Tag Archive for: Myoblast

Isolation, in-vitro expansion, and characterization of human muscle satellite cells from the rectus abdominis muscle

Volume 4 | Issue 1 | January-June 2018 | Page: 16-22 | David Livingstone, Albert A Kota1, Sanjay K Chilbule, Karthikeyan Rajagopal, Sukria Nayak, Vrisha Madhuri

DOI- 10.13107/ijpo.2018.v04i01.005

Authors: David Livingstone, Albert A Kota [1], Sanjay K Chilbule, Karthikeyan Rajagopal, Sukria Nayak [1], Vrisha Madhuri


Department of Orthopaedics, Paediatric Orthopaedics Unit, 1Department of Surgery, Unit IV, Christian Medical College, Vellore, Tamil Nadu, India

Address of Correspondence
Dr. Vrisha Madhuri,
Paediatric Orthopaedics Unit, Christian Medical College, Vellore – 632 009, Tamil Nadu, India.
E-mail: madhuriwalter@cmcvellore.ac.in


Introduction: Satellite cells are a resident population of stem cells beneath the basal lamina of mature skeletal muscle fibers. Their capacity to regenerate muscle makes them a potentially ideal source for human cell therapy with respect to muscle-related diseases such as urinary and fecal incontinence, and others. In this study, we describe a protocol to isolate, expand in-vitro, and characterize human muscle satellite cells from the rectus abdominis muscle. Materials and Methods: Muscle biopsies from human donors were harvested, digested using collagenase type II, and then plated on extracellular matrix-coated plates.
Results: Immunocytochemistry revealed that satellite cells on day 8 were 70–80% Pax7 positive; in contrast, cells expanded until day 12 showed 50–75% positivity for Pax7. The real-time polymerase chain reaction for day 8 culture indicated four-fold increase in Pax3 and Pax7 gene expression, four-fold increase in MyoD gene expression, and five-fold increase in Myf5 gene expression.
Conclusion: These findings suggest that satellite cells can be cultured until day 8 for translational purposes. The protocol described here is modest, operational, and reproducible and involves only basic cell culture equipment.
Keywords: Cell therapy, Human skeletal muscle, Myoblast, Satellite cells, Sphincter injuries, Tissue regeneration


