For Figure 2, templates from Servier Medical Art (https://smart.servier.com/) were used. The FR lab is supported by the Association Fran&#231;aise contre les Myopathies - AFM&#160;via TRANSLAMUSCLE (grants 19507 and 22946), the Fondation pour la Recherche M&#233;dicale - 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.

All experiments complied with French and EU animal regulations at the Institut Mondor de Recherche Biom&#233;dicale (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&#160;cervical dislocation prior to dissection. No interventional procedures were performed during the animals&#39; lifetimes; thus, acquiring approval for the procedure from an Ethics Committee and the French Ministry of Higher Education, Research and Innovation was not necessary. Indeed, no ethics clearance is required for euthanization and post-mortem dissection according to the directive 2010/63/UE. The results presented in this manuscript are from the wild-type C57BL/6NRj line (see Table of Materials) and the transgenic Tg:Pax7-nGFP line12(bred by our team). The protocol was applied to male and female mice aged 8-12 weeks of age.
1. Reagent and equipment preparation pre-digestion
<ol>
	<li>Spray the dissection tools (straight and curved scissors, forceps, see the Table of Materials) with 70% ethanol, and dry them with paper. Coat a cork plate with aluminum foil, and keep 10 cm Petri dishes (one per animal) nearby. Have paper and 70% ethanol in reach. 
	NOTE: At the end of the dissection, rinse the dissection tools with water, then spray them with 70% ethanol, and dry them with paper.</li>
	<li>Set a rotating water bath to 37 &#176;C, and prepare the digestion mix (20 mL/animal) by combining DMEM with 1% penicillin-streptomycin, 0.5 U/mL collagenase, 3 U/mL dispase (see the Table of Materials), and 0.2% BSA in 50 mL tube(s).</li>
	<li>Pass the digestion mix through a 0.22 &#181;m filter in a cell culture hood. 
	NOTE: It is recommended to prepare the digestion mix fresh every time.</li>
</ol>
2. Reagent and equipment preparation post-digestion
<ol>
	<li>Post-digestion, the mix can be frozen or cultured. For freezing, prepare 10% DMSO:90% fetal bovine serum (FBS), as well as a set of cryotubes (1 mL of cell suspension per 2 mL cryotube). For the culture, prepare culture medium (DMEM supplemented with 1% penicillin-streptomycin, 4 ng/mL bFGF, and 20% FBS) and a set of 8-well plates. The plates must be coated before plating the cells (details are provided in step 7.1).</li>
	<li>For the staining, prepare 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) (0.15 mL/well of the 8-well plate) and blocking solution (5% IgG-free bovine serum albumin [BSA] in PBS; 0.15 mL/well of the 8-well plate). 
	CAUTION: Do not breathe in the PFA powder; prepare and handle it under a chemical hood.</li>
</ol>
3. Dissection
<ol>
	<li>Spray the euthanized animal with 70% ethanol. Make a horizontal incision (left side of the body to the right side) with big scissors at the level of the abdomen, and cut around the waist. Pull the skin off of the hindlimbs to reveal the muscles (Figure 1A).</li>
	<li>Place the animal on the aluminum foil-covered cork plate, and pin down the opposite forelimb and hindlimb. Quickly remove all the hindlimb muscles (front and back) into a 10 cm Petri dish placed on ice (Figure 1B,C). Take special care to remove the adipose tissue from the areas around the quadriceps and the posterior muscles. Fascia, nerves, and tendons can also be removed at this point if this does not compromise the overall time spent on dissection. 
	NOTE: An optimal dissection time for both the hindlimbs should be around 15-20 min. It is advised that the dissection time does not exceed 30 min.</li>
	<li>Add drops of DMEM to the muscles occasionally to keep them moist, but not too much, as this will render chopping difficult. Repeat for the other hindlimb. Once all the muscles of one animal are in the Petri dish (Figure 1D), chop them finely with scissors for 7-10 min to obtain a smooth homogenate (Figure 1E). 
	NOTE: In this protocol, DMEM supplemented with L-glutamine, pyruvate, and 4.5 g/L D-glucose is used.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65433/65433fig01.jpg" /> 
