Metabolism of Stem and Progenitor Cells: Proper Methods to Answer Specific Questions

Abstract

Stem cells can stay quiescent for a long period of time or proliferate and differentiate into multiple lineages. The activity of stage-specific metabolic programs allows stem cells to best adapt their functions in different microenvironments. Specific cellular phenotypes can be, therefore, defined by precise metabolic signatures. Notably, not only cellular metabolism describes a defined cellular phenotype, but experimental evidence now clearly indicate that also rewiring cells towards a particular cellular metabolism can drive their cellular phenotype and function accordingly. Cellular metabolism can be studied by both targeted and untargeted approaches. Targeted analyses focus on a subset of identified metabolites and on their metabolic fluxes. In addition, the overall assessment of the oxygen consumption rate (OCR) gives a measure of the overall cellular oxidative metabolism and mitochondrial function. Untargeted approach provides a large-scale identification and quantification of the whole metabolome with the aim to describe a metabolic fingerprinting. In this review article, we overview the methodologies currently available for the study of in vitro stem cell metabolism, including metabolic fluxes, fingerprint analyses, and single-cell metabolomics. Moreover, we summarize available approaches for the study of in vivo stem cell metabolism. For all of the described methods, we highlight their specificities and limitations. In addition, we discuss practical concerns about the most threatening steps, including metabolic quenching, sample preparation and extraction. A better knowledge of the precise metabolic signature defining specific cell population is instrumental to the design of novel therapeutic strategies able to drive undifferentiated stem cells towards a selective and valuable cellular phenotype.

Keywords: cell metabolism; cell reprogramming; indiced Pluripotent Stem Cells (iPSCs); metabolic flux analysis (MFA); metabolomics (OMICS); neural stem cell (NSC); pharmacology; stem cell.

Figure 1

Schematic representation of the different experimental approaches for the study of the metabolism of stem and progenitor cells. In the left panel are depicted the untargeted metabolic analysis, metabolic fingerprint (upper left) and single-cell metabolomics (lower left). The study of the whole metabolite content (untargeted metabolic analysis) of the samples can be performed by using mass spectrometry (MS) and nuclear magnetic resonance (NMR). In the right panel are figured the targeted metabolic approaches, metabolic flux analysis (upper right) and in vivo cell metabolism (lower right), which focus on the study of metabolites possibly involved in a specific pathway. Several techniques can be applied to study targeted metabolic analysis, including MS, NMR, magnetic resonance imaging (MRI), magnetic resonance spectrometry (MRS), positron emission tomography (PET), single photon emission tomography (SPECT), oxygen consumption rate (OCR) and Biosensor.

Figure 2

Graphic summary of recommended quenching workflows for SCs. Schematic representation of the quenching methods for cell in suspension by fast filtration adapted from Bennett et al. (2008) (A) and by cold quenching prior to sample collection as described by Sellick et al. (2009) (B). In Panel (C) is outlined the quenching methods for adherent SCs by culturing cells on glass coverslips as described by Martano et al. (2015).

Figure 3

Summary of selected metabolic pathways detectable non-invasively with radiolabeled compounds applicable with PET imaging in clinical practice. Abbreviations: Cu-ATSM, Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone; F-MISO, fluoromisonidazole; FAZA, Fluoroazomycin Arabinoside; MAT, monoamine transporter; MET, methionine; FET, fluoroethyltyrosine; LAT, large amino acid transporter; F-DOPA, fluoro-3,4-dihydroxyphenylalanine; AA-pool, amino acid pool; FLT, Fluorothymidine; NTs, nucleoside transporter families; TK1, thymidine kinase-1; TMP, thymidine monophosphate; CHO, choline; CHT, choline transporter; ChoK, choline kinase; P-choline, phosphocholine; ACE, acetate; MCT, monocarboxylate transporter; FDG, fluorodeoxyglucose; GLUT, glucose transporter; HK, hexokinase; FDG-6-P, fluorodeoxyglucose-6-phosphatase.