Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-13C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained 13C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis (13C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.
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Support from iNOVA4Health - UID/Multi/04462/2013, a program funded by Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Educação e Ciência and co-funded by FEDER under the PT2020 Partnership Agreement, is acknowledged. This research has also received support from FCT through the project MITP-TB/ECE/0013/2013, from the German research foundation (RTG1331, KoRS-CB) and the German ministry for science (BMBF-DynaMeTox). JV Sá is a recipient of a Ph.D. fellowship from FCT (PD/BD/52474/2014). The expert technical assistance of Lars Evje with GC-MS is gratefully acknowledged. We are also thankful to Nuno Carinhas for his help on fluxome analysis.
S.I. : In honor of Dr. Mary McKenna.
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Sá, J.V., Kleiderman, S., Brito, C. et al. Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis. Neurochem Res 42, 244–253 (2017). https://doi.org/10.1007/s11064-016-1907-z
- Neural stem cells
- Astrocytic differentiation
- Metabolic flux analysis
- Carbon labelling cultures