Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21

Abstract

Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton’s jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

P16 and P21 are involved in the pathogenesis of endometrial thinning: A cross-sectional study

“…Previous findings showed that P16 and P21 jointly regulate fibroblastic growth. [ 29 ] However, our results showed no correlation between P16 and P21 in either group. Further studies are required to explore the underlying mechanism.…”

Cardiac Mesenchymal Stem Cell-like Cells Derived from a Young Patient with Bicuspid Aortic Valve Disease Have a Prematurely Aged Phenotype

“…We observed NANOG expression in both CMSCLC populations; however, there was decreased expression in terms of cell numbers in BAVD-CMSCLC. Furthermore, p16 an inhibitor of cell cycle progression most commonly associated with cell senescence including, tissue resident MSC and cardiomyocytes, was upregulated in BAVD-CMSCLC compared to CAD-CMSCLC [ 30 , 51 , 52 ]. While p16 is a robust marker of human senescence in vivo [ 40 ], the senescent phenotype can be heterogeneric and dependent on cell type and stimuli as such a deeper analysis of senescence-associated gene expression would provide insight into the signature of senescent CMSCLCs [ 53 ].…”

Subcellular structure, heterogeneity, and plasticity of senescent cells

“…These variables are particularly significant because the majority of studies use senescence-enriched cellular populations (SEP), rather than examining a population containing only SnCs (González-Gualda et al, 2021;Jeyapalan & Sedivy, 2013;Liu et al, 2019;Menegotto et al, 2017;Silva et al, 2020) (Figure 3f). To overcome this barrier, techniques designed to isolate SnCs are essential, like cell sorting, scRNA-seq, tissue microdissection, or immunocytochemistry (Bertolo et al, 2020;Gruber et al, 2010;Magkouta et al, 2023;Wallis et al, 2022;Wang et al, 2016).…”

“…Furthermore, different cell types or subpopulations of SnCs can contribute to average levels of senescence markers in a population (Jeyapalan & Sedivy, 2013 ; Sturmlechner et al., 2022 ; Troiani et al., 2022 ), as well as non‐SnCs can also contribute with features attributed to SnCs (Gorgoulis et al., 2019 ), which is especially important in studies using primary cells from human or animal samples (Bahar et al., 2006 ; Delfarah et al., 2021 ; Tuttle et al., 2021 ). These variables are particularly significant because the majority of studies use senescence‐enriched cellular populations (SEP), rather than examining a population containing only SnCs (González‐Gualda et al., 2021 ; Jeyapalan & Sedivy, 2013 ; Liu et al., 2019 ; Menegotto et al., 2017 ; Silva et al., 2020 ) (Figure 3f ). To overcome this barrier, techniques designed to isolate SnCs are essential, like cell sorting, scRNA‐seq, tissue microdissection, or immunocytochemistry (Bertolo et al., 2020 ; Gruber et al., 2010 ; Magkouta et al., 2023 ; Wallis et al., 2022 ; Wang et al., 2016 ).…”