Abstract
Adult skeletal muscle stem cells, satellite cells, remain in an inactive or quiescent state in vivo under physiological conditions. Progression through the cell cycle, including activation of quiescent cells, is a tightly regulated process. Studies employing in vitro culture of satellite cells, primary human myoblasts (PHMs), necessitate isolation myoblasts from muscle biopsies. Further studies utilizing these cells should endeavour to represent their native in vivo characteristics as closely as possible, also considering variability between individual donors. This study demonstrates the approach of utilizing KnockOut™ Serum Replacement (KOSR)-supplemented culture media as a quiescence-induction media for 10 days in PHMs isolated and expanded from three different donors. Cell cycle analysis demonstrated that treatment resulted in an increase in G1 phase and decreased S phase proportions in all donors (p < 0.005). The proportions of cells in G1 and G2 phases differed in proliferating myoblasts when comparing donors (p < 0.05 to p < 0.005), but following KOSR treatment, the proportion of cells in G1 (p = 0.558), S (p = 0.606) and G2 phases (p = 0.884) were equivalent between donors. When cultured in the quiescence-induction media, expression of CD34 and Myf5 remained constant above > 98% over time from day 0 to day 10. In contrast activation (CD56), proliferation (Ki67) and myogenic marker MyoD decreased, indicated de-differentiation. Induction of quiescence was accompanied in all three clones by fold change in p21 mRNA greater than 3.5 and up to tenfold. After induction of quiescence, differentiation into myotubes was not affected. In conclusion, we describe a method to induce quiescence in PHMs from different donors.
Similar content being viewed by others
References
Agley CC, Rowlerson AM, Velloso CP, Lazarus NL, Harridge SDR (2015) Isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle. JoVE J Vis Exp. https://doi.org/10.3791/52049
Asakura A, Hirai H, Kablar B, Morita S, Ishibashi J, Piras BA, Christ AJ, Verma M, Vineretsky KA, Rudnicki MA (2007) Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle. Proc Natl Acad Sci 104:16552–16557. https://doi.org/10.1073/pnas.0708145104
Ashihara T, Baserga R (1979) Cell synchronization. Methods in enzymology, cell culture. Academic Press, Cambridge, pp 248–262. https://doi.org/10.1016/S0076-6879(79)58141-5
Boldrin L, Muntoni F, Morgan JE (2010) Are human and mouse satellite cells really the same? J Histochem Cytochem 58:941–955. https://doi.org/10.1369/jhc.2010.956201
Brunner D, Frank J, Appl H, Schöffl H, Pfaller W, Gstraunthaler G (2010) The serum-free media interactive online database. ALTEX Altern Anim Exp 27:53–62. https://doi.org/10.14573/altex.2010.1.53
Crist CG, Montarras D, Buckingham M (2012) Muscle satellite cells are primed for myogenesis but maintain quiescence with sequestration of myf5 mRNA targeted by microrna-31 in mrnp granules. Cell Stem Cell 11:118–126. https://doi.org/10.1016/j.stem.2012.03.011
De La Garza-Rodea AS, Van Der Velde-Van Dijke I, Boersma H, Gonçalves MAFV, Van Bekkum DW, De Vries AAF, Knaän-Shanzer S (2012) Myogenic properties of human mesenchymal stem cells derived from three different sources. Cell Transplant 21:153–173. https://doi.org/10.3727/096368911X580554
Detela G, Bain OW, Kim H-W, Williams DJ, Mason C, Mathur A, Wall IB (2018) Donor variability in growth kinetics of healthy hmscs using manual processing: considerations for manufacture of cell therapies. Biotechnol J. https://doi.org/10.1002/biot.201700085
Dhawan J, Farmer SR (1990) Regulation of alpha 1 (I)-collagen gene expression in response to cell adhesion in Swiss 3T3 fibroblasts. J Biol Chem 265:9015–9021
Dike LE, Farmer SR (1988) Cell adhesion induces expression of growth-associated genes in suspension-arrested fibroblasts. Proc Natl Acad Sci 85:6792–6796. https://doi.org/10.1073/pnas.85.18.6792
Fuchs E, Tumbar T, Guasch G (2004) Socializing with the neighbors: stem cells and their niche. Cell 116:769–778. https://doi.org/10.1016/S0092-8674(04)00255-7
Fukada S (2011) Molecular regulation of muscle stem cells by “quiescence genes”. Yakugaku Zasshi 131:1329–1332. https://doi.org/10.1248/yakushi.131.1329
Gayraud-Morel B, Chrétien F, Flamant P, Gomès D, Zammit PS, Tajbakhsh S (2007) A role for the myogenic determination gene myf5 in adult regenerative myogenesis. Dev Biol 312:13–28. https://doi.org/10.1016/j.ydbio.2007.08.059
Hindi L, McMillan JD, Afroze D, Hindi SM, Kumar A (2017) Isolation, culturing, and differentiation of primary myoblasts from skeletal muscle of adult mice. Bio Protoc. 7(9):e2248. https://doi.org/10.21769/BioProtoc.2248
Ishii Y, Nhiayi MK, Tse E, Cheng J, Massimino M, Durden DL, Vigneri P, Wang JY (2015) Knockout serum replacement promotes cell survival by preventing BIM from inducing mitochondrial cytochrome C release. PLoS ONE. 10(10):e0140585. https://doi.org/10.1371/journal.pone.0140585
Kitzmann M, Fernandez A (2001) Crosstalk between cell cycle regulators and the myogenic factor MyoD in skeletal myoblasts. Cell Mol Life Sci CMLS 58:571–579. https://doi.org/10.1007/PL00000882
Kuang S, Gillespie MA, Rudnicki MA (2008) Niche regulation of muscle satellite cell self-renewal and differentiation. Cell Stem Cell 2:22–31. https://doi.org/10.1016/j.stem.2007.12.012
