Immunophenotype of replicative senescent mesenchymal stromal cells

A.Yu. Ratushnyy

SSC RF – Institute of Biomedical Problems, Russian Academy of Sciences,
Moscow, 123007 Russia

E-mail: ratushkin@mail.ru

Received October 5, 2022; Accepted November 21, 2022

ORIGINAL ARTICLE

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DOI: 10.26907/2542-064X.2023.2.216-230

For citation: Ratushnyy A.Yu. Immunophenotype of replicative senescent mesenchymal stromal cells. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2023, vol. 165, no. 2, pp. 216–230. doi: 10.26907/2542-064X.2023.2.216-230. (In Russian)

Abstract

Mesenchymal stromal cells (MSCs), a population of progenitor cells in adult tissues, are involved in the processes of physiological tissue renewal and damage-induced regeneration. MSCs have been widely studied as regenerative medicine agents. In this regard, the tissue-specific features of MSC subpopulations should be taken into account. MSCs have many potentially beneficial properties that can alter significantly with age. The ability of a cell to respond to external signals and regulate its functional state is commonly attributed to the repertoire of receptors on the cell membrane. This article considers the surface marker expression of senescent adipose-derived MSCs (AD-MSCs). Replicative senescence was caused by long-term cultivation. An increase in the expression of CD29, CD44, CD54, CD73, CD90, and HLA-ABC on the AD-MSCs was shown. The expression of CD105 and CD51/61 did not change reliably under the experimental conditions. The revealed effects are related not only to the larger cell size or higher autofluorescence, but also to the increased number of markers per unit area of the cell surface. The detected changes may underlie a number of modifications in the properties of senescent MSCs, including migration, adhesion, and immunomodulatory and angiogenic activities.

Keywords: mesenchymal stromal cells (MSCs), cell senescence, immunophenotype, flow cytometry

Acknowledgements. This study was supported by the Russian Science Foundation (project no. 21-75-10117).

Figure Captions

Fig. 1. Signs of MSC senescence and surface marker expression in long-term cultivation: a) – senescence-associated active β-galactosidase (SA-β-gal) in MSCs at early (P5) and late (P21) passages detected by light microscopy; b) – distribution of MSCs by size (FSC-A) and granularity (SSC-A) based on flow cytometry data. The results of a representative experiment are shown; c) – mean fluorescence intensity (MFI), which is associated with antigen expression on the MSC surface, at early (P2–6) and late (P21–23) passages. All data are reported as the mean value ± standard deviation, n ≥ 5, * – p ≤ 0.05.

Fig. 2. Expression of surface markers on MSCs in long-term cultivation at different passages: P5 and P10, as well as P21 (with confirmed senescence). The results of a representative experiment are shown.

Fig. 3. Size gating of MSCs at different passages (gate R3). In this approach, cells outside the specified size range, i.e., either larger or smaller, are excluded from further analysis. Flow cytometry, the results of a representative experiment.

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