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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Published: June 2nd, 2023

DOI:

10.3791/65433

1Univ Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France, 2Ecole nationale vétérinaire d'Alfort, IMRB, F-94700 Maisons-Alfort, France, 3EFS, IMRB, F-94010 Creteil, France, 4AP-HP, Hopital Mondor, Service d'histologie, F-94010 Creteil, France
* These authors contributed equally

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

Movement, breathing, metabolism, body posture, and body temperature maintenance all depend on skeletal muscle, and malfunctions in the skeletal muscle can, thus, cause debilitating pathologies (i.e., myopathies, muscular dystrophies, etc.)1. Given its essential functions and abundance, skeletal muscle has drawn the attention of research labs worldwide that strive to understand the key aspects that support normal muscle function and can serve as therapeutic targets. In addition, skeletal muscle is a widely used model to study regeneration and stem cell function, as healthy muscle can fully self-repair after complete injury ....

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All experiments complied with French and EU animal regulations at the Institut Mondor de Recherche Biomédicale (INSERM U955), notably the directive 2010/63/UE. Animals were kept in a controlled and enriched environment at the animal facilities with certification numbers A94 028 379 and D94-028-028; they were handled only by authorized researchers and animal caretakers, and they were visually inspected by animal housing personnel for signs of discomfort during their lifetime. They were euthanized by cervical dis.......

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This protocol allows for muscle cell culture while preserving the satellite cells and most cells from their endogenous niche. Figure 2 summarizes the main steps of the protocol, while essential parts of the dissection and digestion are presented in Figure 1. Dissection of the hindlimb musculature is recommended (Figure 1A-C), as this group of muscles is well studied and shares a developmental origin.......

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Adult skeletal muscle function is underpinned by a finely orchestrated set of cellular interactions and molecular signals. Here, a method is presented that allows for the study of these parameters in an ex vivo setting that closely resembles the physiological microenvironment.

Several groups have reported in vitro methods to culture myogenic cells. These methods aimed to isolate satellite cells to study their myogenic progenitor properties. Two main approaches are used to iso.......

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For Figure 2, templates from Servier Medical Art (https://smart.servier.com/) were used. The FR lab is supported by the Association Française contre les Myopathies - AFM via TRANSLAMUSCLE (grants 19507 and 22946), the Fondation pour la Recherche Médicale - FRM (EQU202003010217, ENV202004011730, ECO201806006793), the Agence Nationale pour la Recherche - ANR (ANR-21-CE13-0006-02, ANR-19-CE13-0010, ANR-10-LABX-73), and the La Ligue Contre le Cancer (IP/SC-17130). The above funders had no role in the design, collection, analysis, interpretation, or reporting of this study or the writing of this manuscript.

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Name Company Catalog Number Comments
anti-CD31 BD 550274 dilution 1:100
anti-FOSB Santa Cruz sc-7203 dilution 1:200
anti-GFP Abcam ab13970 dilution 1:1000
anti-Ki67 Abcam ab16667 dilution 1:1000
anti-MyHC DSHB MF20-c dilution 1:400
anti-MYOD Active Motif 39991 dilution 1:200
anti-MYOG Santa Cruz sc-576 dilution 1:150
anti-Pax7 Santa Cruz sc-81648 dilution 1:100
anti-PDGFRα Invitrogen PA5-16571 dilution 1:50
b-FGF Peprotech 450-33 concentration 4 ng/mL
bovine serum albumin (BSA) – used for digestion  Sigma Aldrich A7906-1006 concentration 0.2%
BSA IgG-free, protease-free – used for staining Jackson ImmunoResearch 001-000-162 concentration 5%
cell strainer 40 um Dominique Dutscher 352340
cell strainer 70 um Dominique Dutscher 352350
cell strainer 100 um Dominique Dutscher 352360
Collagenase Roche 10103586001 concentration 0.5 U/mL
Dimethyl sulfoxide (DMSO) Euromedex UD8050-05-A
Dispase Roche 4942078001 concentration 3 U/mL
Dissection forceps size 5 Fine Science Tools 91150-20
Dissection forceps size 55 Fine Science Tools 11295-51
Dissection scissors (big, straight) Fine Science Tools 9146-11 ideal for chopping
Dissection scissors (small, curved) Fine Science Tools 15017-10
Dissection scissors (small, straight) Fine Science Tools 14084-08
Dulbecco's Modified Eagle's Medium (DMEM) ThermoFisher 41966-029
EdU Click-iT kit ThermoFisher C10340
Fetal bovine serum – option 1 Eurobio CVF00-01
Fetal bovine serum – option 2 Gibco 10270-106 
Matrigel Corning Life Sciences 354234 coating solution
Parafilm Dominique Dutscher 090261 flexible film
Penicillin streptomycin Gibco 15140-122
Paraformaldehyde – option 1 PanReac AppliChem ITW Reagents 211511.1209 concentration 4%
Paraformaldeyde – option 2 ThermoFisher 28908 concentration 4%
Shaking water bath ThermoFisher TSSWB27
TritonX100 Sigma Aldrich T8532-500 ML concentration 0.5%
Wild-type mice Janvier C57BL/6NRj

