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November 13, 2020

Heart cells made pure without purification

The Yamashita lab reports a new protocol that produces relatively mature cardiomyocytes from iPS cells without a key purification step used in standard protocols.

Researchers at CiRA report a new protocol to generate ventricular cardiomyocytes from human iPS cells. The protocol eliminates the final purification step, significantly maintaining the quality of the cell production. These ventricular cardiomyocytes showed a morphology resembling that of real hearts and, more importantly, responded to drugs with higher fidelity. This new protocol is expected to improve the quality of cardiomyocytes used for research for cell therapies and drug discovery of the heart.

Many experimental drugs will fail not because they do not have a positive effect against the disease, but because they have a toxic effect on an organ, particularly the heart. Even approved drugs like those used for cancer are known to cause arrhythmias. Before a drug is approved for patient risk, cardiotoxic risk is tested on human cardiomyocytes. Since patients are not eager to donate their hearts for these experiments, scientists have looked to other ways to procure cardiomyocytes, such as differentiating iPS cells.

When manufacturing cardiomyocytes from iPS cells, scientists try to follow the natural development of the heart. The iPS cells are stimulated to pass through several embryonic stages. However, the process is not perfect, and some iPS cells will deviate into non-cardiomyocyte lineages, resulting in a heterogenous cell population. Therefore, before the toxicology studies, the cardiomyocytes must be purified.

"The purification process leads to a huge loss of cells, and cells are injured during the process," explained Dr. Miki Yoshioka, one of the researchers involved in the study.

The method proposed in the new study does not dismiss purification outright. Cells representing the paraxial mesoderm, an embryonic stage long gone before birth, are purified by using magnetic activated cell sorting (MACS). Even if some of these cells are damaged, unlike cardiomyocytes, paraxial mesoderm cells can be recovered and then differentiated to cardiomyocytes. Once paraxial mesoderm cells are obtained, the protocol requires no more sorting to acquire cardiomyocytes, which process reduces cardiomyocyte viability and quality.

To promote the differentiation from paraxial mesoderm cells to cardiomyocytes, the protocol adds two inhibitors of canonical Wnt signaling after the MACS, improving the cardiomyocyte yield by a factor of 25. The result was pure cardiomyocytes that could be maintained for a long time (more than 200 days), leading to mature morphology and electrophysiology.

Maturity is an important factor when evaluating drug effects. While iPS cells have been used to produce cardiomyocytes in the past, they are usually immature, resembling those in a fetus. Cardiomyocytes in a patient, however, behave quite differently.

According to Dr. Victor López-Dávila, another scientist who contributed to the work, the maturity was because the highly pure cardiomyocytes could be maintained for more than 200 days.

"To mature the cells, we have to culture them for a long time. However, cardiomyocytes do not expand well, but contaminating cells do. So, unless we have high purity, the cardiomyocytes will be replaced with contaminating cells over time," he said.

To confirm the quality of the cardiomyocytes, the researchers tested known drugs on them, finding the expected changes in their electrophysiology.

Following previous work from his group on other cell types, CiRA Prof. Jun K. Yamashita, who led the study, believes the new system will be attractive to research groups developing new drugs or cell therapies for the heart.

"The long-term purity of induced cells is important for both disease modeling and after transplantation. Eliminating unrelated cells at an earlier stage and potently stimulating differentiation would be a good way to obtain a higher quality cell population from pluripotent stem cells" he said.

Paper Details
  • Journal: PLOS ONE
  • Title: Specific induction and long-term maintenance of high purity ventricular cardiomyocytes from human induced pluripotent stem cells
  • Authors: Hiroyuki Fukushima1, Miki Yoshioka1, Masahide Kawatou1,2,3, Victor López-Dávila1, Masafumi Takeda1,3, Yasunari Kanda4, Yuko Sekino4, Yoshnori Yoshida1 and Jun K. Yamashita1
  • Author Affiliations:
    1. Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
    2. Department of Cardiovascular Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
    3. Institute for Advancement of Clinical and Translation Medicine (iACT), Kyoto University Hospital, Kyoto, Japan
    4. Division of Pharmacology, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan
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