Figure 1: Pre-culture muscle preparation. (A) The skin is removed to reveal the hindlimb muscles, as described in step 3.1. (B,C) All the hindlimb muscles are harvested (B) around and (C) between the bones, as described in step 3.2. (D) The harvested muscles are placed in a 10 cm Petri dish on ice with DMEM drops to keep them moist, as described in step 3.3. (E) The muscles are finely chopped with scissors until a smooth paste is obtained with the consistency depicted in this image. (F) An image of the pellet after the final centrifugation; the blue arrow highlights the pellet, which is against the tube, under the dashed blue line. <a href="https://www.jove.com/files/ftp_upload/65433/65433fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Digestion
NOTE: At the end of the digestion, a centrifuge at 4 &#176;C, a bucket of ice, three cell strainers (100 um, 70 um, 40 um), and three 50 mL tubes (per animal) are needed for section 5.
<ol>
	<li>Prepare and filter the digestion mix as described in step 1.2. Keep the mix on ice.</li>
	<li>Once all muscles are chopped, place the homogenate in a 50 mL tube with 20 mL of digestion mix. Wrap the edges of the lid with flexible film to prevent leaking, and place the tube in a 37 &#176;C shaking water bath at a low to medium speed (50 rpm).</li>
	<li>After 1 h at 37 &#176;C, open the lid, and mix by gently pipetting seven times up and down with a 10 mL pipette to obtain a homogenous mix. Apply new film around the lid, and place it back in the shaking water bath. After 1 h, remove the tube, and turn off the bath. 
	NOTE: For culture, use this incubation time to coat the plates as described in step 7.1 before moving to section 5.</li>
</ol>
5. Filtration
<ol>
	<li>Fill the digestion tube with cold DMEM (supplemented with 1% penicillin-streptomycin) up to 50 mL. Mix by inverting the tube three times. Keep the DMEM in an ice bucket for the next steps.</li>
	<li>Place a 100 &#181;m cell strainer on a new 50 mL tube. Pass the digested mix through the cell strainer into the new tube. Centrifuge at 600 x g for 5 min at 4 &#176;C. Pour out the supernatant into a liquid waste container.</li>
	<li>Resuspend the pellet in 1 mL of cold DMEM (supplemented with 1% penicillin-streptomycin). Fill the tube up to 50 mL with the same DMEM. NOTE: If the centrifuging is skipped, the next pellet will be more difficult to identify and maintain.</li>
	<li>Place a 70 &#181;m cell strainer on a new 50 mL tube. Pass the centrifuged/resuspended mix through the cell strainer into the new tube. Centrifuge at 80 x g for 5 min at 4 &#176;C. 
	NOTE: This step is not mandatory but is recommended to eliminate cell debris.</li>
	<li>Place a 40 &#181;m cell strainer on a new 50 mL tube. Pass the supernatant through the cell strainer into the new tube. Centrifuge at 600 x g for 5 min at 4 &#176;C, pour the supernatant into a liquid waste container, and resuspend the pellet in FBS under the culture hood. The pellet is very small at this step (Figure 1F). 
	NOTE: Filtering through the 40 &#181;m strainer removes debris, which would give a non-specific signal in the later staining of the cultures.</li>
</ol>
6. (Optional) Freezing
NOTE: Section 6 is optional. The protocol can be paused after filtering, but this can reduce the cell survival and culture success.
<ol>
	<li>Add DMSO to obtain a 10% DMSO:90% FBS ratio, and transfer to cryotubes (1 mL of resuspended pellet per 2 mL cryotube).</li>
	<li>Place the cryotube at &#8722;80 &#176;C in a polystyrene box overnight. Move to &#8722;150 &#176;C the next day for long-term storage. 
	NOTE: Short-term storage at &#8722;80 &#176;C is also possible.</li>
	<li>When starting the culture, thaw the cryotube quickly in a 37 &#176;C water bath until the cell suspension has thawed. Mix with 4 mL of DMEM under the culture hood. Spin at 600 x g for 5 min at 4 &#176;C. Pipette out the supernatant, and continue as described in step 7.2.</li>
</ol>
7. Culturing
NOTE: Frozen or fresh cell suspensions can be expected to fill 24-32 wells of three to four 8-well plates.
<ol>
