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Hypoxia increases Sca-1/CD44 co-expression in murine mesenchymal stem cells and enhances their adipogenic differentiation potential

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Abstract

Mesenchymal stem cells (MSCs) are usually cultured under normoxic conditions (21% oxygen). However, in vivo, the physiological “niches” for MSCs have a much lower oxygen tension. Because of their plasticity, stem cells are particularly sensitive to their environments, and oxygen tension is one developmentally important stimulus in stem cell biology and plays a role in the intricate balance between cellular proliferation and commitment towards differentiation. Therefore, we investigated here the effect of hypoxia (2% oxygen) on murine adipose tissue (AT) MSC proliferation and adipogenic differentiation. AT cells were obtained from the omental fat and AT-MSCs were selected for their ability to attach to the plastic dishes, and were grown under normoxic and hypoxic conditions. Prior exposure of MSCs to hypoxia led to a significant reduction of ex vivo expansion time, with significantly increased numbers of Sca-1+ as well as Sca-1+/CD44+double-positive cells. Under low oxygen culture conditions, the AT-MSC number markedly increased and their adipogenic differentiation potential was reduced. Notably, the hypoxia-mediated inhibition of adipogenic differentiation was reversible: AT-MSCs pre-exposed to hypoxia when switched to normoxic conditions exhibited significantly higher adipogenic differentiation capacity compared to their pre-exposed normoxic-cultured counterparts. Accordingly, the expression of adipocyte-specific genes, peroxisome proliferator activated receptor γ (Pparγ), lipoprotein lipase (Lpl) and fatty acid binding protein 4 (Fabp4) were significantly enhanced in hypoxia pre-exposed AT-MSCs. In conclusion, pre-culturing MSCs under hypoxic culture conditions may represent a strategy to enhance MSC production, enrichment and adipogenic differentiation.

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Acknowledgments

The authors thank Dr Gary Warnes, manager of Flow Cytometry Core Facility in the Blizard Institute of Cell and Molecular Science, for his excellent assistance, and Mr George Elia and Mr Luke Gammon for their help, and the sharing of reagents and expertise, and Dr Sarah Howlett and Prof Peter J Dyson for kindly providing NOD mice.

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Authors and Affiliations

  1. Centre for Diabetes, Blizard Institute of Cell and Molecular Science, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK

    M. G. Valorani, C. P. Khoo, W. R. Lin, M. I. Hawa, P. Pozzilli & M. R. Alison

  2. Fondazione Livio Patrizi, Rome, Italy

    M. G. Valorani, A. Germani & M. P. Patrizi

  3. BIOS S.p.A., Rome, Italy

    M. G. Valorani

  4. Histopathology Laboratory, Cancer Research UK, London, UK

    W. R. Otto, W. R. Lin & M. R. Alison

  5. Area di Endocrinologia, Universita’ Campus Bio-Medico, Rome, Italy

    C. P. Khoo & P. Pozzilli

  6. Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science, Barts & The London School of Medicine & Dentistry, Queen Mary University of London, London, UK

    L. Harper, A. Biddle & P. Tropel

  7. Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan

    W. R. Lin

Authors
  1. M. G. Valorani
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  2. A. Germani
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  3. W. R. Otto
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  4. L. Harper
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  7. W. R. Lin
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  8. M. I. Hawa
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  10. M. P. Patrizi
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  11. P. Pozzilli
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  12. M. R. Alison
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Corresponding author

Correspondence to M. G. Valorani.

Additional information

This study was supported by grants from Fondazione Livio Patrizi and BIOS S.p.A, Rome, Italy, from the International PhD programme of Queen Mary University of London/University Campus BioMedico, Rome, Italy, and from the Italian Ministry of Health.

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Supplementary Figure S1

Sca-1+/CDD44+ expression in pancreatic-, testis-tissues and bone marrow derived-MSC cultured under normoxic and hypoxic conditions. FACS analysis showing Sca-1+/CD44+ expression in MSC obtained from pancreas (a,c), and testis tissues (b,d) cultured for 30 and 10 days, respectively, under normoxic (21%) and hypoxic (2%) conditions. Low oxygen levels enhanced the numbers of Sca-1+/CD44+double-positive cells in the MSC fraction obtained from both sources. (e) Bar graphs showing results from FACS analysis of the percentage of Sca1+, CD44+ cells and Sca1+/CD44+ cells (n = 3) in 20 days cultured BM-MSC under normoxic (open bars) and hypoxic (black bars) conditions

Supplementary Figure S1

High resolution Image File (TIFF 7930 kb)

Supplementary Figure S2

Hypoxic culture conditions increased AT-MSC proliferation. Representative images of GM-cultured AT-MSCs after (a) 24 h in normoxia and 3 days either in normoxia (b) or in (c) hypoxia. High magnifications of the insets are shown in (c) and (e)

Supplementary Figure S2

High resolution Image File (TIFF 2996 kb)

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Valorani, M.G., Germani, A., Otto, W.R. et al. Hypoxia increases Sca-1/CD44 co-expression in murine mesenchymal stem cells and enhances their adipogenic differentiation potential. Cell Tissue Res 341, 111–120 (2010). https://doi.org/10.1007/s00441-010-0982-8

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  • DOI: https://doi.org/10.1007/s00441-010-0982-8

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