Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations

Jennifer Turner; Lake-Ee Quek; Drew Titmarsh; Jens O Krömer; Li-Pin Kao; Lars Nielsen; Ernst Wolvetang; Justin Cooper-White

doi:10.1371/journal.pone.0112757

Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations

PLoS One. 2014 Nov 20;9(11):e112757. doi: 10.1371/journal.pone.0112757. eCollection 2014.

Authors

Jennifer Turner¹, Lake-Ee Quek², Drew Titmarsh¹, Jens O Krömer³, Li-Pin Kao⁴, Lars Nielsen³, Ernst Wolvetang⁴, Justin Cooper-White⁵

Affiliations

¹ Tissue Engineering and Microfluidics Laboratory, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia; Stem Cell Engineering Group, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia.
² Centre for Systems and Synthetic Biotechnology, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia.
³ Centre for Systems and Synthetic Biotechnology, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia; Metabolomics Australia, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia.
⁴ Stem Cell Engineering Group, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia.
⁵ Tissue Engineering and Microfluidics Laboratory, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia; School of Chemical Engineering, The University of Queensland, St Lucia, Queensland, Australia.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Count
Cell Culture Techniques / methods
Cell Hypoxia
Cell Proliferation
Cells, Cultured
Culture Media / metabolism
Gene Expression Regulation
Glycolysis
Human Embryonic Stem Cells / cytology
Human Embryonic Stem Cells / physiology*
Humans
Metabolome*
Metabolomics / methods*
Mitochondria / physiology
Oxidative Phosphorylation
Oxygen / metabolism*

Substances

Culture Media
Oxygen

Grants and funding

The authors would like to acknowledge the financial support of the Australian Research Council Special Research Initiative Stem Cells Australia (SR110001002), and the support of the Australian Stem Cell Centre's Core hESC Laboratories (Stem Core) for providing cell culture and support services. The authors would also like to acknowledge the Queensland Brain Institute Flow Cytometry Facility for assistance sorting cells. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.