title: Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations.
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.
authors.affiliation: 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.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Turner
authors.affiliation: Centre for Systems and Synthetic Biotechnology, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Lake-Ee
authors.initials: LE
authors.lastname: Quek
authors.affiliation: 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.
authors.forename: Drew
authors.initials: D
authors.lastname: Titmarsh
authors.affiliation: 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.
authors.forename: Jens O
authors.initials: JO
authors.lastname: Krömer
authors.affiliation: Stem Cell Engineering Group, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Li-Pin
authors.initials: LP
authors.lastname: Kao
authors.affiliation: 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.
authors.forename: Lars
authors.initials: L
authors.lastname: Nielsen
authors.affiliation: Stem Cell Engineering Group, The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Ernst
authors.initials: E
authors.lastname: Wolvetang
authors.affiliation: 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.
authors.forename: Justin
authors.initials: J
authors.lastname: Cooper-White
mesh_terms: D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism; D005786:Gene Expression Regulation; D006019:Glycolysis; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D008928:Mitochondria / Q000502:physiology; D010085:Oxidative Phosphorylation; D010100:Oxygen / Q000378:metabolism*
chemical_list: D003470:Culture Media; D010100:Oxygen