, Volume 61, Issue 3, pp 93–107 | Cite as

Enhanced proliferation of human skeletal muscle precursor cells derived from elderly donors cultured in estimated physiological (5%) oxygen

  • Sheree D. Martin
  • Fiona M. Collier
  • Mark A. Kirkland
  • Ken Walder
  • Nicole StupkaEmail author
Original Paper


Human skeletal muscle precursor cells (myoblasts) have significant therapeutic potential and are a valuable research tool to study muscle cell biology. Oxygen is a critical factor in the successful culture of myoblasts with low (1–6%) oxygen culture conditions enhancing the proliferation, differentiation, and/or viability of mouse, rat, and bovine myoblasts. The specific effects of low oxygen depend on the myoblast source and oxygen concentration; however, variable oxygen conditions have not been tested in the culture of human myoblasts. In this study, muscle precursor cells were isolated from vastus lateralis muscle biopsies and myoblast cultures were established in 5% oxygen, before being divided into physiological (5%) or standard (20%) oxygen conditions for experimental analysis. Five percent oxygen increased proliferating myoblast numbers, and since low oxygen had no significant effect on myoblast viability, this increase in cell number was attributed to enhanced proliferation. The proportion of cells in the S (DNA synthesis) phase of the cell cycle was increased by 50%, and p21Cip1 gene and protein expression was decreased in 5 versus 20% oxygen. Unlike in rodent and bovine myoblasts, the increase in myoD, myogenin, creatine kinase, and myosin heavy chain IIa gene expression during differentiation was similar in 5 and 20% oxygen; as was myotube hypertrophy. These data indicate for the first time that low oxygen culture conditions stimulate proliferation, whilst maintaining (but not enhancing) the viability and the differentiation potential of human primary myoblasts and should be considered as optimum conditions for ex-vivo expansion of these cells.


Human primary myoblasts Oxygen Proliferation p21Cip1 Differentiation Cell cycle Species differences 





Creatine kinase


Lactate dehydrogenase


Myosin heavy chain


Skeletal muscle growth medium


Skeletal muscle differentiation medium



We thank Assoc Prof David Cameron-Smith for his expertise with primer design; Prof Geoff C Nicholson for his assistance with the human ethics applications; the Orthopedic Surgeons—Drs David Bainbridge, Rick Angliss, and Rob Wood—for collection of the muscle biopsy samples; and Lisa Coleman, the Orthopedic Liaison Nurse, for her participation in subject recruitment. We gratefully acknowledge all of their contributions towards the completion of this project. The work presented in this report was supported by a NHMRC Postdoctoral Fellowship to NS and by the Deakin University Central Research Grants Scheme.


