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.
Highlights
The pluripotent nature of human embryonic stem cells along with their capacity for unlimited self-renewal makes them ideal candidates for use in regenerative medicine
The second stage utilised the experimental data to develop a fluxomic model of human embryonic stem cells (hESCs) metabolism capable of resolving fluxes through internal metabolic pathways
Human embryonic stem cells are considered to be highly metabolically active with a highly glycolytic nature [14,15,16]. They are known to have fewer mitochondria than terminally differentiated cells [18] and possess mitochondria that appear immature and lack normal cristae [16,19]. Together this has led to the preposition that the mitochondria are more inactive in hESCs than in differentiated cells and that energy generation by oxidative phosphorylation (OXPHOS) in the mitochondria is reduced in hESCs [16]
Summary
The pluripotent nature of human embryonic stem cells (hESCs) along with their capacity for unlimited self-renewal makes them ideal candidates for use in regenerative medicine. Before this potential can truly be realised expansion of hESCs to clinically relevant numbers must be based on a more detailed understanding of their metabolic and growth characteristics compared with workhorse lines such as CHO cells [1]. There has been very little exploration into the fundamental metabolic requirements necessary to support cell expansion in a pluripotent state. Such data would enable the rational design of hESC expansion systems. This work, which describes an in-depth study of hESC metabolism during the exponential growth phase, addresses this deficit
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