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The Impact of Long-Term In Vitro Expansion on the Senescence-Associated Markers of Human Adipose-Derived Stem Cells

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Abstract

Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P < 0.05). The data showed that growth of ASCs reduced in long-term culture and the ß-galactosidase activity was significantly increased at later passage (P20). The morphology of ASCs in long-term culture showed the manifestation of senescent feature at P15 and P20. Significant alteration in the senescence-associated genes expression levels was observed in MMP1, p21, Rb and Cyclin D1 at P15 and P20. Significant increase in CD45 and HLA DR DQ DP surface marker was observed at P20. While cell cycle analysis showed significant decrease in percentage of ASCs at S and G2/M phase at later passage (P15). Our data showed ASCs cultured beyond P10 favours the senescence pathway and its clinical usage in cell-based therapy may be limited.

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Abbreviations

ASCs:

Human adipose-derived stem cells

MSCs:

Mesenchymal stem cells

CD:

Cluster differentiation

MMP1:

Matrix metalloproteinase 1

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HLA:

Human leukocyte antigen

MHC:

Major histocompatibility complex

IL6:

Interleukin 6

TERT:

Telomerase reverse transcriptase

Rb:

Retinoblastoma

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Acknowledgements

This study was supported by Ministry of Science, Technology and Innovation (MOSTI; Grant No. 02-01-02 SF0290) and Ministry of Higher Education Malaysia (MOHE; Grant No. UKM-FF-03-FRGS0004-2009). Special thanks to Ms. Nurhazira Abdul Rahim and Ms. Azalina Zainuddin for providing the gene primers and the staff of the Biochemistry Department for their technical assistance.

Competing Interests

The author(s) declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

    Wan Kamarul Zaman Wan Safwani & Kien Hui Chua

  2. Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

    Suzana Makpol

  3. Subang Jaya Medical Centre, Selangor, Malaysia

    Somasundaram Sathapan

  4. Department of Physiology, Faculty of Medicine, Jalan Raja Muda Abdul Aziz, Universiti Kebangsaan Malaysia, 50300, Kuala Lumpur, Malaysia

    Kien Hui Chua

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  1. Wan Kamarul Zaman Wan Safwani
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  4. Kien Hui Chua
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Correspondence to Kien Hui Chua.

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Safwani, W.K.Z.W., Makpol, S., Sathapan, S. et al. The Impact of Long-Term In Vitro Expansion on the Senescence-Associated Markers of Human Adipose-Derived Stem Cells. Appl Biochem Biotechnol 166, 2101–2113 (2012). https://doi.org/10.1007/s12010-012-9637-4

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