  1. Berggren JR, Tanner CJ, Koves TR, Muoio DM, Houmard JA (2005) Glucose uptake in muscle cell cultures from endurance-trained men. Med Sci Sports Exerc 37:579–584CrossRefGoogle Scholar
  2. Besson A, Gurian-West M, Chen X, Kelly-Spratt KS, Kemp CJ, Roberts JM (2006) A pathway in quiescent cells that control p27Kip1 stability, subcellular localization, and tumour suppression. Genes Dev 20:47–64.CrossRefGoogle Scholar
  3. Capkovic KL, Stevenson S, Johnson MC, Thelen JJ, Cornelison DDW (2008) Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation. Exp Cell Res 314:1553–1565CrossRefGoogle Scholar
  4. Chakravarthy MV, Spangenburg EE, Booth FW (2001) Culture in low levels of oxygen enhances in vitro proliferation potential of satellite cells from old skeletal muscle. Cell Mol Life Sci 58:1150–1158CrossRefGoogle Scholar
  5. Chargé SBP, Rudnicki MA (2004) Cellular and molecular regeneration. Physiol Rev 84:209–238CrossRefGoogle Scholar
  6. Conboy IM, Rando TA (2005) Aging, stem cells and tissue regeneration: lessons from muscle. Cell Cycle 4:407–410Google Scholar
  7. Cornelison DDW (2008) Context matters: in vivo and in vitro influences on muscle satellite cell activity. J Cell Biochem 105:663–669CrossRefGoogle Scholar
  8. Csete M (2005) Oxygen in the cultivation of stem cells. Ann N Y Acad Sci 1049:1–8CrossRefGoogle Scholar
  9. Csete M, Walikonis J, Slawny N, Wei Y, Korsnes S, Doyle JC, Barbara W (2001) Oxygen-mediated regulation of skeletal muscle satellite cell proliferation and adipogenesis in culture. J Cell Physiol 189:189–196CrossRefGoogle Scholar
  10. Decary S, Mouly V, Hamida BC, Barbet JP, Butler-Browne GS (1997) Replicative potential of telomere length in human skeletal muscle: implications for satellite cell-mediated gene therapy. Hum Gene Ther 8:1429–1438CrossRefGoogle Scholar
  11. Fehrer C, Brunauer R, Laschober G, Unterluggauer H, Reitinger S, Kloss F, Gülly C, Gassner R, Lepperdinger G (2007) Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell 6:745–757CrossRefGoogle Scholar
  12. Gaster M, Beck-Nielsen H, Schrøder HD (2001) Proliferation conditions for human satellite cells: the fractional content of satellite cells. APMIS 109:726–734CrossRefGoogle Scholar
  13. Hansen JM, Klass M, Harris C, Csete M (2007) A reducing redox environment promotes C2C12 myogenesis: implications for regeneration in aged muscle. Cell Biol Int 31:546–553CrossRefGoogle Scholar
  14. Kitzmann M, Fernandez A (2001) Crosstalk between cell cycle regulators and the myogenic factor MyoD in skeletal myoblasts. Cell Mol Life Sci 58:571–579CrossRefGoogle Scholar
  15. Kook S-H, Son Y-O, Lee K-Y, Lee H-J, Chung W-T, Choi K-C, Lee J-C (2008) Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of myoD. Cell Biol Int 32:871–878CrossRefGoogle Scholar
  16. Lawson-Smith MJ, McGeachie JK (1998) The identification of myogenic cells in skeletal muscle, with emphasis on the use of tritiated thymidine autoradiography and desmin antibodies. J Anat 192:161–171CrossRefGoogle Scholar
  17. Lees SJ, Childs TE, Booth FW (2008) p21Cip1 expression is increased in ambient oxygen, compared to estimated physiological (5%) levels in rat muscle precursor cells. Cell Prolif 41:193–207CrossRefGoogle Scholar
  18. Li X, Zhu L, Chen X, Fan M (2007) Effects of hypoxia on proliferation and differentiation of myoblasts. Med Hypotheses 69:629–636CrossRefGoogle Scholar
  19. McAinch AJ, Steinberg GR, Mollica J, O’Brien PE, Dixon JB, Macaulay SL, Kemp BE, Cameron-Smith D (2006) Differential regulation of adiponectin receptor gene expression by adiponectin and leptin in myotubes derived from obese and diabetic individuals. Obesity 14:1898–1904CrossRefGoogle Scholar
  20. McGeachie JK, Grounds MD (1999) The timing between skeletal muscle myoblast replication and fusion into myotubes, and the stability of regenerated dustrophic myofibres: an autoradiographic study in mdx mice. J Anat 194:287–295CrossRefGoogle Scholar
  21. Messina G, Blasi C, La Rocca SA, Pompili M, Calconi A, Grossi M (2005) p27Kip1 acts downstream of N-cadherin-mediated cell adhesion to promote myogenesis beyond cell cycle regulation. Mol Biol Cell 16:1469–1480CrossRefGoogle Scholar
  22. Mouly V, Aamiri A, Bigot A, Cooper RN, Di Donna S, Furling D, Gidaro T, Jacquemin V, Mamchaoui K, Negroni E, Périé S, Renault V, Silva-Barbosa SD, Butler-Browne GS (2006) The mitotic clock in skeletal muscle regeneration, disease and cell mediated gene therapy. Acta Physiol Scand 184:3–15CrossRefGoogle Scholar
  23. Pavlath GK, Gussoni E (2005) Human myoblasts and muscle derived SP cells. Methods Mol Med 107:97–110Google Scholar
  24. Péault B, Rudnicki MA, Torrente Y, Cossu G, Tremblay JP, Partridge T, Gussoni E, Kunkel LM, Huard J (2007) Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Mol Therapy 15:867–872CrossRefGoogle Scholar
  25. Rathbone CR, Booth FW, Lees SJ (2009) Sirt1 increases skeletal muscle precursor cell proliferation. Eur J Cell Biol 88:35–44CrossRefGoogle Scholar
  26. Smogorzewska A, de Lange T (2002) Different telomere damage signalling pathways in human and mouse cells. EMBO J 21:4338–4348CrossRefGoogle Scholar
  27. Stewart JD, Masi TL, Cumming AE, Molnar GM, Wentworth BM, Sampath K, McPherson JM, Yaeger PC (2003) Characterization of proliferating human skeletal muscle-dervived cells in vitro: differential modulation of myoblast markers by TGF-b2. J Cell Physiol 196:70–78CrossRefGoogle Scholar
  28. Zhang P, Wong C, Liu D, Finegold M, Harper WJ, Elledge SJ (2008) p21CIP1 and p57KIP2 control muscle differentiation at the myogenin step. Genes Dev 13:213–224CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Sheree D. Martin
    • 1
  • Fiona M. Collier
    • 2
  • Mark A. Kirkland
    • 2
  • Ken Walder
    • 1
    • 3
  • Nicole Stupka
    • 1
    Email author
  1. 1.Institute for Technology, Research and InnovationDeakin UniversityWaurn PondsAustralia
  2. 2.Barwon Biomedical Research, Barwon HealthGeelongAustralia
  3. 3.School of MedicineDeakin UniversityWaurn PondsAustralia

Personalised recommendations