Chick embryonic cells as a source for generating in vitro model of muscle cell dystrophy

  • Verma Urja
  • Kashmira Khaire
  • Suresh Balakrishnan
  • Gowri Kumari UgginiEmail author


Chick embryonic cells can be used to develop an easy and economical in vitro model for conducting studies on the disease muscle dystrophy (MD). For this, the limb myoblasts from 11th day chick embryo were isolated and cultured. To this muscle cell culture, anti-dystroglycan antibody (IIH6) was added so as to target the α-dystroglycan and disrupt the connection between the cytoskeletal proteins and the extracellular matrix (which is a characteristic feature of MD). Cells were allowed to differentiate further and the morphometrics and mRNA expression were studied. The IIH6-treated muscle cells displayed changes in morphometry, contractibility, and also atrophy was observed when compared to the control cultures. Concomitant gene expression studies showed an upregulation in TGF-β expression, while the muscle sculpture genes MYOD1, MYF5, LAMA2 and MYOG were downregulated resembling the MD in vivo. This simple and cost-effective method can be useful in studies to further understand the disease mechanism and also in conducting initial studies on effect of novel pharmacological agents.


Chick embryo Embryonic cells Muscle dystrophy In vitro model α-dystroglycan 



This work was supported by University Grants Commission, New Delhi, grant number F.No.43–593/2014(SR).


