Over the last several years, cell therapy has emerged as an increasingly promising treatment for the world’s leading cause of death: heart disease. At the Institute for Stem Cell and Regenerative Medicine (ISCRM), researchers in multiple labs are using stem cell technology to pioneer novel approaches to treating heart disease in ways that address the root causes of this chronic disease, rather than manage its symptoms.
There is good reason for optimism. One emerging approach involves regenerating damaged or lost heart tissue by engrafting stem cell-derived cardiomyocytes (heart muscle cells) to help prevent heart failure in heart attack patients. However, key questions about this cutting-edge treatment remain.
One set of questions being explored by ISCRM researchers centers on how cell therapy helps damaged hearts become remuscularized. One theory is that it is driven primarily by proliferation – in other words, transplanted cardiomyocytes divide and replicate, creating large colonies of new muscle cells. Another theory is that engrafted cardiomyocytes undergo hypertrophy and grow bigger and stronger. Uncertainty lingers, however, because the tools used to examine the grafts have so far lacked the precision to deliver a definitive verdict.
Now, a new paper published in the journal Circulation details the use of rainbow cell technology to demonstrate that injected cells do proliferate, a finding that could help researchers enhance the efficacy of cell therapy for heart disease and perhaps conditions impacting other organs in the human body. The study was co-led by ISCRM faculty members Jen Davis, PhD, Associate Professor of Lab Medicine and Pathology and Bioengineering and Nate Sniadecki, PhD, Professor of Mechanical Engineering.
The findings come at an exciting time for heart research. In 2018, a study led by ISCRM Director Dr. Charles Murry demonstrated that stem cell-derived cardiomyocytes could be used to regenerate heart tissue in non-human primates by forming grafts of healthy heart muscle cells, preventing the scarring that inhibits heart functioning after heart attacks while promoting significant recovery. Rainbow reporter technology gives researchers like Murry new ways to better understand how cell therapy works – and how to improve it.
The technology used in the investigation was developed by the paper’s lead author, Danny El-Nachef, PhD, now a senior scientist as Sana Biotechnology. As a postdoc in the Davis and Sniadecki labs, El-Nachef engineered human stem cells embedded with color-coding genes found naturally in jellyfish, sea anemones, and coral. When activated, those genes turn on proteins that become fluorescent tracers, which in turn, make it possible to follow individual cells as they move and divide in laboratory environments. Just as importantly, the first generation of color-coded cells pass their unique fluorescence barcode on to their progeny, enabling lineage tracing of individual cells.
“There’s not a lot of tools out there to see if these cells are growing, dividing, and proliferating,” says Davis. “That’s why we built the lines of rainbow cells. We can inject a mixture of individually colored cells into a rat heart. If the cells didn’t proliferate, you would just see red, blue, and green all by itself. But if you see a cluster of red or a cluster of blue, that means those cells all came from the original single-colored cell. We saw conclusively that some percentage of the graft is due to proliferation.”
The experiments were conducted in rats and in laboratory dishes. Davis explains the advantages of both models. “We needed to know how these myocytes with the rainbow colors were going to behave in a highly controlled environment, which is the dish. But ultimately the point was to see if they proliferate in vivo, where the cues are different than they are in a dish, and which is more relevant to translational questions.”
Kevin Beussman, a PhD student in the Sniadecki Lab, and Darrian Bugg, a PhD student in the Davis Lab, performed much of the hands-on work in the study. Bugg conducted validation experiments in the rats after the rainbow cells were injected, including detailed histology and imaging. Beussman led the computational analysis effort, helping the team interpret the color patterns and determine the precise number of cells present.
The experiments were challenging, but rewarding, says Beussman. “Injecting a new cell line into a living tissue and hoping it will survive and engraft was a little nerve-wracking. It was exciting to see that some of the rainbow cells definitely proliferated in vivo. Going forward, it’ll be interesting to use techniques like spatial transcriptomics to understand what genetic factors might be responsible for that proliferation.”
Understanding how and why certain cells continue to grow and make new muscle in the heart and why others do not starts with the nature of cell identity. As a cell grows up to perform a specific function, like contracting, secreting hormones, or forming scars, it passes through various states of plasticity. Youthful cells have a greater proliferative potential, while more mature cells divide more slowly or not at all. Insights into the nature of cell proliferation could help researchers carefully control the process, improving outcomes for patients.
Sniadecki adds that the rainbow reporter tool described in the paper could also have implications for another challenge inherent in cell therapy: manufacturing. “Successfully growing a graft to heal a heart requires more than half a billion cells. That’s staggering from a manufacturing standpoint. It goes to reason the more we understand how our cells proliferate, the better we are going to be at growing them at the scale we need.”
While the rainbow reporter technology is designed to help researchers answer questions about the heart and other organs, it can also help pose new questions. “As a cardiac muscle biologist, I want to know what’s making those cells proliferate,” says Davis. “Is it something like genes being expressed inside the cell or is it something from the extra cellular environment that’s causing them to expand?”
“It opens a lot more questions about what’s contributing to the growth of the grafts,” says Sniadecki. “Is it just the cells themselves? Is it the environment that they’re in? I’m really curious about some of the questions that we can ask with this tool.”
This work was supported by National Institutes of Health grants HL141187 and HL142624 (Dr Davis), National Science Foundation grant CMMI-1661730 (Dr Sniadecki), National Institutes of Health grant F32HL143851 (Dr El-Nachef), National Institutes of Health grant T32AG066574 (D. Bugg), and a Gree Family Gift (Dr Davis, Dr Sniadecki, and Dr Murry). Dr Murry was also supported by National Institutes of Health grants R01HL128362, U54DK107979, R01HL128368, R01HL141570, and R01HL146868 and a grant from the Foundation Leducq Transatlantic Network of Excellence.