  1. Frontera, W. R., Ochala, J. Skeletal muscle: A brief review of structure and function. Calcified Tissue International. 96 (3), 183-195 (2015).
  2. Forcina, L., Cosentino, M., Musarò, A. Mechanisms regulating muscle regeneration: Insights into the interrelated and time-dependent phases of tissue healing. Cells. 9 (5), 1297 (2020).
  3. Mauro, A. Satellite cell of skeletal muscle fibers. Journal of Biophysical and Biochemical Cytology. 9 (2), 493-495 (1961).
  4. Lepper, C., Partridge, T. A., Fan, C. -. M. An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Development. 138 (17), 3639-3646 (2011).
  5. McCarthy, J. J., et al. Effective fiber hypertrophy in satellite cell-depleted skeletal muscle. Development. 138 (17), 3657-3666 (2011).
  6. Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A., Kardon, G. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development. 138 (17), 3625-3637 (2011).
  7. Sambasivan, R., et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development. 138 (17), 3647-3656 (2011).
  8. Hicks, M. R., Pyle, A. D. The emergence of the stem cell niche. Trends in Cell Biology. 33 (22), 112-123 (2022).
  9. Relaix, F., et al. Perspectives on skeletal muscle stem cells. Nature Communications. 12 (1), 692 (2021).
  10. Gama, J. F. G., et al. Role of regulatory T cells in skeletal muscle regeneration: A systematic review. Biomolecules. 12 (6), 817 (2022).
  11. Loreti, M., Sacco, A. The jam session between muscle stem cells and the extracellular matrix in the tissue microenvironment. NPJ Regenerative Medicine. 7 (1), 16 (2022).
  12. Sambasivan, R., et al. Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates. Developmental Cell. 16 (6), 810-821 (2009).
  13. Pereira, P. D., et al. Quantification of cell cycle kinetics by EdU (5-ethynyl-2'-deoxyuridine)-coupled-fluorescence-intensity analysis. Oncotarget. 8 (25), 40514-40532 (2017).
  14. Bismuth, K., Relaix, F. Genetic regulation of skeletal muscle development. Experimental Cell Research. 316 (18), 3081-3086 (2010).
  15. Yin, H., Price, F., Rudnicki, M. A. Satellite cells and the muscle stem cell niche. Physiological Reviews. 93 (1), 23-67 (2013).
  16. Lertkiatmongkol, P., Liao, D., Mei, H., Hu, Y., Newman, P. J. Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31). Current Opinion in Hematology. 23 (3), 253-259 (2016).
  17. Scholzen, T., Gerdes, J. The Ki-67 protein: From the known and the unknown. Journal of Cellular Physiology. 182 (3), 311-322 (2000).
  18. Abou-Khalil, R., Le Grand, F., Chazaud, B. Human and murine skeletal muscle reserve cells. Stem Cell Niche. 1035, 165-177 (2013).
  19. Pasut, A., Oleynik, P., Rudnicki, M. A. Isolation of muscle stem cells by fluorescence activated cell sorting cytometry. Methods in Molecular Biology. 798, 53-64 (2011).
  20. Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nature Protocols. 10 (10), 1612-1624 (2015).
  21. Montarras, D., et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science. 309 (5743), 2064-2067 (2005).
  22. Qu, Y., Edwards, K., Barrow, J. Isolation, culture, and use of primary murine myoblasts in small-molecule screens. STAR Protocols. 4 (2), 102149 (2023).
  23. Danoviz, M. E., Yablonka-Reuveni, Z. Skeletal muscle satellite cells: Background and methods for isolation and analysis in a primary culture system. Methods in Molecular Biology. 798, 21-52 (2011).
  24. Saclier, M., Theret, M., Mounier, R., Chazaud, B. Effects of macrophage conditioned-medium on murine and human muscle cells: analysis of proliferation, differentiation, and fusion. Methods in Molecular Biology. 1556, 317-327 (2017).
  25. Giordani, L., et al. High-dimensional single-cell cartography reveals novel skeletal muscle-resident cell populations. Molecular Cell. 74 (3), 609-621 (2019).
  26. Tabula Muris Consortium et al. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature. 562 (7727), 367-372 (2018).
  27. Brunetti, J., Koenig, S., Monnier, A., Frieden, M. Nanopattern surface improves cultured human myotube maturation. Skeletal Muscle. 11 (1), 12 (2021).
  28. Denes, L. T., et al. Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation. Skeletal Muscle. 9 (1), 17 (2019).
  29. LaFramboise, W. A., et al. Effect of muscle origin and phenotype on satellite cell muscle-specific gene expression. Journal of Molecular and Cellular Cardiology. 35 (10), 1307-1318 (2003).
  30. Azhar, M., Wardhani, B. W. K., Renesteen, E. The regenerative potential of Pax3/Pax7 on skeletal muscle injury. Journal of Generic Engineering and Biotechnology. 20 (1), 143 (2022).
  31. Hardy, D., et al. Comparative study of injury models for studying muscle regeneration in mice. PLoS One. 11 (1), e0147198 (2016).

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