International Journal of Stem Cells

Korean Society for Stem Cell Research (한국줄기세포학회)
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Energy Metabolism in Human Pluripotent Stem and Differentiated Cells Compared Using a Seahorse XF96 Extracellular Flux Analyzer

  • Hyun Kyu Kim (Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University) ;
  • Yena Song (Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University) ;
  • Minji Kye (Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University) ;
  • Byeongho Yu (Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University) ;
  • Sang Beom Park (Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University) ;
  • Ji Hyeon Kim (Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University) ;
  • Sung-Hwan Moon (Department of Animal Science and Technology College of Biotechnology, Chung-Ang University) ;
  • Hyungkyu Choi (Department of Animal Science and Technology College of Biotechnology, Chung-Ang University) ;
  • Jong-Seok Moon (Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University) ;
  • Jae Sang Oh (Department of Neurosurgery, Uijeonbu St. Mary's Hospital, College of Medicine, The Catholic University of Korea) ;
  • Man Ryul Lee (Soonchunhyang Institute of Medi-bio Science (SIMS), Soon Chun Hyang University)
  • Received : 2023.10.17
  • Accepted : 2023.12.11
  • Published : 2024.05.30
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Abstract

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous, a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult, resulting in inaccuracies. In this study, we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts, human induced PSCs, and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time, with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2~4 and 2~3 hours after starvation, respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs, focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types, including undifferentiated PSCs, differentiated cells, and cells undergoing cellular reprogramming, and addresses critical issues, such as differences in basal metabolic levels and sensitivity to normalization, providing valuable insights into cellular energetics.

Keywords

Acknowledgement

We express our sincere gratitude to Davide Cacciarelli from Broad Institute for generously providing us with the hiF-T cell line used in this study for human reprogramming research. We would also like to thank the Soonchunhyang Biomedical Research Core Facility of the Korea Basic Science Institute for their help with microscopy. Special thanks go to STEMOPIA.

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