Researchers from the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS) say that it is now possible for the first time to monitor the functional development and maturation of cardiomyocytes, the cells responsible for regulating the heartbeat through synchronized electrical signals, on the single-cell level using tissue-embedded nanoelectronic devices.

The flexible and stretchable devices can integrate with living cells to create “cyborgs,” according to their article “Tissue-embedded stretchable nanoelectronics reveal endothelial cell–mediated electrical maturation of human 3D cardiac microtissues” in Science Advances.

“Clinical translation of stem cell therapies for heart disease requires electrical integration of transplanted cardiomyocytes. Generation of electrically matured human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) is critical for electrical integration. Here, we found that hiPSC-derived endothelial cells (hiPSC-ECs) promoted the expression of selected maturation markers in hiPSC-CMs,” write the investigators.

“Using tissue-embedded stretchable mesh nanoelectronics, we achieved a long-term stable map of human three-dimensional (3D) cardiac microtissue electrical activity. The results revealed that hiPSC-ECs accelerated the electrical maturation of hiPSC-CMs in 3D cardiac microtissues. Machine learning–based pseudotime trajectory inference of cardiomyocyte electrical signals further revealed the electrical phenotypic transition path during development.

Single-cell RNA sequencing

“Guided by the electrical recording data, single-cell RNA sequencing identified that hiPSC-ECs promoted cardiomyocyte subpopulations with a more mature phenotype, and multiple ligand-receptor interactions were up-regulated between hiPSC-ECs and hiPSC-CMs, revealing a coordinated multifactorial mechanism of hiPSC-CM electrical maturation.

“Collectively, these findings show that hiPSC-ECs drive hiPSC-CM electrical maturation via multiple intercellular pathways.

“These mesh-like nanoelectronics, designed to stretch and move with growing tissue, can continuously capture long-term activity within individual stem-cell derived cardiomyocytes of interest,” says Jia Liu, PhD, co-senior author on the paper, who is an assistant professor of bioengineering at SEAS, where he leads a lab dedicated to bioelectronics.

Liu’s team, which specializes in engineering nanoelectronics to bridge the gap between living tissue and electronics, has developed several mesh-like, minimally invasive flexible nanoelectronic sensors designed to be embedded with natural tissues without disturbing normal cellular grown or function.

“Nature showed us the solution to monitoring tissue in 3D,” Liu says. “We were inspired by the way neural tubes fold during development, stretching as cells migrate and take shape into tissue volume.”

First cyborg organoid created four years ago

His team created their first cyborg organoid in 2019 to test the idea of using a mesh-like nanoelectronic structure, and has previously demonstrated that these types of flexible nanoelectronics can be safely implanted into living mice without disrupting the function of nearby cells.

In their latest study, Liu’s lab joined forces with Richard Lee and his team at Harvard Stem Cell Institute and used the nanoelectronics to monitor electrical activity of stem-cell derived cardiomyocytes. To do so, the researchers cultured cells onto a sheet made of commercially available cellular matrix known as “Matrigel” and the mesh-like nanoelectronic sensor (which contains a flexible grid of microelectrodes).

The nanoelectronic sensors (yellow, blue) are embedded with natural tissues (red, green). [Liu Lab, Harvard SEAS]
As the cells grew and developed into a small organoid structure, the researchers observed that the sheet easily stretched and accommodated the stem-cell derived tissues as they proliferated and expanded in 3D.

Using these techniques in in vitroexperiments, the team discovered that endothelial cells play a previously underestimated but crucial role in the rapid and functional maturation of stem-cell derived cardiomyocytes. When cultured together in a 3D cardiac tissue matrix, cardiomyocytes underwent “extraordinary electrical maturation” in the presence of endothelial cells, according to Liu.

Over the course of seven weeks of monitoring the developing organoids, the team observed that proximity to endothelial cells had a direct impact. Cardiomyocytes cultured next to endothelial cells matured faster compared to cardiomyocytes located farther away from endothelial cells, and they also displayed electrical characteristics typically found in healthy heart tissue.

The new insight is a leap forward for engineering stem-cell derived cardiac tissues, say the scientists. Experimental preclinical research in animals with human-like hearts has proven it’s difficult to engineer and transplant stem-cell derived cardiomyocytes that can beat in tandem with surrounding heart tissue for extended periods of time. Immature cardiomyocytes transplanted into an animal’s heart tend to beat to their own drum, and this electrical misfire can cause dangerous irregular heartbeats.

That’s why the discovery that co-culturing stem-cell-derived cardiomyocytes with endothelial cells can create more functionally mature cardiomyocytes is so significant, notes Liu.

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