1. Chargé SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004;84:209-38.
2. Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 2005;122:289-301.
3. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 1961;9:493-5.
4. Kuang S, Rudnicki MA. The emerging biology of satellite cells and their therapeutic potential. Trends Mol Med 2008;14:82-91.
5. Holterman CE, Rudnicki MA. Molecular regulation of satellite cell function. Semin Cell Dev Biol 2005;16:575-84.
6. Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single transplanted muscle stem cells. Nature 2008;456:502-6.
7. Usas A, Huard J. Muscle-derived stem cells for tissue engineering and regenerative therapy. Biomaterials 2007;28:5401-6.
8. Ostrovidov S, Hosseini V, Ahadian S, Fujie T, Parthiban SP, Ramalingam M et al. Skeletal muscle tissue engineering: Methods to form skeletal myotubes and their applications. Tissue Eng Part B Rev 2014;20:403-36.
9. Liao H, Zhou GQ. Development and progress of engineering of skeletal muscle tissue. Tissue Eng Part B Rev 2009;15:319-31.
10. Danoviz ME, Yablonka-Reuveni Z. Skeletal muscle satellite cells: Background and methods for isolation and analysis in a primary culture system. Methods Mol Biol 2012;798:21-52.
11. Keire P, Shearer A, Shefer G, Yablonka-Reuveni Z. Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 2013;946:431-68.
12. Bischoff R. Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 1986;115:129-39.
13. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Pax7 is required for the specification of myogenic satellite cells. Cell 2000;102:777-86.
14. Boldrin L, Muntoni F, Morgan JE. Are human and mouse satellite cells really the same? J Histochem Cytochem 2010;58:941-55.
15. Cornelison D, Wold BJ. Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 1997;191:270-83.
16. Otto A, Collins‐Hooper H, Patel K. The origin, molecular regulation and therapeutic potential of myogenic stem cell populations. J Anat 2009;215:477-97.
17. Nierobisz LS, Cheatham B, Buehrer BM, Sexton JZ. High-content screening of human primary muscle satellite cells for new therapies for muscular atrophy/dystrophy. Curr Chem Genom Transl Med 2013;7:21-9.
18. Kajbafzadeh AM, Elmi A, Payabvash S, Salmasi AH, Saeedi P, Mohamadkhani A et al. Transurethral autologous myoblast injection for treatment of urinary incontinence in children with classic bladder exstrophy. J Urol 2008;180:1098-105.
19. Frudinger A, Kölle D, Schwaiger W, Pfeifer J, Paede J, Halligan S. Muscle-derived cell injection to treat anal incontinence due to obstetric trauma: pilot study with 1 year follow-up. Gut 2010;59:55-61.
20. Nikolavasky D, Stangel-Wójcikiewicz K, Stec M, Chancellor MB. Stem cell therapy: A future treatment of stress urinary incontinence. Semin Reprod Med 2011;29:61-70.
21. Gerullis H, Eimer C, Georgas E, Homburger M, El-Baz AG, Wishahi M et al. Muscle-derived cells for treatment of iatrogenic sphincter damage and urinary incontinence in men. ScientificWorldJournal 2012;2012:898535.
22. Bean AC, Huard J. Tissue Engineering Applications in Orthopedic Surgery. In: Mayer U, editor. Fundamentals of Tissue Engineering and Regenerative Medicine. Berlin: Springer-Berlin Heidelberg; 2009. p. 913-9.
23. Rajagopal K, Chilbule SK, Madhuri V. Viability, proliferation and phenotype maintenance in cryopreserved human iliac apophyseal chondrocytes. Cell Tissue Bank 2014;15:153-63.
24. Harel I, Nathan E, Tirosh-Finkel L, Zigdon H, Guimarães-Camboa N, Evans SM, Tzahor E. Distinct origins and genetic programs of head muscle satellite cells. Dev Cell 2009;16:822-32.
25. Halum SL, Naidu M, Delo DM, Atala A, Hingtgen CM. Injection of autologous muscle stem cells (myoblasts) for the treatment of vocal fold paralysis: A pilot study. Laryngoscope 2007;117:917-22.
26. Koning M, Harmsen MC, van Luyn MJ, Werker PM. Current opportunities and challenges in skeletal muscle tissue engineering. J Tissue Eng Regen Med 2009;3:407-15.
27. Sirabella D, De Angelis L, Berghella L. Sources for skeletal muscle repair: From satellite cells to reprogramming. J Cachexia Sarcopenia Muscle 2013;4:125-36.
28. Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005;309:2064-7.
29. Scott IC, Tomlinson W, Walding A, Isherwood B, Dougall IG. Large-scale isolation of human skeletal muscle satellite cells from post-mortem tissue and development of quantitative assays to evaluate modulators of myogenesis. J Cachexia Sarcopenia Muscle 2013;4:157-69.
30. Boldrin L, Morgan JE. Human satellite cells: identification on human muscle fibres. PLoS Curr 2012;3:1-14.
31. Sharifiaghdas F, Taheri M, Moghadasali R. Isolation of human adult stem cells from muscle biopsy for future treatment of urinary incontinence. Urol J 2011;8:54-9.
32. Zammit PS, Relaix F, Nagata Y, Ruiz AP, Collins CA, Partridge TA, Beauchamp JR. Pax7 and myogenic progression in skeletal muscle satellite cells. J Cell Sci 2006;119:1824-32.
33. Christov C, Chrétien F, Abou-Khalil R, Bassez G, Vallet G, Authier F-J et al. Muscle satellite cells and endothelial cells: close neighbors and privileged partners. Mol Biol Cell 2007;18:1397-409.
34. Kuang S, Gillespie MA, Rudnicki MA. Niche regulation of muscle satellite cell self-renewal and differentiation. Cell Stem Cell 2008;2:22-31.
35. Akhyari P, Kamiya H, Haverich A, Karck M, Lichtenberg A. Myocardial tissue engineering: The extracellular matrix. Eur J Cardiothorac Surg 2008;34:229-41.
36. Chaturvedi V, Dye DE, Coombe DR, Grounds MD. Bioactive scaffolds in skeletal muscle regeneration and tissue engineering. Australian Biochemist 2011;42:8-10.
37. Aicher A, Heeschen C, Sasaki K, Urbich C, Zeiher AM, Dimmeler S. Low-energy shock wave for enhancing recruitment of endothelial progenitor cells: A new modality to increase efficacy of cell therapy in chronic hind limb ischemia. Circulation 2006;114:2823-30.
38. Wang CJ, Huang HY, Pai CH. Shock wave-enhanced neovascularization at the tendon-bone junction: An experiment in dogs. J Foot Ankle Surg 2002;41:16-22.

How to Cite this Article:  Livingstone D, Kota AA, Chilbule SK, Rajagopal K, Nayak S, Madhuri V | Isolation, in-vitro expansion, and characterization of human muscle satellite cells from the rectus abdominis muscle | January-June 2018; 4(1): 16-22.


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