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Cited by 240Mesenchymal stem cell researchPeriodontal Regeneration and TreatmentsOsteoarthritis Treatment and Mechanisms

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The Influence of Aging on the Regenerative Potential of Human Adipose Derived Mesenchymal Stem Cells

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Marędziak, Monika, Marycz, Krzysztof, Tomaszewski, Krzysztof A., Kornicka, Katarzyna, Henry, Brandon Michael·2016·Stem Cells International
DOI 10.1155/2016/2152435PMC PMC4749808
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Abstract

Tissue regeneration using human adipose derived mesenchymal stem cells (hASCs) has significant potential as a novel treatment for many degenerative bone and joint diseases. Previous studies have established that age negatively affects the proliferation status and the osteogenic and chondrogenic differentiation potential of mesenchymal stem cells. The aim of this study was to assess the age‐related maintenance of physiological function and differentiation potential of hASCs in vitro. hASCs were isolated from patients of four different age groups: (1) >20 years ( n = 7), (2) >50 years ( n = 7), (3) >60 years ( n = 7), and (4) >70 years ( n = 7). The hASCs were characterized according to the number of fibroblasts colony forming unit (CFU‐F), proliferation rate, population doubling time (PDT), and quantified parameters of adipogenic, chondrogenic, and osteogenic differentiation. Compared to younger cells, aged hASCs had decreased proliferation rates, decreased chondrogenic and osteogenic potential, and increased senescent features. A shift in favor of adipogenic differentiation with increased age was also observed. As many bone and joint diseases increase in prevalence with age, it is important to consider the negative influence of age on hASCs viability, proliferation status, and multilineage differentiation potential when considering the potential therapeutic applications of hASCs.

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Figures

Figure 1 Characterization of MSCs. (a) Flow cytometry analysis of MSC positive (CD44, CD73b, CD90, and CD105) and negative (CD34 and CD45) surface markers. (b) Flow cytometry analysis determined the percentage of specific markers in total analyzed hASCs from patients differing in age. Quantification of markers revealed no significant differences between groups. (c) Alizarin Red staining of calcium deposits, Safranin O staining for proteoglycans, and accumulation of lipid droplets dyed with Oil Red O following differentiation into osteoblast, chondroblasts, and adipocyte lineages.
Figure 1 Characterization of MSCs. (a) Flow cytometry analysis of MSC positive (CD44, CD73b, CD90, and CD105) and negative (CD34 and CD45) surface markers. (b) Flow cytometry analysis determined the percentage of specific markers in total analyzed hASCs from patients differing in age. Quantification of markers revealed no significant differences between groups. (c) Alizarin Red staining of calcium deposits, Safranin O staining for proteoglycans, and accumulation of lipid droplets dyed with Oil Red O following differentiation into osteoblast, chondroblasts, and adipocyte lineages.CC BY · PubMed Central
Figure 2 Comparison of hASCs morphology from different age groups, evaluated after seven days of propagation. Scale bars: (a–d) 250 μm; (e–h) 300 μm; (i–l) 10 μm.
Figure 2 Comparison of hASCs morphology from different age groups, evaluated after seven days of propagation. Scale bars: (a–d) 250 μm; (e–h) 300 μm; (i–l) 10 μm.CC BY · PubMed Central
Figure 3 Comparison of cell proliferation in different age groups. (a) Cell proliferation assay, (b) population doubling time, and (c) fibroblast colony-forming unit efficiency over seven days. Results expressed as mean ± SD. ∗ p value <0.05.
Figure 3 Comparison of cell proliferation in different age groups. (a) Cell proliferation assay, (b) population doubling time, and (c) fibroblast colony-forming unit efficiency over seven days. Results expressed as mean ± SD. ∗ p value <0.05.CC BY · PubMed Central
Figure 4 Characterization of age-dependent apoptosis. (a) Cells stained for senescence showing cells with β-galactosidase accumulation (A–D), Calcein-AM (E–H), and propidium iodide (I–L) for live-dead cells identification. (b) β-galactosidase absorbance measurement. (c) Percentage of apoptotic cells calculated from Calcein-AM/propidium iodide staining, (d) evaluation of p53, and (e) p21 on the mRNA level. Results expressed as mean ± SD. ∗ p value <0.05.
Figure 4 Characterization of age-dependent apoptosis. (a) Cells stained for senescence showing cells with β-galactosidase accumulation (A–D), Calcein-AM (E–H), and propidium iodide (I–L) for live-dead cells identification. (b) β-galactosidase absorbance measurement. (c) Percentage of apoptotic cells calculated from Calcein-AM/propidium iodide staining, (d) evaluation of p53, and (e) p21 on the mRNA level. Results expressed as mean ± SD. ∗ p value <0.05.CC BY · PubMed Central
Figure 5 Levels of oxidative stress in hASCs. (a) Reactive oxygen species, (b) nitric oxide, and (c) superoxide dismutase levels. Results expressed as mean ± SD. ∗ p value <0.05.
Figure 5 Levels of oxidative stress in hASCs. (a) Reactive oxygen species, (b) nitric oxide, and (c) superoxide dismutase levels. Results expressed as mean ± SD. ∗ p value <0.05.CC BY · PubMed Central
Figure 6 Characterization of age related changes in hASCs chondrogenic differentiation potential. (a) Morphology of cells cultured in chondrogenic medium visualized by Safranin O staining (A–D) and SEM (E–H). Quantitative ELISA analysis and gene expression of (b) Col-II and (c) ACAN. Results expressed as mean ± SD. ∗ p value <0.05.
Figure 6 Characterization of age related changes in hASCs chondrogenic differentiation potential. (a) Morphology of cells cultured in chondrogenic medium visualized by Safranin O staining (A–D) and SEM (E–H). Quantitative ELISA analysis and gene expression of (b) Col-II and (c) ACAN. Results expressed as mean ± SD. ∗ p value <0.05.CC BY · PubMed Central
Figure 7 Characterization of age related changes in hASCs osteogenic differentiation potential. (a) Morphology of cells cultured in osteogenic medium visualized by (A–D) Alizarin Red staining and SEM (E–H). (b) SEM quantitative evalutaion of ALP secretion.
Figure 7 Characterization of age related changes in hASCs osteogenic differentiation potential. (a) Morphology of cells cultured in osteogenic medium visualized by (A–D) Alizarin Red staining and SEM (E–H). (b) SEM quantitative evalutaion of ALP secretion.CC BY · PubMed Central
Figure 8 Gene expression and ELISA anaylsis of osteogenic markers. (a) Gene expression analysis and (b) quantitative ELISA analysis of OPN, Col-I, and OCL. Results expressed as mean ± SD. ∗ p value <0.05.
Figure 8 Gene expression and ELISA anaylsis of osteogenic markers. (a) Gene expression analysis and (b) quantitative ELISA analysis of OPN, Col-I, and OCL. Results expressed as mean ± SD. ∗ p value <0.05.CC BY · PubMed Central
Figure 9 Characterization of age related changes in hASCs adipogenic differentiation potential. (a) Morphology of cells cultured in adipogenic medium visualized by Oil Red O staining (A–D) and SEM (E–H). Quantitative ELISA analysis and gene expression of LEP (b) and ADIQ (c). Gene expresion analysis of PPAR-γ (d). Results expressed as mean ± SD. ∗ p value <0.05.
Figure 9 Characterization of age related changes in hASCs adipogenic differentiation potential. (a) Morphology of cells cultured in adipogenic medium visualized by Oil Red O staining (A–D) and SEM (E–H). Quantitative ELISA analysis and gene expression of LEP (b) and ADIQ (c). Gene expresion analysis of PPAR-γ (d). Results expressed as mean ± SD. ∗ p value <0.05.CC BY · PubMed Central

