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. 2022 Dec 10;11(24):4001.
doi: 10.3390/cells11244001.

Detection of De Novo Dividing Stem Cells In Situ through Double Nucleotide Analogue Labeling

Affiliations

Detection of De Novo Dividing Stem Cells In Situ through Double Nucleotide Analogue Labeling

Sheed Itaman et al. Cells. .

Abstract

Tissue-specific somatic stem cells are characterized by their ability to reside in a state of prolonged reversible cell cycle arrest, referred to as quiescence. Maintenance of a balance between cell quiescence and division is critical for tissue homeostasis at the cellular level and is dynamically regulated by numerous extrinsic and intrinsic factors. Analysis of the activation of quiescent stem cells has been challenging because of a lack of methods for direct detection of de novo dividing cells. Here, we present and experimentally verify a novel method based on double labeling with thymidine analogues to detect de novo dividing stem cells in situ. In a proof of concept for the method, we show that memantine, a drug widely used for Alzheimer's disease therapy and a known strong inducer of adult hippocampal neurogenesis, increases the recruitment into the division cycle of quiescent radial glia-like stem cells-primary precursors of the adult-born neurons in the hippocampus. Our method could be applied to assess the effects of aging, pathology, or drug treatments on the quiescent stem cells in stem cell compartments in developing and adult tissues.

Keywords: adult hippocampal neurogenesis; memantine; proliferation; radial glia-like stem cells; stem cell quiescence; thymidine analogues.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Labeling scheme for the unequivocal detection of de novo dividing stem cells. G1, S, G2 and M: phases of the cell cycle. G0: a state outside the cell cycle. t1: duration of cumulative labeling with the first nucleotide analogue. ∆t1–2: a time window between the termination of cumulative labeling with the first nucleotide analogue and pulse labeling with the second nucleotide analogue. ∆t1-A: a time window between pulse labeling with the second nucleotide analogue and the collection of samples for analysis. Details are in the main text.
Figure 2
Figure 2
Validation of the double labeling method for the detection of de novo dividing RGL stem cells in the hippocampal dentate gyrus in adult mice. (A) Scheme of double labeling for the detection of de novo dividing cells. (B) Representative image of the DG sample with a de novo dividing RGL stem cell (EdU+BrdU with the phenotype GFP+GFAP+process+) in its first S phase (arrow) (see also Figure S1). The image is an optical section captured with a confocal microscope. Arrowheads indicate an apical process of the de novo dividing RGL stem cell. Scale bars: 100 µm. (C) Total number of cells in S phase and the number of de novo dividing cells (cells that had incorporated EdU but not BrdU). Data are presented as mean ± SEM. (D) Phenotyping of de novo dividing cells indicated that most were RGL stem cells (Figure S2 and Table S1).
Figure 3
Figure 3
Memantine enhances proliferation in the DG of the adult brain via recruitment of quiescent RGL stem cells. (A) Scheme of the experiment, demonstrating delivery of two labels and treatment with memantine. Memantine significantly induces proliferation (B) and increases the numbers of proliferating RGLs (C) and de novo dividing cells (D) in the DG. (E) Phenotyping of EdU+BrdU cells, indicating that memantine significantly increases the numbers of de novo dividing bona fide (GFP+GFAP+process+) and presumptive (GFP+GFAP+process) RGLs. Quantitative data are presented as mean ± SEM. Statistics: vehicle n = 4, memantine n = 6; Mann–Whitney test, * p < 0.05, ** p < 0.01.

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