Title of Research:
22-1-04

Establishment of Human iPSC Reporter-Based Developmental
Toxicity Assay that Detects FGF Signal Disruption
Principal Investigator:
Junji Fukuda (PhD, Professor, Yokohama National University, Faculty of Engineering)79-5 Tokiwadai, Hodogaya Ward, Yokohama, Kanagawa 240-8501, Japan
Collaborators:
Yusuke Okubo (PhD, Principle Investigator, National Institute of Health Sciences)
3-25-26 Tono-machi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan
Yoshihiro Nakajima (PhD, Principle Investigator, National Institute of Advanced Industrial Scienceand Technology)2217-14 Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan
Summary of Research:
In this study, a real-time luminescence measurement system was introduced to improve theaccuracy of the kinetic assay, which has been a hallmark of the signal disruption assay. Although
this system enabled continuous measurements over a 3-day period and captured more detailed
changes in developmental toxicity than conventional manual measurements, it also raised severalissues. The measurement system revealed the edge effect of the culture plate and experimental
errors in each run. Therefore, a protocol to reduce these problems was investigated in detail andreconstructed in this study. This includes optimization of the pre-culture schedule, reduction ofevaporation of culture medium, and concentration of luminescent substrates. In the Wnt signal
disruption test, we prepared Wnt signal reporter cells and examined for the signal disruption assay.
In addition, we tested Wnt ligands to FGF/SRF signal reporter cells. In particular, the latter showedthat intracellular signaling network links may be responsible for the high probability of teratogendetection in this assay, suggesting that the number of signaling pathways in the future battery
assay could be reduced.
Timeline: March 1, 2023 –Topics:
Y. Okubo and J. Fukuda, Evaluation of developmental toxicity analyzing by dynamics ofsignal disruption, Seikagaku Mini Review, 95, 2, 1-6, 2023, Doi: 10.14952/SEIKAGAKU.2023.95
Presentation Award, K. Mizota, Y. Okubo, M. Shibata, R. Ohara, Y. Nakajima, J. Fukuda:
Developmental toxicity testing of chemicals based on long-term signal disruption using human iPS
reporter cells, 61st Japanese Society for Artificial Organs, Nov. 9-11, Tokyo
Publications:
Invited talk. Y. Okubo: Developmental toxicity detection via dynamics of FGF-SRF signal
disruption in human iPSC-based assay. 13TH Global Summit on Regulatory Science (GSRS23)
In-Person Annual conference．Sep. 27, 2023.
Y. Okubo, K. Mizota, M. Shibata, R. Ohara, S. Kitajima, Y. Hirabayashi, Y. Nakajima, J. Fukuda:
Developmental toxicity test using human iPS cells based on signal disruptions induced bychemical substances. 12th World Congress on Alternatives and Animal Use in the Life Sciences(WC12), Niagara Falls, Canada. (Aug. 30, 2023). Oral presentation.
K. Mizota, Y. Okubo, M. Shibata, R. Ohara, S. Kitajima, Y. Hirabayashi, Y. Nakajima, J. Fukuda:
Developmental toxicity testing of chemicals based on long-term signal disruption using human iPS
reporter cells. 12th World Congress on Alternatives and Animal Use in the Life Sciences (WC12),
Niagara Falls, Canada. (Aug. 30, 2023). Poster presentation.