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  1. 10.1016/0003-2697(87)90021-2 · DOI
  2. 10.1172/jci200420427 · DOI

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Metadata

Authors
Marędziak, M., Marycz, K., Tomaszewski, K. A., Kornicka, K., & Henry, B. M.
Affiliations
  • Wrocław University of Environmental and Life Sciences (PL)
  • Jagiellonian University (PL)
Corresponding author
Marycz, Krzysztof
ORCID
Venue
Stem Cells International
Publisher
Wiley
Published
2016-01
Volume
2016
Issue
1
Pages
2152435-2152435
ISSN
  • 1687-966X (ISSN-L)
  • 1687-9678
DOI
10.1155/2016/2152435
PMC
PMC4749808
OpenAlex
W2286064488
Cited by
225
References
54
Citations by year
2026: 8 · 2025: 15 · 2024: 15 · 2023: 13 · 2022: 19 · 2021: 34 · 2020: 31 · 2019: 29 · 2018: 24 · 2017: 22 · 2016: 13
FWCI
21.28
Type
journal article
Language
en
Open access
Licence
Locations
  • Stem Cells International (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Jagiellonian University Repository (Jagiellonian University) (submittedVersion, cc-by-nc-nd)
  • Homo Politicus (Academy of Humanities and Economics in Lodz) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Wrocław Centre of Biotechnology
  • Foundation for Polish Science
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Health Sciences › Dentistry › Periodontics › Periodontal Regeneration and Treatments
  • Health Sciences › Medicine › Rheumatology › Osteoarthritis Treatment and Mechanisms
Keywords
human adipose-derived mesenchymal stem cells, aging, cellular senescence, multilineage differentiation, osteogenic differentiation, chondrogenic differentiation, adipogenesis, cell proliferation, population doubling time, CFU-F, tissue regeneration

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