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. 2020 Feb 21;10(2):340. doi: 10.3390/biom10020340

Table 1.

Markers of MSC senescence and techniques most frequently used for their detection.

Senescence Marker Techniques for Detection Senescent Features, Pros and Cons
Cell morphology graphic file with name biomolecules-10-00340-i001.jpg graphic file with name biomolecules-10-00340-i002.jpg Microscopy [185]
Flow cytometry [186]
(FSC for size, SSC for granularity)		 Senescent MSCs show enlarged and granular cell morphology.
Microscopic assessment is easy but only qualitative.
Flow cytometric assessment is quantitative
CFU graphic file with name biomolecules-10-00340-i003.jpg Colony formation
in agar culture [187] 		The CFU number is a measure of cell clonogeneity and decreases with MSC age. CFU assessment requires careful plating at low density.
Sa-β-gal graphic file with name biomolecules-10-00340-i004.jpg Microscopy (colorimetric activity assay with X-gal) [188]
Flow cytometry (fluorimetric activity assay with C12FDG) [189]
IHC, IF or WB with specific Abs (protein expression) [190]		 Senescent cells at low density express a lysosomal β-galactosidase active at pH 6.0, detectable either with activity assays or with a specific antibody.
The activity assays can give altered results on high density cultures [191]
8-oxo-dG graphic file with name biomolecules-10-00340-i005.jpg IHC, IF, ELISA [192],
HPLC [192]-MS/MS		 8-oxodG is a DNA base derivative, robust marker of oxidative DNA and RNA damage
γH2AX Inline graphic Inline graphic Inline graphic IF
Flow cytometry [193]
WB		 Histone H2AX phosphorylation is an indirect measure of DNA double strand breaks due to physical, chemical, oxidative stress.
It indicates that cells organize a DNA damage response, but its persistence sustains senescence of the cells.
Telomeres Inline graphic Inline graphic Southern blotting [170]
Flow FISH [194]
Real-time PCR [194]
STELA [195]		 Telomere attrition is directly correlated to replicative senescence, but it also occurs after exposure to oxidative damage. The subpopulation heterogeneity must be taken into account and may be addressed with emerging techniques such as STELA, detecting individual telomere length.
MSI graphic file with name biomolecules-10-00340-i011.jpg PCR followed by gel
or capillary
electrophoresis [196]		 Repeated sequences variations are an indirect indication of genomic instability and deficient DNA repair due to replicative or oxidative stress. They increase with cell aging.
Gene expression
of senescent
markers at
mRNA level		 graphic file with name biomolecules-10-00340-i012.jpg Real-time RT-PCR [33]
Microarray
RNAseq		 Expression of genes related to senescence. Several pathways can be analyzed, but gene expression analysis prevalently focuses on p53 and cyclin dependent kinase inhibitors (p16 and p21)
Expression
of senescent
markers at
protein level		 Inline graphic Inline graphic WB [197]
IHC
IF
Flow cytometry		 Evaluation of the expression levels of proteins related to senescence (p16, p21, p53, etc.)
Global methylation graphic file with name biomolecules-10-00340-i015.jpg NGS after bisulfite treatment [198] Genome wide analysis of methylated cytosines.

FSC: forward scatter; SSC: side scatter; CFU: colony forming unit; X-gal: galactosidase substrate; C12FDG: fluorogenic galactosidase substrate; IHC: immunohistochemistry; IF: immunofluorescence; WB: western blotting; ELISA: enzyme-linked immunosorbent assay; HPLC-MS: high performance liquid chromatography-mass spectrometry; 8-oxo-gG: 8-Dihydro-8-oxo-2’-deoxyguanosine; γH2AX: phosphorylated H2A histone family member X; FISH: fluorescence in situ hybridization; STELA: single telomere length analysis; PCR: polymerase chain reaction; RT-PCR: reverse transcriptase PCR; NGS: next generation sequencing.