Quantifying Senescence-Associated Phenotypes in Primary Multipotent Mesenchymal Stromal Cell Cultures

  • Stéphanie Nadeau
  • Anastasia Cheng
  • Inés Colmegna
  • Francis Rodier
Part of the Methods in Molecular Biology book series (MIMB, volume 2045)


Cellular senescence is a tumor suppressor mechanism that removes potentially neoplastic cells from the proliferative pool. Senescent cells naturally accumulate with advancing age; however, excessive/aberrant accumulation of senescent cells can disrupt normal tissue function. Multipotent mesenchymal stromal cells (MSCs), which are actively evaluated as cell-based therapy, can undergo replicative senescence or stress-induced premature senescence. The molecular characterization of MSCs senescence can be useful not only for understanding the clinical correlations between MSCs biology and human age or age-related diseases but also for identifying competent MSCs for therapeutic applications. Because MSCs are involved in regulating the hematopoietic stem cell niche, and MSCs dysfunction has been implicated in age-related diseases, the identification and selective removal of senescent MSC may represent a potential therapeutic target. Cellular senescence is generally defined by senescence-associated (SA) permanent proliferation arrest (SAPA) accompanied by persistent DNA damage response (DDR) signaling emanating from persistent DNA lesions including damaged telomeres. Alongside SA cell cycle arrest and DDR signaling, a plethora of phenotypic hallmarks help define the overall senescent phenotype including a potent SA secretory phenotype (SASP) with many microenvironmental functions. Due to the complexity of the senescence phenotype, no single hallmark is alone capable of identifying senescent MSCs. This protocol highlights strategies to validate MSCs senescence through the measurements of several key SA hallmarks including lysosomal SA Beta-galactosidase activity (SA-βgal), cell cycle arrest, persistent DDR signaling, and the inflammatory SASP.


Senescence Stem cells Immunofluorescence Senescence-associated beta-galactosidase Sandwich enzyme-linked immunosorbent assay Senescence-associated secretory phenotype DNA damage foci Mesenchymal stromal stem cell Multipotent mesenchymal stromal cell 



We thank Dr. Rodier and Dr. Colmegna’s laboratory members for their valuable comments and discussions. This effort was supported by the Institut du cancer de Montréal (ICM to FR), the Israel Cancer Research Foundation (ICRF to FR), and the Canadian Institute for Health Research (CIHR MOP114962 to FR and MOP287233 to IC/FR). FR is a researcher at CRCHUM/ICM, which receives support from the Fonds de recherche du Québec – Santé (FRQS). FR is supported by a FRQS junior I–II career awards (22624, 33070). IC is a Chercheur Boursier Senior from FRQS. SN is supported by a FRQS PhD scholarship and a Canderel-ICM excellence award.


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Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  • Stéphanie Nadeau
    • 1
  • Anastasia Cheng
    • 2
  • Inés Colmegna
    • 2
    • 3
  • Francis Rodier
    • 1
    • 4
  1. 1.CRCHUM et Institut du cancer de MontréalMontrealCanada
  2. 2.Research Institute of the McGill University Health Centre (MUHC)MontrealCanada
  3. 3.Division of Rheumatology, Department of MedicineMcGill UniversityMontrealCanada
  4. 4.Université de Montréal, Département de Radiologie, Radio-Oncologie et Médicine NucléaireMontrealCanada

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