, Volume 19, Issue 5, pp 401–414 | Cite as

Detecting senescent fate in mesenchymal stem cells: a combined cytofluorimetric and ultrastructural approach

  • Manuela Dicarlo
  • Gabriella Teti
  • Iolanda Iezzi
  • Giorgia Cerqueni
  • Sandra Manzotti
  • Mirella Falconi
  • Monica Mattioli-BelmonteEmail author
Research Article


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.


Periosteal stem cells Flow cytometry Quiescence Senescence TEM Tissue engineering 



Alkaline phosphatase staining


Alizarin Red S staining


Forward scatter


Forward scatter coefficient of variation


Microtubule-associated protein light chain


Mesenchymal stem cells


Nitric oxide


Population doubling time


Periosteum-derived progenitor cells


Reactive oxygen species

SA βgal

Senescence-associated β-galactosidase


Senescence-associate secretory phenotype


Side scatter


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.

Ethical standards

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.

Supplementary material

10522_2018_9766_MOESM2_ESM.pdf (119 kb)
Supplementary material 1 (PDF 414 kb)
10522_2018_9766_MOESM1_ESM.pdf (415 kb)
Supplementary material 2 (PDF 118 kb)


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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Clinical and Molecular Sciences-DISCLIMOUniversità Politecnica delle MarcheAnconaItaly
  2. 2.Department of Biomedical and Neuromotor Sciences-DBNSUniversità di BolognaBolognaItaly

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