	<li>Coat 8-well plates with the coating solution, which should be thawed at 4 &#176;C or on ice (stock coating solution is normally kept at &#8722;20 &#176;C). Add 0.4 mL of coating solution to one well, and pipette it from well to well. After transferring the coating solution through all the wells, it can be recollected and refrozen for future cultures. Keep the coated plates at 37 &#176;C for 30 min before plating the cells.</li>
	<li>Add DMEM (supplemented with 1% penicillin-streptomycin) supplemented with 4 ng/mL bFGF (see Table of Materials) to the FBS-cell suspension to obtain a 20% FBS:80% DMEM ratio. 
	NOTE: Even though the addition of bFGF can be beneficial in primary myoblast cultures and in satellite-like cell production in bulk cultures, its addition is optional, as its omission in bulk cultures of ~7 days does not severely compromise the cell yields.</li>
	<li>Plate 0.4 mL of the suspension per well (from step 7.2) in the coated 8-well plates. 
	NOTE: Calculate 30 cm2 of culture per animal for frozen and fresh preparations.</li>
	<li>Incubate the cultures at 37 &#176;C with 5% CO2 for up to 10 days, changing the medium every day after the culture begins to change to a yellowish color (usually 5-7 days). 
	NOTE: To quantify cells in the S phase of the cell cycle13, add 10&#160;&#181;M EdU 2 h before fixation. To capture the first S phase, add 10 &#181;M EdU from the plating, and fix at 40 h of culture.</li>
</ol>
8. Fixation
NOTE: Sections 8-10 should be conducted at room temperature unless otherwise stated.
<ol>
	<li>Pipette out the culture medium, and fix the cells with 4% PFA (0.15 mL/well). 
	CAUTION: Add PFA under a chemical hood. 
	NOTE: If all the wells are fixed at the same time, incubate with PFA at room temperature for 10 min. If the wells are fixed at different time points, add PFA to the wells to be fixed, and keep the plate in the incubator at 37 &#176;C for 5 min.</li>
	<li>Pipette out the PFA, and add PBS for 10 s (0.15 mL/well). Pipette out the PBS, and add fresh PBS for 5 min (0.15 mL/well). 
	NOTE: If all the wells are fixed at the same time, incubate with PBS at room temperature. If the wells are fixed at different time points, add PBS to the fixed wells, and keep the plate in the incubator at 37 &#176;C for 5 min. Then, add 0.4 mL of PBS, and keep the plate in the incubator for up to 1 week.</li>
</ol>
9. Permeabilization and blocking
<ol>
	<li>When ready to stain, pipette out the PBS, and permeabilize with 0.5% TritonX 100 in PBS (0.15 mL/well) for 8 min. Pipette out the TritonX 100, rinse with PBS for 10 s (0.15 mL/well), pipette out the PBS, and wash with PBS for 5 min (0.15 mL/well).</li>
	<li>Block with 5% IgG-free BSA in PBS for 30-60 min (0.15 mL/well).</li>
</ol>
10. Staining
<ol>
	<li>Pipette out the BSA, and add the primary antibody mix diluted in PBS (0.15 mL/well) (see the Table of Materials; dilutions: anti-CD31 1:100, anti-FOSB 1:200, anti-GFP 1:1,000, anti-KI67 1:1,000, anti-MyHC 1:400, anti-MYOD 1:200, anti-MYOG 1:150, anti-PAX7 1:100, anti-PDGFRa 1:50) for overnight incubation at 4 &#176;C. 
	NOTE: After the antibody incubation, collect the antibody mix, add sodium azide, and keep at 4 &#176;C or &#8722;20 &#176;C (as per the antibody manufacturer&#39;s instructions) for future reuse.</li>
	<li>Pipette out the antibody mix, rinse with PBS for 10 s (0.15 mL/well), pipette out the PBS, and wash with PBS for 5 min (0.15 mL/well).</li>
	<li>Pipette out the washing PBS, add the secondary antibody mix (goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555, goat anti-rat Alexa Fluor 647, goat anti-mouse Alexa Fluor 555, goat anti-chicken Alexa Fluor 488, all used at dilutions of 1:500-1,000) and nucleus marker (e.g., DAPI) diluted in PBS (0.15 mL/well) (see Table of Materials), and incubate for 1 h at room temperature, protected from light.</li>
	<li>Pipette out the secondary antibody mix, rinse with PBS for 10 s (0.15 mL/well), pipette out the PBS, wash with PBS for 5 min (0.15 mL/well), pipette out the PBS, and mount. 
	NOTE: If 8-well plates with removable separators are used, peel off the separators before mounting.</li>
</ol>

