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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2´-deoxyuridine incorporation assay.

Abstract

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2´-deoxyuridine (EdU) incorporation assay.

Introduction

Skeletal muscle has a high capacity for regeneration due to the presence of muscle stem cells (MuSCs). MuSCs are located on the myofibers, underneath the basal lamina, and reside in a quiescent state of prolonged, reversible cell cycle exit1,2,3,4. Upon injury, MuSCs activate and enter the cell cycle to give rise to amplifying progenitors that can differentiate and fuse to form new myofibers2,5. Previous work has showed that MuSCs are absolutely essential for muscle regeneration

Protocol

The present protocol was performed in accordance with animal care guidelines at Aarhus University and local ethics regulations.

NOTE: Make sure to comply with the regulations of the local ethical committee for animal experimentation and handling of post-mortem rodent samples. Mice are a potential source of allergens; if available, turn on exhaust ventilation and place it over the workspace to avoid excessive exposure to allergens. Alternatively, wear a face mask if the experiment is carried ou.......

Representative Results

Following the protocol for individual skeletal muscle isolation (Figure 2), the gracilis, TA, EDL, GA, soleus, triceps, masseter, and diaphragm muscles were isolated from three Swiss male outbred mice that had been discontinued from a local breeding program (Figure 2). Following tissue dissociation and antibody staining, MuSCs and FAPs from the individual muscles were purified by FACS (Figure 3). The initial gating was obtained with.......

Discussion

Several steps are key in the execution of this protocol to achieve good yields. The individual muscles have a small volume compared to the amount of muscle used in bulk isolation protocols. This results in a risk of the muscle drying out during the dissection, which reduces yield. To prevent this, it is important to add medium to the muscles immediately after dissection. In addition, if dissection is taking longer, the skin can be removed from one limb at a time to reduce the time of exposure of the muscles to air. The s.......

Acknowledgements

Cell sorting was performed at the FACS Core Facility, Aarhus University, Denmark. Figures were created using Biorender.com. We thank Dr. J. Farup for sharing the rabbit anti-PDGFRa antibody. This work was supported by an AUFF Starting Grant to E.P. and Start Package grants from NovoNordiskFonden to E.P. (0071113) and to A.D.M. (0071116).

