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Cited by 127Mesenchymal stem cell researchTelomeres, Telomerase, and SenescencePluripotent Stem Cells Research

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Antioxidants inhibit cell senescence and preserve stemness of adipose tissue-derived stem cells by reducing ROS generation during long-term in vitro expansion

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Liao, Naishun, Shi, Yingjun, Zhang, Cuilin, Zheng, Youshi, Wang, Yingchao, Zhao, Bixing, Zeng, Yongyi, Liu, Xiaolong, Liu, Jingfeng·2019·Stem Cell Research & Therapy
DOI 10.1186/s13287-019-1404-9PMC PMC6798439
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Abstract

Background: Adipose tissue-derived mesenchymal stem cells (ADSCs) are promising candidates for regenerative medicine. However, long-term in vitro passaging leads to stemness loss and cell senescence of ADSCs, resulting in failure of ADSC-based therapy. Methods: In this study, ADSCs were treated with low dose of antioxidants (reduced glutathione and melatonin) with anti-aging and stem cell protection properties in the in vitro passaging, and the cell functions including stem cell senescence, cell migration, cell multidirectional differentiation potential, and ROS content were carefully analyzed. Results: We found that GSH and melatonin could maintain ADSC cell functions through reducing cell senescence and promoting cell migration, as well as by preserving stemness and multidirectional differentiation potential, through inhibiting ROS generation during long-term expansion of ADSCs. Conclusions: Our results suggested that antioxidant treatment could efficiently prevent the dysfunction and preserve cell functions of ADSCs after long-term passaging, providing a practical strategy to facilitate ADSC-based therapy.

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Fig. 1 Antioxidants promote ADSC cell proliferation during long-term in vitro expansion. The CCK-8 assay was applied to study the proliferation of ADSCs at passage 3 (P3), passage 6 (P6), and passage 9 after 10 μM GSH or melatonin treatment, or the combination of GSH and melatonin treatment, when the cells were cultured for 24, 48, and 96 h, respectively (n = 5 per group; *p < 0.05). ADSCs, adipose tissue-derived stem cells; GSH, reduced glutathione
Fig. 1 Antioxidants promote ADSC cell proliferation during long-term in vitro expansion. The CCK-8 assay was applied to study the proliferation of ADSCs at passage 3 (P3), passage 6 (P6), and passage 9 after 10 μM GSH or melatonin treatment, or the combination of GSH and melatonin treatment, when the cells were cultured for 24, 48, and 96 h, respectively (n = 5 per group; *p < 0.05). ADSCs, adipose tissue-derived stem cells; GSH, reduced glutathioneCC BY · PubMed Central
Fig. 2 Antioxidants inhibit ADSC cell senescence during long-term in vitro expansion. After treatment with 10 μM GSH or melatonin, the ADSCs cultured for passage 3 (P3), passage 6 (P6), and passage 9 (P9) were used in the following analysis. a SA-β-gal staining of ADSCs (× 200 magnification; scale bar, 50 μm). The relative gene expression of p16 (b), p21 (c), and p53 (d) of ADSCs with or without antioxidant treatment in vitro expansion (n = 3 per group; *p < 0.05; **p < 0.01). ADSCs, adipose tissue-derived stem cells; GSH, reduced glutathione
Fig. 2 Antioxidants inhibit ADSC cell senescence during long-term in vitro expansion. After treatment with 10 μM GSH or melatonin, the ADSCs cultured for passage 3 (P3), passage 6 (P6), and passage 9 (P9) were used in the following analysis. a SA-β-gal staining of ADSCs (× 200 magnification; scale bar, 50 μm). The relative gene expression of p16 (b), p21 (c), and p53 (d) of ADSCs with or without antioxidant treatment in vitro expansion (n = 3 per group; *p < 0.05; **p < 0.01). ADSCs, adipose tissue-derived stem cells; GSH, reduced glutathioneCC BY · PubMed Central
Fig. 3 Antioxidants promote ADSC cell migration during long-term in vitro expansion. After treatment with 10 μM GSH or melatonin, the ADSCs cultured for passage 3 (P3), passage 6 (P6), and passage 9 (P9) were used in the following analysis. a Motility of ADSCs (× 100 magnification; scale bar, 100 μm). b Quantification of the cell motility. c Migration of passaged ADSCs (× 200 magnification; scale bar, 50 μm). d Quantification of the number of migrated cells (n = 5 per group; **p < 0.01). e The relative CXCR4 expression of passaged ADSCs with or without antioxidant treatment (n = 3 per group; *p < 0.05). ADSCs, adipose tissue-derived stem cells, GSH, reduced glutathione
Fig. 3 Antioxidants promote ADSC cell migration during long-term in vitro expansion. After treatment with 10 μM GSH or melatonin, the ADSCs cultured for passage 3 (P3), passage 6 (P6), and passage 9 (P9) were used in the following analysis. a Motility of ADSCs (× 100 magnification; scale bar, 100 μm). b Quantification of the cell motility. c Migration of passaged ADSCs (× 200 magnification; scale bar, 50 μm). d Quantification of the number of migrated cells (n = 5 per group; **p < 0.01). e The relative CXCR4 expression of passaged ADSCs with or without antioxidant treatment (n = 3 per group; *p < 0.05). ADSCs, adipose tissue-derived stem cells, GSH, reduced glutathioneCC BY · PubMed Central
Fig. 4 Antioxidants preserve ADSC cell stemness and multidirectional differentiation potential during long-term in vitro expansion. After treatment with 10 μM GSH or melatonin, the ADSCs cultured for passage 3 (P3), passage 6 (P6), and passage 9 (P9) were used in the following analysis. a Osteogenesis differentiation of passaged ADSCs (Alizarin Red S staining; scale bar, 50 μm). b Adipogenesis differentiation of passaged ADSCs (Oil Red O staining; scale bar, 50 μm). c Western blot analysis for RUNX-2 in osteogenic cells. d Western blot analysis for perilipin A in adipogenic cells. e Chondrogenesis differentiation of passaged ADSCs (Alcian blue staining; scale bar, 50 μm). f Western blot analysis for SOX-9 in chondrogenic cells. g Western blot analysis for SOX-2, OCT-4, and β-actin in ADSCs. ADSCs, adipose tissue-derived stem cells; GSH, reduced glutathione
Fig. 4 Antioxidants preserve ADSC cell stemness and multidirectional differentiation potential during long-term in vitro expansion. After treatment with 10 μM GSH or melatonin, the ADSCs cultured for passage 3 (P3), passage 6 (P6), and passage 9 (P9) were used in the following analysis. a Osteogenesis differentiation of passaged ADSCs (Alizarin Red S staining; scale bar, 50 μm). b Adipogenesis differentiation of passaged ADSCs (Oil Red O staining; scale bar, 50 μm). c Western blot analysis for RUNX-2 in osteogenic cells. d Western blot analysis for perilipin A in adipogenic cells. e Chondrogenesis differentiation of passaged ADSCs (Alcian blue staining; scale bar, 50 μm). f Western blot analysis for SOX-9 in chondrogenic cells. g Western blot analysis for SOX-2, OCT-4, and β-actin in ADSCs. ADSCs, adipose tissue-derived stem cells; GSH, reduced glutathioneCC BY · PubMed Central
Fig. 5 Antioxidants inhibit ROS generation of ADSCs during long-term in vitro expansion. After treatment with 10 μM GSH or melatonin, the ADSCs cultured for passage 3 (P3), passage 6 (P6), and passage 9 (P9) were used in the following analysis. a Confocal images of ROS content in ADSCs by DCFH-DA labeling (× 400 magnification; scale bar, 50 μm). b Quantification of ROS fluorescence intensity (n = 3 per group; **p < 0.01). c Superoxide level in ADSCs (n = 3 per group; **p < 0.01). d NOXs content of ADSCs (n = 3 per group; *p < 0.05; **p < 0.01). ADSCs, adipose tissue-derived stem cells; GSH, reduced glutathione; ROS, reactive oxygen species
Fig. 5 Antioxidants inhibit ROS generation of ADSCs during long-term in vitro expansion. After treatment with 10 μM GSH or melatonin, the ADSCs cultured for passage 3 (P3), passage 6 (P6), and passage 9 (P9) were used in the following analysis. a Confocal images of ROS content in ADSCs by DCFH-DA labeling (× 400 magnification; scale bar, 50 μm). b Quantification of ROS fluorescence intensity (n = 3 per group; **p < 0.01). c Superoxide level in ADSCs (n = 3 per group; **p < 0.01). d NOXs content of ADSCs (n = 3 per group; *p < 0.05; **p < 0.01). ADSCs, adipose tissue-derived stem cells; GSH, reduced glutathione; ROS, reactive oxygen speciesCC BY · PubMed Central

