Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

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 exit^1,2,3,4. Upon injury, MuSCs activate and enter the cell cycle to give rise to amplifying progenitors that can differentiate and fuse to form new myofibers^2,5. 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 out regularly. This protocol involves working with sharps, and researchers are recommended to familiarize themselves with the procedures and logistics f....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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/S	72.706.700	1.5 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/S	62.554.502	15 mL tube
5 mL polystyrene round-bottom tube	Falcon, Fisher Scientific	352054	FACS tube without strainer cap
5 mL polystyrene Round-bottom tube with cell-strainer cap	Falcon, Fisher Scientific	352235	FACS tube with strainer cap
5 mL tube (PP, non sterile autoclavable)	VWR collection	525.0946	5 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/S	62.547.254	50 mL tube
Alexa Fluor 555 Donkey anti-rabbit IgG (H+L)	Invitrogen, Thermo Fisher	Lot: 2387458 (Cat # A31572)
Alexa Fluor 647 donkey-anti mouse IgG (H+L)	Invitrogen, Thermo Fisher	Lot: 2420713 (Cat#A31571)
ARIA 3	BD		FACS, Core facility Aarhus University
Centrifuge 5810	eppendorf	EP022628188	Centrifuge
Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye	Invitrogen, Thermo Fisher	Lot: 2387287 (Cat# C10337)	Cell Proliferation Kit
Collagen from calf-skin	Bioreagent, Sigma Aldrich	Source: SLCK6209 (Cat# C8919)
Collagenase type II	Worthington, Fisher Scientific	Lot: 40H20248 (cat# L5004177 )	Collagenase
Dispase	Gibco, Fisher Scientific	Lot: 2309415 (cat# 17105-041 )	Dispase
Donkey serum (non-sterile)	Sigma Aldrich, Merck	Lot: 2826455 (Cat# S30-100mL)
Dumont nr. 5, 110 mm	Dumont, Hounisen Laboratorieudstyr A/S	1606.327	Straight forceps with fine tips
Dumont nr. 7, 115 mm	Dumont, Hounisen Laboratorieudstyr A/S	1606.335	Curved forceps
F-10 Nutrient mixture (Ham) (1x), +L-glutamine	Gibco, Fisher Scientific	Lot. 2453614 (cat# 31550-023)
FITC anti-mouse CD31	BioLegend, NordicBioSite	MEC13.3 (Cat # 102506)
FITC Anti-mouse CD45	BioLegend, NordicBioSite	30-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 serum	Gibco, Fisher Scientific	Lot. 2482639 (cat# 10368902 )
Isotemp SWB 15	FisherBrand, Fisher Scientific	15325887	Shaking water bath
MS2 mini-shaker	IKA		Vortex unit
Needle 20 G (0.9 mm x 25 mm)	BD microlance, Fisher Scientific	304827	20G needle
Neutral formalin buffer 10%	CellPath, Hounisen Laboratorieudstyr A/S	Lot: 03822014 (Cat # HOU/1000.1002)
Non-pyrogenic cell strainer (40 µM)	Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S	83.3945.040	Cell strainer
Pacific Blue anti-mouse Ly-6A/E (Sca-1)	BioLegend, NordicBioSite	D7 (Cat# 108120)
Pax7 primary antibody	DSHB	Lot: 2/3/22-282ug/mL (Cat# AB 528428)
PBS 10x powder concentrate	Fisher BioReagents, Fisher Scientific	BP665-1
PE/Cy7 anti-mouse CD106 (VCAM1)	BioLegend, NordicBioSite	429 (MVCAM.A) (Cat # 105720)
Pen/strep	Gibco, Fisher Scientific	Lot. 163589 (cat# 11548876 )
Pipette tips p10	Art tips, self sealing barrier, Thermo Scientific	2140-05	Low retention, pre-sterilized, filter tips
Pipette tips p1000	Art tips, self sealing barrier, Thermo Scientific	2279-05	Low retention, pre-sterilized, filter tips
Pipette tips p20	Art tips, self sealing barrier, Thermo Scientific	2149P-05	Low retention, pre-sterilized, filter tips
Pipette tips p200	Art tips, self sealing barrier, Thermo Scientific	2069-05	Low retention, pre-sterilized, filter tips
Protective underpad	Abena	ACTC-7712	60 x 40cm, 8 layers
Rainin, pipet-lite XLS	Mettler Toledo, Thermo Scientific	2140-05, 2149P-05, 2279-05, 2069-05	Pipettes (P10, P20, P200, P1000)
Recombinant anti-PDGFR-alpha	RabMAb, abcam	AB134123
Scalpel (shaft no. 3)	Hounisen, Hounisen Laboratorieudstyr A/S	1902.502	Scalpel
Scalpel blade no. 11	Heinz Herenz, Hounisen Laboratorieudstyr A/S	1902.0911	Scalpel
Scanlaf mars	Labogene	class 2 cabinet: Mars	Flow bench
ScanR	Olympus		Microscope, Core facility Aarhus University
Scissors	FST	14568-09
Series 8000 DH	Thermo Scientific	3540-MAR	Incubator
Serological pipette 10 mL	VWR	612-3700	Sterile, non-pyrogenic
Serological pipette 5 mL	VWR, Avantor delivered by VWR	612-3702	Sterile, non-pyrogenic
Syringe 5 mL, Luer tip (6%), sterile	BD Emerald, Fisher Scientific	307731	Syringe
TC Dish 100, standard	Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S	83.3902	Petri dish
Tissue Culture (TC)-treated surface, black polystyrene, flat bottom, sterile, lid, pack of 20	Corning, Sigma Aldrich	3764	96-well Half bottom plate
Triton X-100	Sigma Aldrich, Merck	Source: SLCJ6163 (Cat # T8787)