcher on this study. This curiosity led to the discovery that differences in response to calcium in these early stages can be a key driver in cardiomyocyte differentiation: results show that the amount of calcium present in the cell culture medium produced a significant change in the maturation of iPSC-CMs.
Growing Mature Heart Tissues
To further advance the maturation of iPSC-CMs, researchers tested whether the combination of electrical ramp pacing and an increase in free calcium ions, Ca2+, in culture could produce a scalable improvement in the maturation of EHTs compared to the standard maturation protocol.
Four groups of EHTs—high-Ca2+ non-paced (HC-NP), low-Ca2+ non-paced (LC-NP), high-Ca2+ ramp-paced (HC-RP), and low-Ca2+ ramp-paced (LC-RP)—were studied to determine the effects of electrical pacing and calcium level on their own and in conjunction. The team performed the ramp pacing at frequencies higher than that of a regular human heart rate. “The regular human heart rate would fit between one to two hertz, so putting it at three hertz is like putting a YouTube video at two times speed,” Stephanie Shao, an undergraduate researcher on this study, said. “You can really increase the frequency and speed it up without harming it. In this case, it benefits it because it makes the EHTs mature at an accelerated rate.”
To test the functionality of these heart tissues, a variety of metrics were measured, ultimately showing that the HC-RP group performed much better than the other groups. One of the most notable improvements was the force-frequency relationship (FFR). FFR reflects increases in the contractile force of the heart with increasing frequency stimulus. “This is one of the key results to determine whether our experiments are successful because healthy humans need this particular phenomenon to function, […] and one of the hallmarks of a cardiac disease is the lack of increase in force,” Shen said.
FFR is measured using a mechanical testing apparatus that measures force in response to 0.25-Hz increases in frequency from 1 to 3 Hz. While high calcium marginally improved the FFR of the groups with no ramp pacing, both groups still showed a negative FFR, meaning the force continuously decreased rather than increasing to a systolic peak force and dropping again. However, once the researchers induced a ramp pacing, they saw a positive FFR in the group with high calcium, whereas the FFR of the low calcium group was still negative. Healthy human myocardium has a positive FFR of up to 2-2.5 Hz, similar to that of the HC-RP group, which exhibited a FFR around 2 Hz.
Other metrics that measure heart tissue functionality are time-to-peak (TTP) and time to-fifty percent relaxation (RT50). TTP is the time it takes for the tissue to reach its peak contractile force, and RT50 is the time it takes for the tissue to reach fifty percent relaxation after its peak force. Human tissues exhibit TTP and RT50 values at around 200 ms and 120 ms, respectively. The HC-RP group showed a similar TTP of around 290 ms and RT50 of around 120 ms, which is significantly faster than the other EHT groups.
The effect of isoproterenol (ISO) on the EHTs was also observed by measuring FFR after exposure to ISO. ISO is a drug that increases the contractility of the heart. It is used for patients with weakened hearts to improve cardiac output. The increase in the systolic peak force for the HC-RP group was much more significant compared to the increase in the systolic peak force for the LC-RP group. These results indicate that the HC-RP group behaves more similarly to actual mature heart tissue in the presence of ISO.
On top of these metrics, western blots and RNA sequencing were performed to analyze changes in protein and RNA levels on a molecular level that may have influenced improvements seen in the different groups. The results showed that markers associated with mature heart tissues were elevated in the HC-RP group. They also found that the genes expressed in the HC-NP and LC-RP groups were not the same, indicating that both electrical ramp pacing and high calcium are needed to produce the mature characteristics achieved in the EHTs.
With this improved protocol, the maturation of iPSC-CMs can be significantly shortened relative to previously published techniques. While EHTs cannot be used to grow hearts directly from stem cells, this advancement has significant implications for future research. Given that many researchers around the world are using iPSC-CMs for a variety of purposes, this technique has the potential to find widespread use and make mature, functional EHTs more readily available.
“For something like a drug study, a lot less compound would be used,” Shao said. “Especially if it’s a drug that’s not out yet, you have to have a chemist make it, and that’s not easy to make large quantities of, which is what you would need for an in vivo study.”
Another major advantage of using EHTs is that they are grown from stem cells derived from a specific patient. This means that any testing a patient may need to undergo can be performed on an EHT grown from their stem cells, which will more accurately represent the characteristics of their own heart.
This study is a catalyst for future cardiac research exploring the vast applications of EHTs. “There’s a large segment of work that’s being done targeted towards implanting cells back into patients to repair the heart to replace or regenerate heart muscle,” Campbell said. “I would hope that the field takes notice of our protocol because if you’re repairing the heart, for instance, you want to generate a lot of mature cells, so someone’s going to have to decide how to treat those cells so that they’re as mature as possible.” Campbell hopes this paper will contribute to the growing body of literature for the most optimal maturing conditions.
Moving forward, there are still other factors to investigate that may further improve the maturation of stem cells for EHT growth. “Something that we haven’t tried is combining a realistic pattern of mechanical loading, or maturation of mechanical loading—so what a fetus experiences in terms of the fetal heart versus a newborn versus an adult heart—modulating the mechanical load in conjunction with those heart rate changes”, says Campbell. While growing a functioning adult human heart is not yet in the cards, we are getting closer to a future where that is possible.
The author would like to thank Professor Campbell, Dr. Shi Shen, and Stephanie Shao for their time and enthusiasm in sharing their research.
Shen, S., Sewanan, L. R., Shao, S., Halder, S. S., Stankey, P., Li, X., & Campbell, S. G. (2022). Physiological calcium combined with electrical pacing accelerates maturation of human engineered heart tissue. Stem Cell Reports, 17(9), 2037–2049. https://doi.org/10.1016/j.stemcr.2022.07.006