Detecting senescent fate in mesenchymal stem cells: a combined cytofluorimetric and ultrastructural approach
Senescence can impair the therapeutic potential of stem cells. In this study, senescence-associated morphofunctional changes in periosteum-derived progenitor cells (PDPCs) from old and young individuals were investigated by combining cytofluorimetry, immunohistochemistry, and transmission electron microscopy. Cell cycle analysis demonstrated a large number of G0/G1 phase cells in PDPCs from old subjects and a progressive accumulation of G0/G1 cells during passaging in cultures from young subjects. Cytofluorimetry documented significant changes in light scattering parameters and closely correlated with the ultrastructural features, especially changes in mitochondrial shape and autophagy, which are consistent with the mitochondrial-lysosomal axis theory of ageing. The combined morphological, biofunctional, and ultrastructural approach enhanced the flow cytometric study of PDPC ageing. We speculate that impaired autophagy, documented in replicative senescent and old PDPCs, reflect a switch from quiescence to senescence. Its demonstration in a tissue with limited turnover—like the cambium layer of the periosteum, where reversible quiescence is the normal stem cell state throughout life—adds a new piece to the regenerative medicine jigsaw in an ageing society.
KeywordsPeriosteal stem cells Flow cytometry Quiescence Senescence TEM Tissue engineering
Alkaline phosphatase staining
Alizarin Red S staining
Forward scatter coefficient of variation
Microtubule-associated protein light chain
Mesenchymal stem cells
Population doubling time
Periosteum-derived progenitor cells
Reactive oxygen species
- SA βgal
Senescence-associate secretory phenotype
Side scatter coefficient of variation
The authors are grateful to Dr. Caterina Licini for her valid collaboration in Western Blot analysis.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
- Georgakopoulou EA, Tsimaratou K, Evangelou K et al (2013) Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues. Aging 5:37–50. https://doi.org/10.18632/aging.100527 CrossRefPubMedGoogle Scholar
- Lorenzini A, Maier AB (2016) Influence of donor age and species longevity on replicative cellular senescence. In: Cellular ageing and replicative senescence. Springer, Cham, pp 49–70Google Scholar