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Review
. 2021 Nov;297(5):101345.
doi: 10.1016/j.jbc.2021.101345. Epub 2021 Oct 28.

Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview

Georgy M Solius  1 , Dmitry I Maltsev  2 , Vsevolod V Belousov  3 , Oleg V Podgorny  4
Affiliations
Review

Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview

Georgy M Solius et al. J Biol Chem. 2021 Nov.

Abstract

Detection of thymidine analogues after their incorporation into replicating DNA represents a powerful tool for the study of cellular DNA synthesis, progression through the cell cycle, cell proliferation kinetics, chronology of cell division, and cell fate determination. Recent advances in the concurrent detection of multiple such analogues offer new avenues for the investigation of unknown features of these vital cellular processes. Combined with quantitative analysis, temporal discrimination of multiple labels enables elucidation of various aspects of stem cell life cycle in situ, such as division modes, differentiation, maintenance, and elimination. Data obtained from such experiments are critically important for creating descriptive models of tissue histogenesis and renewal in embryonic development and adult life. Despite the wide use of thymidine analogues in stem cell research, there are a number of caveats to consider for obtaining valid and reliable labeling results when marking replicating DNA with nucleotide analogues. Therefore, in this review, we describe critical points regarding dosage, delivery, and detection of nucleotide analogues in the context of single and multiple labeling, outline labeling schemes based on pulse-chase, cumulative and multilabel marking of replicating DNA for revealing stem cell proliferative behaviors, and determining cell cycle parameters, and discuss preconditions and pitfalls in conducting such experiments. The information presented in our review is important for rational design of experiments on tracking dividing stem cells by marking replicating DNA with thymidine analogues.

Keywords: BrdU (5-bromo-2′-deoxyuridine); EdU (5-ethynyl-2′-deoxyuridine); click chemistry; immunohistochemistry; proliferation; stem cells; thymidine analogues.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Key staining stages for the concurrent detection of three nucleotide analogues: CldU, IdU, and EdU. Here, the nonfluorogenic second click reaction is necessary for blocking nonspecific binding of anti-CldU and anti-IdU antibodies to residual EdU, which has not reacted with a fluorescent azide.
Figure 2
Figure 2
Major labeling schemes used to reveal the fate of dividing cells.A, pulse-chase labeling. B, window labeling. C, label retaining assay.
Figure 3
Figure 3
Labeling schemes for revealing progression through the cell cycle and determining S-phase duration and cell cycle length.A, cumulative labeling (adapted from Nowakowski et al. (101)). B, percent labeled mitoses method (adapted from Cai et al. (100)). C, double labeling (adapted from Encinas et al. (64)). G1, S, G2, and M, phases of the cell cycle; Tcell cycle, cell cycle duration; TS-phase, S-phase duration; Δt, an intercept between the linear regression line of the slope and the plateau, Δt = Tcell cycle−TS-phase; y0, an intercept between the y-axis and the continuation of the slope, y0 = TS-phase/Tcell cycle.
Figure 4
Figure 4
Labeling schemes for the discrimination between proliferating and quiescent subpopulations of daughter cells.A, double labeling in separate experimental groups (adapted from Takahashi et al. (16, 117)). B, triple labeling in the same experimental group (adapted from Podgorny et al. (41)). G1, S, G2, and M, phases of the cell cycle; P, proliferative fraction; Q, quiescent fraction; t, the time interval during which the second label is repeatedly delivered; t3, the time point when the third label is delivered; Tcell cycle, cell cycle length; TS-phase, S-phase duration; Δt1−2, a time interval between the first and the second label pulses.
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