Harvesting Mouse Skeletal Muscle to Culture Stem Cells and Their Niche

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The authors declare no conflicts of interest.

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...

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.

<table><tbody><tr><td>anti-CD31</td><td>BD</td><td>550274</td><td>dilution 1:100</td></tr><tr><td>anti-FOSB</td><td>Santa Cruz</td><td>sc-7203</td><td>dilution 1:200</td></tr><tr><td>anti-GFP</td><td>Abcam</td><td>ab13970</td><td>dilution 1:1000</td></tr><tr><td>anti-Ki67</td><td>Abcam</td><td>ab16667</td><td>dilution 1:1000</td></tr><tr><td>anti-MyHC</td><td>DSHB</td><td>MF20-c</td><td>dilution 1:400</td></tr><tr><td>anti-MYOD</td><td>Active Motif</td><td>39991</td><td>dilution 1:200</td></tr><tr><td>anti-MYOG</td><td>Santa Cruz</td><td>sc-576</td><td>dilution 1:150</td></tr><tr><td>anti-Pax7</td><td>Santa Cruz</td><td>sc-81648</td><td>dilution 1:100</td></tr><tr><td>anti-PDGFR&amp;alpha;</td><td>Invitrogen</td><td>PA5-16571</td><td>dilution 1:50</td></tr><tr><td>b-FGF</td><td>Peprotech</td><td>450-33</td><td>concentration 4 ng/mL</td></tr><tr><td>Bovine serum albumin (BSA) &amp;ndash; used for digestion&amp;nbsp;</td><td>Sigma Aldrich</td><td>A7906-1006</td><td>concentration 0.2%</td></tr><tr><td>BSA IgG-free, protease-free &amp;ndash; used for staining</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td>concentration 5%</td></tr><tr><td>Cell strainer 40 um</td><td>Dominique Dutscher</td><td>352340</td><td></td></tr><tr><td>Cell strainer 70 um</td><td>Dominique Dutscher</td><td>352350</td><td></td></tr><tr><td>Cell strainer 100 um</td><td>Dominique Dutscher</td><td>352360</td><td></td></tr><tr><td>Collagenase</td><td>Roche</td><td>10103586001</td><td>concentration 0.5 U/mL</td></tr><tr><td>Culture plate</td><td>Sarstedt</td><td>94.6140.802</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Euromedex</td><td>UD8050-05-A</td><td></td></tr><tr><td>Dispase</td><td>Roche</td><td>4942078001</td><td>concentration 3 U/mL</td></tr><tr><td>Dissection forceps size 5</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Dissection forceps size 55</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Dissection scissors (big, straight)</td><td>Fine Science Tools</td><td>9146-11</td><td>ideal for chopping</td></tr><tr><td>Dissection scissors (small, curved)</td><td>Fine Science Tools</td><td>15017-10</td><td></td></tr><tr><td>Dissection scissors (small, straight)</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>ThermoFisher</td><td>41966-029</td><td></td></tr><tr><td>EdU Click-iT kit</td><td>ThermoFisher</td><td>C10340</td><td></td></tr><tr><td>Fetal bovine serum &amp;ndash; option 1</td><td>Eurobio</td><td>CVF00-01</td><td></td></tr><tr><td>Fetal bovine serum &amp;ndash; option 2</td><td>Gibco</td><td>10270-106&amp;nbsp;</td><td></td></tr><tr><td>Matrigel</td><td>Corning Life Sciences</td><td>354234</td><td>coating solution</td></tr><tr><td>Parafilm</td><td>Dominique Dutscher</td><td>090261</td><td>flexible film</td></tr><tr><td>Paraformaldehyde &amp;ndash; option 1</td><td>PanReac AppliChem ITW Reagents</td><td>211511.1209</td><td>concentration 4%</td></tr><tr><td>Paraformaldeyde &amp;ndash; option 2</td><td>ThermoFisher</td><td>28908</td><td>concentration 4%</td></tr><tr><td>Penicillin streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Shaking water bath</td><td>ThermoFisher</td><td>TSSWB27</td><td></td></tr><tr><td>TritonX100</td><td>Sigma Aldrich</td><td>T8532-500 ML</td><td>concentration 0.5%</td></tr><tr><td>Wild-type mice</td><td>Janvier</td><td>C57BL/6NRj</td><td></td></tr></tbody></table>

unfractionated bulk culture of mouse skeletal muscle to recapitulate niche and stem cell quiescence

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.

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...

Watch this Scientific Journal Video about Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components at JoVE.com

Author Spotlight: Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components 

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.

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

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...

unfractionated-bulk-culture-mouse-skeletal-muscle-to-recapitulate

Research

JoVE Journal

Biology

1.2K Views.  Univ Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France. Ons onderzoek omvat de studie van gezonde en zieke skeletspieren, met speciale nadruk op de satellietcellen. Een van de fundamentele aspecten die we onderzoeken is de identificatie van transcriptionele enhancers die celspecifieke genexpressie coördineren. Dit protocol stelt ons in staat om sneltesten uit te voeren om de specificiteit te bepalen.Op het gebied van skeletspieronderzoek worden single-cell en single nucleus RNA-sequencing-benaderingen vaak gebruikt om verschillende celpopu...

Video: Auteur in de schijnwerpers: Ex Vivo-protocol voor het kweken van slapende spierstamcellen met nichecomponenten 

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Developmental Biology

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Medicine

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

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

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