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 and degeneration, mostly due to its resident stem cells2; these are also called satellite cells and are localized under the basal lamina in the periphery of the muscle fibers3.
The core cells of adult skeletal muscle are the myofibers (long syncytial multinuclear cells) and the satellite cells (stem cells with myogenic potential that are quiescent until an injury activates them). The latter cells are the central cells of muscle regeneration, and this process cannot occur in their absence4,5,6,7. In their immediate microenvironment, there are multiple cell types and molecular factors that signal to them. This niche is gradually established throughout development and until adulthood8. Adult muscle contains multiple cell types (endothelial cells, pericytes, macrophages, fibro-adipogenic progenitors-FAPs, regulatory T cells, etc.)9,10 and extracellular matrix components (laminins, collagens, fibronectin, fibrillins, periostin, etc.)11 that interact with each other and with the satellite cells in the context of health, disease, and regeneration.
Preserving this complex niche in experimental settings is fundamental but challenging. Equally difficult is to maintain or return to quiescence, a cell state that is critical for satellite cells9. Several methods have been introduced to partially tackle these challenges, each with its advantages and disadvantages (detailed in the discussion section). Here, a method is presented that can partially overcome these two barriers. Muscles are initially harvested and then broken down mechanically and enzymatically before the heterogenous cell mixture is put into culture. Over the course of the culture, many cell types of the niche are detected, and satellite cells that have returned to quiescence are observed. As a last step of the protocol, the immunofluorescence steps that allow for the detection of each cell type through the use of universally accepted markers, are presented.
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…
The authors have nothing to disclose.
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.
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 |