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Materials

NameCompanyCatalog NumberComments
1.5 mL tube( PCR performance tested, PP, 30,000 xg, DNA/DNase-/RNase-free, Low DNA binding, Sterile )Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S72.706.7001.5 mL tube
15 mL tube (PP/HD-PE, 20,000 xg, IVD/CE, IATA, DNA/DNase-/RNase-free, Non-cytotoxic, pyrogen free, Sterile)Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S62.554.50215 mL tube
5 mL polystyrene round-bottom tubeFalcon, Fisher Scientific 352054FACS tube without strainer cap
5 mL polystyrene Round-bottom tube with cell-strainer capFalcon, Fisher Scientific  352235FACS tube with strainer cap
5 mL tube (PP, non sterile autoclavable)VWR collection525.09465 mL tube
50 mL tube( PP/HD-PE, 20,000 xg, IVD/CE, ADR, DNA/DNase-/RNase-free, non-cytotoxic, pyrogen free, Sterile)Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S62.547.25450 mL tube
Alexa Fluor 555 Donkey anti-rabbit IgG (H+L)Invitrogen, Thermo FisherLot: 2387458 (Cat # A31572)
Alexa Fluor 647 donkey-anti mouse IgG (H+L)Invitrogen, Thermo FisherLot: 2420713 (Cat#A31571)
ARIA 3BDFACS, Core facility Aarhus University
Centrifuge 5810eppendorfEP022628188Centrifuge
Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dyeInvitrogen, Thermo FisherLot: 2387287 (Cat# C10337)Cell Proliferation Kit
Collagen from calf-skin Bioreagent, Sigma Aldrich Source: SLCK6209 (Cat# C8919)
Collagenase type IIWorthington, Fisher Scientific Lot: 40H20248 (cat# L5004177 )Collagenase
DispaseGibco, Fisher Scientific Lot: 2309415 (cat# 17105-041 )Dispase
Donkey serum (non-sterile)Sigma Aldrich, MerckLot: 2826455 (Cat# S30-100mL)
Dumont nr. 5, 110 mmDumont, Hounisen Laboratorieudstyr A/S1606.327Straight forceps with fine tips
Dumont nr. 7, 115 mmDumont, Hounisen Laboratorieudstyr A/S1606.335Curved forceps
F-10 Nutrient mixture (Ham) (1x), +L-glutamineGibco, Fisher Scientific Lot. 2453614 (cat# 31550-023)
FITC anti-mouse CD31BioLegend, NordicBioSiteMEC13.3 (Cat # 102506)
FITC Anti-mouse CD45BioLegend, NordicBioSite30-F11 (Cat# 103108)
Glacial acetic acid (100%)EMSURE, Merck  K44104563 9Cat # 1000631000)
Head over head mini-tube rotator Fisher Scientific 15534080 (Model no. 88861052)Head over head mini-tube rotator
Horse serumGibco, Fisher Scientific Lot. 2482639 (cat# 10368902 )
Isotemp SWB 15FisherBrand, Fisher Scientific15325887Shaking water bath
MS2 mini-shaker IKA Vortex unit
Needle 20 G (0.9 mm x 25 mm)BD microlance, Fisher Scientific 30482720G needle 
Neutral formalin buffer 10%CellPath, Hounisen Laboratorieudstyr A/SLot: 03822014 (Cat # HOU/1000.1002)
Non-pyrogenic cell strainer (40 µM)Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S83.3945.040Cell strainer 
Pacific Blue anti-mouse Ly-6A/E (Sca-1)BioLegend, NordicBioSiteD7 (Cat# 108120)
Pax7 primary antibodyDSHBLot: 2/3/22-282ug/mL (Cat# AB 528428)
PBS 10x powder concentrateFisher BioReagents, Fisher ScientificBP665-1
PE/Cy7 anti-mouse CD106 (VCAM1)BioLegend, NordicBioSite429 (MVCAM.A) (Cat # 105720)
Pen/strepGibco, Fisher Scientific Lot. 163589 (cat# 11548876 )
Pipette tips p10Art tips, self sealing barrier, Thermo Scientific2140-05Low retention, pre-sterilized, filter tips
Pipette tips p1000Art tips, self sealing barrier, Thermo Scientific2279-05Low retention, pre-sterilized, filter tips
Pipette tips p20Art tips, self sealing barrier, Thermo Scientific2149P-05Low retention, pre-sterilized, filter tips
Pipette tips p200Art tips, self sealing barrier, Thermo Scientific2069-05Low retention, pre-sterilized, filter tips
Protective underpadAbena ACTC-7712 60 x 40cm, 8 layers
Rainin, pipet-lite XLSMettler Toledo, Thermo Scientific 2140-05, 2149P-05, 2279-05, 2069-05Pipettes (P10, P20, P200, P1000)
Recombinant anti-PDGFR-alphaRabMAb, abcamAB134123
Scalpel (shaft no. 3)Hounisen, Hounisen Laboratorieudstyr A/S1902.502Scalpel
Scalpel blade no. 11Heinz Herenz, Hounisen Laboratorieudstyr A/S1902.0911Scalpel
Scanlaf marsLabogeneclass 2 cabinet: MarsFlow bench
ScanROlympusMicroscope, Core facility Aarhus University
ScissorsFST14568-09
Series 8000 DHThermo Scientific3540-MARIncubator
Serological pipette 10 mLVWR612-3700Sterile, non-pyrogenic
Serological pipette 5 mLVWR, Avantor delivered by VWR612-3702Sterile, non-pyrogenic
Syringe 5 mL, Luer tip (6%), sterile BD Emerald, Fisher Scientific307731Syringe
TC Dish 100, standardSarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S83.3902Petri dish 
Tissue Culture (TC)-treated surface, black polystyrene, flat bottom, sterile, lid, pack of 20Corning, Sigma Aldrich376496-well Half bottom plate
Triton X-100Sigma Aldrich, MerckSource: SLCJ6163 (Cat # T8787)

References

  1. Mauro, A. Satellite cell of skeletal muscle fibers. The Journal of Biophysical and Biochemical Cytology. 9 (2), 493-495 (1961).
  2. Relaix, F., et al. Perspectives on skeletal muscle stem cells. Nature Communications. 12 (1), ....

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Stem CellSkeletal MuscleIsolationQuiescentRegenerationHomeostasisDisease ProgressionGracilisTibialis Anterior TAExtensor Digitorum Longus EDLDissectionForcepsScalpelTendonTissue Isolation

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