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Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations

As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirem...

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Autores principales: Turner, Jennifer, Quek, Lake-Ee, Titmarsh, Drew, Krömer, Jens O., Kao, Li-Pin, Nielsen, Lars, Wolvetang, Ernst, Cooper-White, Justin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239018/
https://www.ncbi.nlm.nih.gov/pubmed/25412279
http://dx.doi.org/10.1371/journal.pone.0112757
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author Turner, Jennifer
Quek, Lake-Ee
Titmarsh, Drew
Krömer, Jens O.
Kao, Li-Pin
Nielsen, Lars
Wolvetang, Ernst
Cooper-White, Justin
author_facet Turner, Jennifer
Quek, Lake-Ee
Titmarsh, Drew
Krömer, Jens O.
Kao, Li-Pin
Nielsen, Lars
Wolvetang, Ernst
Cooper-White, Justin
author_sort Turner, Jennifer
collection PubMed
description As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion.
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spelling pubmed-42390182014-11-26 Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations Turner, Jennifer Quek, Lake-Ee Titmarsh, Drew Krömer, Jens O. Kao, Li-Pin Nielsen, Lars Wolvetang, Ernst Cooper-White, Justin PLoS One Research Article As human embryonic stem cells (hESCs) steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic) and atmospheric (normoxic) oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that can be used in rational bioreactor design and in the development of novel culture media for hESC maintenance and expansion. Public Library of Science 2014-11-20 /pmc/articles/PMC4239018/ /pubmed/25412279 http://dx.doi.org/10.1371/journal.pone.0112757 Text en © 2014 Turner et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Turner, Jennifer
Quek, Lake-Ee
Titmarsh, Drew
Krömer, Jens O.
Kao, Li-Pin
Nielsen, Lars
Wolvetang, Ernst
Cooper-White, Justin
Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations
title Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations
title_full Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations
title_fullStr Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations
title_full_unstemmed Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations
title_short Metabolic Profiling and Flux Analysis of MEL-2 Human Embryonic Stem Cells during Exponential Growth at Physiological and Atmospheric Oxygen Concentrations
title_sort metabolic profiling and flux analysis of mel-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239018/
https://www.ncbi.nlm.nih.gov/pubmed/25412279
http://dx.doi.org/10.1371/journal.pone.0112757
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