  1. Abmayr SM, Pavlath GK (2012) Myoblast fusion: lessons from flies and mice. Development 139(4):641–656CrossRefGoogle Scholar
  2. Asmundson VS, Julian LM (1956) Inherited muscle abnormality in the domestic fowl. J Heredity 47(5):248–252CrossRefGoogle Scholar
  3. Bentzinger CF, Wang YX, Rudnicki MA (2012) Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol 4(2):a008342CrossRefGoogle Scholar
  4. Biondi O, Villemeur M, Marchand A, Chretien F, Bourg N, Gherardi RK, Richard I, Authier FJ (2013) Dual effects of exercise in dysferlinopathy. Am J Pathol 182(6):2298–2309CrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254CrossRefGoogle Scholar
  6. Braun T, Bober E, Buschhausen-Denker G, Kohtz S, Grzeschik KH, Arnold HH, Kotz S (1989) Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products. The EMBO J 8(12):3617–3625CrossRefGoogle Scholar
  7. Brown SC, Fassati A, Popplewell L, Page AM, Henry MD, Campbell KP, Dickson G (1999) Dystrophic phenotype induced in vitro by antibody blockade of muscle alpha-dystroglycan-laminin interaction. J Cell Sci 112(2):209–216PubMedGoogle Scholar
  8. Campbell KP, Kahl SD (1989) Association of dystrophin and an integral membrane glycoprotein. Nature 338(6212):259–262CrossRefGoogle Scholar
  9. Davis RL, Weintraub H, Lassar AB (1987) Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51(6):987–1000CrossRefGoogle Scholar
  10. Dennis JE, Shimizu T, Reinach FC, Fischman DA (1984) Localization of C-protein isoforms in chicken skeletal muscle: ultrastructural detection using monoclonal antibodies. The J Cell Biol 98(4):1514–1522CrossRefGoogle Scholar
  11. Emery AE, Muntoni F, Quinlivan RC (2015) Duchenne muscular dystrophy. OUP Oxford, England UKCrossRefGoogle Scholar
  12. Friedrich O, Von Wegner F, Chamberlain JS, Fink RH, Rohrbach P (2008) L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle. PLoS One 3(3):e1762CrossRefGoogle Scholar
  13. Grodzki AC, Berenstein E (2010) antibody purification: affinity chromatography–protein a and protein G Sepharose. In: Immunocytochemical methods and protocols. Humana Press, New York, United States, pp 33–41Google Scholar
  14. Godinho RO (2006) In vitro development of skeletal muscle fiber. Braz J Morphol Sci 23:173–186Google Scholar
  15. Goldstein JA, McNally EM (2010) Mechanisms of muscle weakness in muscular dystrophy. J Gen Physiol 136(1):29–34CrossRefGoogle Scholar
  16. Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88(1):49–92CrossRefGoogle Scholar
  17. Henry MD, Campbell KP (1999) Dystroglycan inside and out. Curr Opin Cell Biol 11(5):602–607CrossRefGoogle Scholar
  18. Hernández-Ochoa EO, Pratt SJ, Garcia-Pelagio KP, Schneider MF, Lovering RM (2015) Disruption of action potential and calcium signaling properties in malformed myofibers from dystrophin-deficient mice. Physiol Rep 3(4)CrossRefGoogle Scholar
  19. Hutson MR, Kirby ML (2007) Model systems for the study of heart development and disease: cardiac neural crest and conotruncal malformations. Semin Cell Dev Biol 18(1):101–110CrossRefGoogle Scholar
  20. Imamura M, Nakamura A, Mannen H, Takeda SI (2015) Characterization of WWP1 protein expression in skeletal muscle of muscular dystrophy chickens. J Biochem 159(2):171–179CrossRefGoogle Scholar
  21. Isaacs ER, Bradley WG, Henderson G (1973) Longitudinal fibre splitting in muscular dystrophy: a serial cinematographic study. J Neurol Neurosurg Psychiatry 36(5):813–819CrossRefGoogle Scholar
  22. Jimenez-Mallebrera C, Brown SC, Sewry CA, Muntoni F (2005) Congenital muscular dystrophy: molecular and cellular aspects. Cell Mol Life Sci 62(7–8):809–823CrossRefGoogle Scholar
  23. Li Y, Foster W, Deasy BM, Chan Y, Prisk V, Tang Y, Cummins J, Huard J (2004) Transforming growth factor-β1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis. Am J Pathol 164(3):1007–1019CrossRefGoogle Scholar
  24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4):402–408CrossRefGoogle Scholar
  25. Manning J, O’Malley D (2015) What has the mdx mouse model of duchenne muscular dystrophy contributed to our understanding of this disease? J Muscle Res Cell Motil 36(2):155–167CrossRefGoogle Scholar
  26. Masaki T (1974) Immunochemical comparison of myosins from chicken cardiac, fast white, slow red, and smooth muscle. J Biochem 76(2):441–449CrossRefGoogle Scholar
  27. McColl R, Nkosi M, Snyman C, Niesler C (2016) Analysis and quantification of in vitro myoblast fusion using the LADD multiple stain. Biotechniques 61:323–326CrossRefGoogle Scholar
  28. McGreevy JW, Hakim CH, McIntosh MA, Duan D (2015) Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Dis Model Mech 8(3):195–213CrossRefGoogle Scholar
  29. Nunes VA, Gozzo AJ, Cruz-Silva I, Juliano MA, Viel TA, Godinho RO, Meirelles FV, Sampaio MU, Sampaio CA, Araujo MS (2005) Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells. Comp Biochem Physiol C Toxicol Pharmacol 141(3):225–240CrossRefGoogle Scholar
  30. Paulino N, Dantas AP, Bankova V, Longhi DT, Scremin A, de Castro SL, Calixto JB (2003) Bulgarian propolis induces analgesic and anti-inflammatory effects in mice and inhibits in vitro contraction of airway smooth muscle. J Pharmacol Sci 93(3):307–313CrossRefGoogle Scholar
  31. Ribble D, Goldstein NB, Norris DA, Shellman YG (2005) A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol 5(1):12CrossRefGoogle Scholar
  32. Salani S, Donadoni C, Rizzo F, Bresolin N, Comi GP, Corti S (2012) Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies. J Cell Mol Med 16(7):1353–1364CrossRefGoogle Scholar
  33. Schmalbruch H (1976) Muscle fibre splitting and regeneration in diseased human muscle. Neuropathol Appl Neurobiol 2(1):3–19CrossRefGoogle Scholar
  34. Strober W (2015) Trypan blue exclusion test of cell viability. Curr Protoc Immunol 111(1):A3-BGoogle Scholar
  35. Theadom A, Rodrigues M, Roxburgh R, Balalla S, Higgins C, Bhattacharjee R, Jones K, Krishnamurthi R, Feigin V (2014) Prevalence of muscular dystrophies: a systematic literature review. Neuroepidemiology 43(3–4):259–268CrossRefGoogle Scholar
  36. Vieira NM, Elvers I, Alexander MS, Moreira YB, Eran A, Gomes JP, Marshall JL, Karlsson EK, Verjovski-Almeida S, Lindblad-Toh K, Kunkel LM (2015) Jagged 1 rescues the Duchenne muscular dystrophy phenotype. Cell 163(5):1204–1213CrossRefGoogle Scholar
  37. Williamson RA, Henry MD, Daniels KJ, Hrstka RF, Lee JC, Sunada Y, Ibraghimov-Beskrovnaya O, Campbell KP (1997) Dystroglycan is essential for early embryonic development: disruption of Reichert's membrane in Dag1-null mice. Hum Mol Genet 6(6):831–841CrossRefGoogle Scholar
  38. Wilson BW, Randall WR, Patterson GT, Entrikin RK (1979) Major physiologic and histochemical characteristics of inherited dystrophy of the chicken. Ann N Y Acad Sci 317(1):224–246CrossRefGoogle Scholar
  39. Wright WE, Sassoon DA, Lin VK (1989) Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56(4):607–617CrossRefGoogle Scholar
  40. Ziv I, Melamed E, Nardi N, Luria D, Achiron A, Offen D, Barzilai A (1994) Dopamine induces apoptosis-like cell death in cultured chick sympathetic neurons—a possible novel pathogenetic mechanism in Parkinson’s disease. Neurosci Lett 170(1):136–140CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  • Verma Urja
    • 1
  • Kashmira Khaire
    • 1
  • Suresh Balakrishnan
    • 1
  • Gowri Kumari Uggini
    • 1
    Email author
  1. 1.Department of Zoology, Faculty of ScienceThe Maharaja Sayajirao University of BarodaVadodaraIndia

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