Purification of Myogenic Progenitors from Human Muscle Using Fluorescence-Activated Cell Sorting (FACS)

  • Anna Pakula
  • Janelle M. Spinazzola
  • Emanuela GussoniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1889)


Primary myoblasts derived from human tissue are a valuable tool in research of muscle disease and pathophysiology. However, skeletal muscle biopsies, especially from diseased muscle, contain a plethora of non-myogenic cells, necessitating purification of the myogenic cell population. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study.

Key words

Skeletal muscle Myoblast isolation Tissue dissociation Fluorescence-activated cell sorting (FACS) CD82 CD56 Immunostaining Pax7 



This work is supported by a grant from the Muscular Dystrophy Association #479606 (EG) and by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number 1R01AR069582-01 (EG). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This protocol was modified from previous work, specifically from the listed references [10, 11].


  1. 1.
    Geiger RS, Garvin JS (1957) Pattern of regeneration of muscle from progressive muscular dystrophy patients cultivated in vitro as compared to normal human skeletal muscle. J Neuropathol Exp Neurol 16(4):523–543CrossRefGoogle Scholar
  2. 2.
    Herrmann H, Konigsberg UR, Robinson G (1960) Observations on culture in vitro of normal and dystrophic muscle tissue. Proc Soc Exp Biol Med 105:217–221CrossRefGoogle Scholar
  3. 3.
    Goyle S, Kalra SL, Singh B (1967) The growth of normal & dystrophic human skeletal muscle in tissue culture. Neurol India 15(4):149–151PubMedGoogle Scholar
  4. 4.
    Bishop A, Gallup B, Skeate Y, Dubowitz V (1971) Morphological studies on normal and diseased human muscle in culture. J Neurol Sci 13(3):333–350CrossRefGoogle Scholar
  5. 5.
    Witkowski JA (1977) Diseased muscle cells in culture. Biol Rev Camb Philos Soc 52(4):431–476CrossRefGoogle Scholar
  6. 6.
    Hauschka SD (1974) Clonal analysis of vertebrate myogenesis. II. Environmental influences upon human muscle differentiation. Dev Biol 37(2):329–344CrossRefGoogle Scholar
  7. 7.
    Blau HM, Webster C (1981) Isolation and characterization of human muscle cells. Proc Natl Acad Sci U S A 78(9):5623–5627CrossRefGoogle Scholar
  8. 8.
    Webster C, Pavlath GK, Parks DR, Walsh FS, Blau HM (1988) Isolation of human myoblasts with the fluorescence-activated cell sorter. Exp Cell Res 174(1):252–265CrossRefGoogle Scholar
  9. 9.
    Walsh FS, Ritter MA (1981) Surface antigen differentiation during human myogenesis in culture. Nature 289(5793):60–64CrossRefGoogle Scholar
  10. 10.
    Alexander MS, Rozkalne A, Colletta A, Spinazzola JM, Johnson S, Rahimov F, Meng H, Lawlor MW, Estrella E, Kunkel LM, Gussoni E (2016) CD82 is a marker for prospective isolation of human muscle satellite cells and is linked to muscular dystrophies. Cell Stem Cell 19(6):800–807. Scholar
  11. 11.
    Lapan AD, Gussoni E (2012) Isolation and characterization of human fetal myoblasts. Methods Mol Biol 798:3–19. Scholar
  12. 12.
    Yaffe D, Saxel O (1977) A myogenic cell line with altered serum requirements for differentiation. Differentiation 7(3):159–166CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Anna Pakula
    • 1
    • 2
  • Janelle M. Spinazzola
    • 1
    • 2
  • Emanuela Gussoni
    • 1
    • 2
    • 3
    Email author
  1. 1.Division of Genetics and GenomicsBoston Children’s HospitalBostonUSA
  2. 2.Department of PediatricsHarvard Medical SchoolBostonUSA
  3. 3.The Stem Cell Program at Boston Children’s HospitalBostonUSA

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