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Metadata

Authors
Liao, N., Shi, Y., Zhang, C., Zheng, Y., Wang, Y., Zhao, B., Zeng, Y., Liu, X., & Liu, J.
Affiliations
Fujian Medical University (CN)
Corresponding author
  • Liu, Xiaolong
  • Liu, Jingfeng
ORCID
Venue
Stem Cell Research & Therapy
Publisher
Springer Science and Business Media LLC
Published
2019-10-17
Volume
10
Issue
1
Pages
306-306
ISSN
1757-6512 (ISSN-L)
DOI
10.1186/s13287-019-1404-9
PMC
PMC6798439
OpenAlex
W2980985101
Cited by
127
References
40
Citations by year
2026: 7 · 2025: 18 · 2024: 25 · 2023: 25 · 2022: 24 · 2021: 19 · 2020: 8
FWCI
7.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Stem Cell Research & Therapy (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • the National Natural Science Foundation of China (81672376)
  • the Youth Scientific Research Project of Fujian Provincial Health and Family Planning Commission (2017-1-85, 2017-2-60, 2018-1-93)
  • the Fuzhou Health and Family Planning Science and Technology Project (2017-S-wt2)
  • the Startup Fund for scientific research, Fujian Medical University (2016QH080, 2016QH081, 2018QH1198)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
Keywords
adipose-derived stem cells, cellular senescence, stemness maintenance, antioxidants, reduced glutathione, melatonin, reactive oxygen species, cell migration, multilineage differentiation, anti-aging
MeSH terms
Adipose Tissue, Animals, Antioxidants, Cell Differentiation, Cell Movement, Cells, Cultured, Glutathione, Male, Melatonin, Mice, Inbred C57BL, Stem Cells, Cellular Senescence, Reactive Oxygen Species, Stem Cell Transplantation, Regenerative Medicine, Cell Proliferation, Mice, Mesenchymal Stem Cells

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