Langan TJ, Chou RC (2011) Synchronization of mammalian cell cultures by serum deprivation. In: Banfalvi G (ed) cell cycle synchronization: methods and protocols, methods in molecular biology. Humana Press, Totowa, pp 75–83. https://doi.org/10.1007/978-1-61779-182-6_5
Li J, Han S, Cousin W, Conboy IM (2015) Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells. Stem cells (Dayton, Ohio) 33(3):951–961. https://doi.org/10.1002/stem.1908
Liu L, Cheung TH, Charville GW, Hurgo BMC, Leavitt T, Shih J, Brunet A, Rando TA (2013) Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging. Cell Rep 4(1):189–204. https://doi.org/10.1016/j.celrep.2013.05.043
Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495
Megeney LA, Kablar B, Garrett K, Anderson JE, Rudnicki MA (1996) MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev 10:1173–1183. https://doi.org/10.1101/gad.10.10.1173
Milasincic DJ, Dhawan J, Farmer SR (1996) Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts. Vitro Cell Dev Biol Anim 32:90–99. https://doi.org/10.1007/BF02723040
Olson EN (1990) MyoD family: a paradigm for development? Genes Dev 4:1454–1461. https://doi.org/10.1101/gad.4.9.1454
Ono Y, Masuda S, Nam H, Benezra R, Miyagoe-Suzuki Y, Takeda S (2012) Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle. J Cell Sci 125:1309–1317. https://doi.org/10.1242/jcs.096198
Rando TA, Blau HM (1994) Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125(6):1275–1287. https://doi.org/10.1083/jcb.125.6.1275
Sabourin LA, Girgis-Gabardo A, Seale P, Asakura A, Rudnicki MA (1999) Reduced differentiation potential of primary MyoD−/− myogenic cells derived from adult skeletal muscle. J Cell Biol 144:631–643. https://doi.org/10.1083/jcb.144.4.631
Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM (2008) Self-renewal and expansion of single transplanted muscle stem cells. Nature 456:502–506. https://doi.org/10.1038/nature07384
Schultz E (1996) Satellite cell proliferative compartments in growing skeletal muscles. Dev Biol 175:84–94. https://doi.org/10.1006/dbio.1996.0097
Sellathurai J, Cheedipudi S, Dhawan J, Schrøder HD (2013) A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts. PLoS ONE. https://doi.org/10.1371/journal.pone.0064067
Shahini A, Vydiam K, Choudhury D, Rajabian N, Nguyen T, Lei P, Andreadis ST (2018) Efficient and high yield isolation of myoblasts from skeletal muscle. Stem Cell Res. 30:122–129. https://doi.org/10.1016/j.scr.2018.05.017
Siddappa R, Licht R, van Blitterswijk C, de Boer J (2007) Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering. J Orthop Res Off Publ Orthop Res Soc 25:1029–1041. https://doi.org/10.1002/jor.20402
Smith C, Kruger MJ, Smith RM, Myburgh KH (2008) The inflammatory response to skeletal muscle injury. Sports Med 38:947–969. https://doi.org/10.2165/00007256-200838110-00005
Spinazzola J, Gussoni E (2017) Isolation of primary human skeletal muscle cells. Bio-Protoc 7:1–19. https://doi.org/10.21769/BioProtoc.2591
Steyn PJ, Dzobo K, Smith RI, Myburgh KH (2019) Interleukin-6 induces myogenic differentiation via JAK2-STAT3 signaling in mouse C2C12 myoblast cell line and primary human myoblasts. Int J Mol Sci. https://doi.org/10.3390/ijms20215273
Vousden KH, Prives C (2009) Blinded by the light: the growing complexity of p53. Cell 3:413–431. https://doi.org/10.1016/j.cell.2009.04.037
Xiang C, Jia BY, Quan GB, Zhang B, Shao QY, Zhao ZY, Hong QH (2019) Wu GQ (2019) Effect of Knockout serum replacement during postwarming recovery culture on the development and quality of vitrified parthenogenetic porcine blastocysts. Biopreserv Biobank. 17(4):342–351. https://doi.org/10.1089/bio.2018.0132
Acknowledgements
The authors wish to thank Mrs Lize Engelbrecht and Ms Rozanne Adams from the Central Analytical Facility at Stellenbosch University for their technical assistance with imaging and flow cytometry.
Funding
This study was funded by the National Research Foundation: South African Research Chairs Initiative (SARChI). Grant number: SARCI150212114075
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interests
The authors declare no conflicts of interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
10616_2019_365_MOESM1_ESM.docx
Supplementary material 1. Supplementary Figure 1: Representative images (A, B) of cell cycle analysis by flow cytometry of PHMs. Cell cycle analysis of proliferation (A) and quiescence (B) of S6.3 N (1) is represented to illustrate the method by which the data was acquired. Supplementary Table 1: qPCR instrument settings used to perform analysis on acquired samples. Supplementary Table 2: Details of the antibodies used in the flow cytometric assessment of quiescent PHMs including nuclear and cell surface markers. Supplementary Table 3 (A, B, C): Raw data Ct values of qPCR of PHMs from 3 subjects (3A - S6.3; 3B - KH1; 3C - KH3). (DOCX 58 kb)
Rights and permissions
About this article
Cite this article
Gudagudi, K.B., d’Entrèves, N.P., Woudberg, N.J. et al. In vitro induction of quiescence in isolated primary human myoblasts. Cytotechnology 72, 189–202 (2020). https://doi.org/10.1007/s10616-019-00365-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-019-00365-8