Int J Stem Cells.  2024 May;17(2):194-203. 10.15283/ijsc23167.

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

• Kim HK
• Song Y
• Kye M
• Yu B
• Park SB
• Kim JH
• Moon SH
• Choi H
• Moon JS
• Oh JS
• Lee MR

• KMID: 2556237
• DOI: http://doi.org/10.15283/ijsc23167

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.

Keyword

Pluripotent stem cells; Glycolysis; Oxidative phosphorylation; Mitochondria; Glucose

Figure

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  Fig. 1 Optimization of cell number is essential for the Seahorse cell metabolic assay. (A) Standard curves of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) according to cell number. (B) The appropriate number of cells is represented by a constant OCR/ECAR ratio. iPSC: induced pluripotent stem cell.

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  Fig. 2 Determination of the appropriate starvation period. (A) Oxygen consumption rate (OCR) response to glucose after glucose starvation. (B) Extracellular acidification rate (ECAR) response to glucose after glucose starvation. (C) Changes in OCR and ECAR responses to glucose in different cell types after glucose starvation. Student’s t-test: *p<0.05. iPSCs: induced pluripotent stem cells, hESCs: human embryonic stem cells.

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  Fig. 3 Linear regression of total protein concentration and cellular metabolism according to cell number. (A) Differences in size between fibroblasts (MRC5 and hiF-T) and pluripotent stem cells (TRA-1-60 ＋ and human embryonic stem cells [hESCs]) (hiF-T-derived TRA-1-60 ＋ and hESCs. (B) Linear regression of protein concentration according to cell type. (C) Linear regression of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) according to cell number.

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  Fig. 4 Differences in the measurement of metabolic levels according to normalization method. (A) Real-time adenosine triphosphate (ATP) rate assay of somatic and pluripotent stem cells. (B) Non-normalized ATP production rate (pmol/min). (C) Normalized ATP production rate (pmol/min) by total protein, oxygen consumption rate (OCR), and extracellular acidification rate (ECAR). Student’s t-test: *p<0.05. R/A: rotenone & antimycin A, hESCs: human embryonic stem cells.

Reference

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