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Energy Metabolism in Human Pluripotent Stem and Differentiated Cells Compared Using a Seahorse XF96 Extracellular Flux Analyzer

Authors
Kim, Hyun KyuSong, YenaKye, MinjiYu, ByeonghoPark, Sang BeomKim, Ji HyeonMoon, Sung-HwanChoi, HyungkyuMoon, Jong-SeokOh, Jae SangLee, Man Ryul
Issue Date
Apr-2024
Publisher
Korean Society for Stem Cell Research
Keywords
Pluripotent stem cells; Glycolysis; Oxidative phosphorylation; Mitochondria; Glucose
Citation
International Journal of Stem Cells
Journal Title
International Journal of Stem Cells
URI
http://scholarworks.bwise.kr/kbri/handle/2023.sw.kbri/1176
DOI
10.15283/ijsc23167
ISSN
2005-3606
2005-5447
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.
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