A Reversible Neural Stem Cell Quiescence and Activation Culture System for Metabolic Study

Abstract

Stem cells in vivo can transit between quiescence and activation, two metabolically distinct states. It is increasingly appreciated that cell metabolism assumes profound roles in stem cell maintenance and tissue homeostasis. However, the lack of suitable models greatly hinders our understanding of the metabolic control of stem cell quiescence and activation. In the present study, we have utilized classical signaling pathways and developed a cell culture system to model reversible NSC quiescence and activation. Unlike activated ones, quiescent NSCs manifested distinct morphology characteristics, cell proliferation, and cell cycle properties but retained the same cell proliferation and differentiation potentials once reactivated. Further transcriptomic analysis revealed that extensive metabolic differences existed between quiescent and activated NSCs. Subsequent experimentations confirmed that NSC quiescence and activation transition was accompanied by a dramatic yet coordinated and dynamic shift in RNA metabolism, protein synthesis, and mitochondrial and autophagy activity. The present work not only showcases the broad utilities of this powerful in vitro NSC quiescence and activation culture system but also provides timely insights for the field and warrants further investigations.

Keywords: activation; cell cycle; cell metabolism; neural stem cell; quiescence.

Figure 1.

Establishment of a quiescent NSC culture system. (A) Morphological distinctions of activated and quiescent HCN cells. (B) Flow cytometry cell cycle analysis of activated and quiescent HCN cell cultures. (C) Ki67 and BrdU staining of activated and quiescent HCN cell cultures. (D) Quantitative flow cytometry cell division speed analysis of activated and quiescent HCN cell cultures over time with Celltrace Violet staining. (E and F) Quantitative flow cytometry cell quiescence analysis of activated and quiescent HCN cell cultures over time with Pyronin Y/Hoechst staining to distinguish G0 from G1. P-values are denoted as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.

Reversible NSC quiescence and activation transition in vitro. (A and B) Representative images and quantification of Ki67/BrdU positive cells in activated and reactivated HCN cell culture over time (n = 3). (C and D) Quantification of proliferation rates and G0 in activated (A), quiescent (Q), and reactivated (R) with Ki67 immunostaining (n = 3) and with Pyronin Y/Hoechst staining (n = 3). All data are presented as the mean ± SEM values. (E) Assessment of cell-differentiation capability to neurons, oligodendrocytes, and astrocytes (Tuj1: red, RIP: green, GFAP: red). P-values are denoted as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Transcriptomic profiling of quiescent and activated NSCs. (A) Circular heatmap illustrating differentially regulated gene expression from RNA-seq analysis between activated and quiescent NSCs. Gene ontology (GO) enrichment analysis of genes upregulated and downregulated in these two states of NSCs (P < 0.05). Genes upregulated and downregulated are shown in red and green, respectively. Values are presented as the log2 of tag counts. (B) Heatmap of representative DNA replication and cell cycle signaling genes in activated and quiescent HCN cells. (C) Heatmap of BMP, Notch, bFGF, and Wnt signaling genes in activated and quiescent HCN cells.

Figure 4.

Low metabolic activity and high stress resistance with quiescent NSCs. (A) Organelle (Golgi apparatus, lysosome, mitochondria)-specific fluorescent staining of activated and quiescent HCN cells. (B) Representative images of activated and quiescent HCN cells cultured with different concentrations of glucose (0 mM, 0.5 mM, 1.0 mM, 2.5 mM, 10 mM, 25 mM, and the standard high glucose control) over time.

Figure 5.

Global anabolic and catabolic differences between quiescent and activated NSCs. (A) Expression of representative genes related to RNA metabolism, protein synthesis, and mitochondrial and autophagy activity. (B) Comparison of RNA metabolism dynamics between activated and quiescent HCN cell cultures shown by BrUTP staining and its quantification (n = 5). (C) Comparison of protein synthesis dynamics between activated and quiescent HCN cell cultures shown by OPP incorporation and its quantification (activated, n = 53; quiescent, n = 50). (D) Mitochondrial morphological distinctions between activated and quiescent HCN cells revealed by transmission electron microscopy. (E) Comparison of autophagy dynamics between activated and quiescent HCN cell cultures shown by the Adv-mRFP-GFP-LC3 reporter (activated, n = 18; quiescent, n = 32). P-values are denoted as follows: *P < 0.05; **P < 0.01; ***P < 0.001.