While cardiovascular disease remains the leading cause of death in the United States, there are barriers to studying and modeling heart disease in the laboratory. The specialized muscle cells that make up a beating heart, called cardiomyocytes, are difficult to grow and maintain using traditional cell culture techniques.
New research published in the journal Biophotonics Discovery by scientists at the University of Wisconsin-Madison describes an imaging method to observe stem-cell derived cardiomyocytes grown in a variety of biosynthetic hydrogels and assess the ideal conditions for successful growth.
Danielle Desa, a postdoctoral fellow at the Morgridge Institute for Research, adapted optical imaging techniques she learned in the lab of Melissa Skala, a professor of biomedical engineering and Carol Skornicka Chair in Biomedical Imaging at Morgridge, to support stem cell biology research in the Bioinspired Materials Lab led by William Murphy, professor of biomedical engineering and orthopedics and rehabilitation.
“The goal is having something that will be tunable and reproducible,” Desa says. “The dream application of using these synthetic materials would be for biomanufacturing, because you’d want something robust and repeatable.”
To grow cardiomyocytes from induced pluripotent stem cells, researchers have traditionally used a composite called Matrigel, a mixture of proteins derived from mouse tumors that form an extracellular-like matrix to culture the cells. However, this method is highly variable, prone to contamination, and degrades easily over time.
Instead, the Murphy Lab experiments with different compositions of synthetic polyethylene glycol-based hydrogels to induce stem cell differentiation and maturation. They can fine-tune the composition of each gel to vary the concentration of peptides and the stiffness of the gel to create conditions best suited for cardiomyocyte development. Their work has resulted in hundreds of different formulations, this technique allows for each one to be highly reproducible and uniform.
To screen the cells to determine their overall maturity, the typical assays used often require processing a stain with harsh chemicals that ultimately damage the cells. The Skala Lab specializes in label-free imaging techniques that detect metabolic changes in the cells without destroying them.
“It’s complementary to those other kinds of assays,” says Desa. “And having the metabolic information gives us a look into another facet of maturation.”
The optical imaging technique relies on detecting metabolic molecules involved in many of the complex biological pathways that provide stem cells with energy to differentiate and mature into specialized cardiomyocytes. In particular, the system targets the autofluorescent signals from two molecules, NAD(P)H and FAD. These signals are used together to calculate the optical redox ratio — which represents the metabolic changes and shifts in energy production and consumption.
“We wanted to do repeated imaging on the same gels over time and see if the technique was sensitive to any metabolic changes,” Desa says.
They found that the redox ratio decreased in cardiomyocytes grown in all the synthetic hydrogel formulations during differentiation, early maturation, and late maturation. This indicates that metabolic pathways shift from glycolysis (breaking down glucose to access energy quickly) to oxidative phosphorylation (using oxygen to produce energy).
Desa teamed up with graduate student Margot Amitrano to ultimately screen cardiomyocytes grown in the hydrogels out to 100 days post-differentiation.
“It was useful to see that they didn’t degrade in that time,” Desa adds. “I think that’s kind of a selling point for the Murphy Lab’s gels. And we did continue to see the metabolic changes over that time.”
Additionally, they observed metabolic differences in low and high efficiency differentiation batches of cells. Desa says the sensitivity of the imaging technique could serve as an early predictor for which batch of cells might be more effective at maturing into functional cardiomyocytes. This suggests that optical redox imaging could be a useful screening tool for cell manufacturing.
This summer, Desa finished her postdoctoral fellowship with the Skala Lab and is beginning the next chapter in her career as an assistant scientist in the lab of Randy Bartels, a professor of biomedical engineering and Morgridge investigator, where she will lean further into optical engineering design and customization. She hopes her new role will provide new opportunities for collaboration and to share her knowledge of these imaging tools.
“I like being in this place where I can talk to people who have the interesting biological questions and show them instrumentation that they didn’t know existed and that could answer those questions,” Desa says.
This story was originally published by the Morgridge Institute for Research. Top photo: Morgridge Postdoctoral Fellow Danielle Desa.