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.
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.
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 regeneration6,7,8. Moreover, a single MuSC can engraft and generate both new stem cells and new myofibers9. Skeletal muscle also harbors a population of mesenchymal stromal cells called fibro-adipogenic progenitors (FAPs), which play a crucial role in supporting MuSC function during muscle regeneration6,10,11,12.
Due to their potential to coordinate muscle regeneration, there has been tremendous interest in understanding how MuSCs and FAPs work. Quiescent MuSCs are marked by expression of the transcription factors Pax7 and Sprouty1, and the cell surface protein calcitonin receptor, whereas quiescent FAPs are marked by the cell surface protein platelet-derived growth factor receptor alpha (PDGFRa)10,12,13,14,15. Previous studies have showed that MuSCs and FAPs could be purified from skeletal muscles using cell surface markers and fluorescence-activated cell sorting (FACS)9,15,16,17,18,19,20,21. While these protocols have greatly advanced the ability to study MuSCs and FAPs, one drawback is that most of these protocols require the isolation of MuSCs from a pool of different muscle tissues. Recent work from us and others have revealed differences in cell phenotype and gene expression levels between MuSCs isolated from different tissues22,23. MuSCs from the diaphragm, triceps, and gracilis show faster activation than MuSCs from lower hindlimb muscles22, while MuSCs from extraocular muscle show faster differentiation than MuSCs from the diaphragm and lower hindlimb muscles23.
This protocol describes the isolation of MuSCs and FAPs from individual skeletal muscles (Figure 1). This includes the dissection of the diaphragm, triceps, gracilis, tibialis anterior (TA), soleus, extensor digitorum longus (EDL), gastrocnemius (GA), and masseter muscles. Dissected muscles are subsequently dissociated by enzymatic digestion using collagenase II (a protease that specifically targets the Pro-X-Gly-Pro amino sequence in collagen, enabling the degradation of connective tissue and tissue dissociation24) and dispase (a protease that cleaves fibronectin and collagen IV, enabling further cell dissociation25). MuSCs and FAPs are isolated from single-cell suspensions by FACS. As examples of downstream assays for cell analysis, stem cell activation is determined by assaying 5-ethynyl-2´-deoxyuridine (EdU) incorporation, while cell purity is determined by immunofluorescence staining for the cell-type specific markers Pax7 and PDGFRa.
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 for applying first aid in the case of a cut.
1. Preparation (1-2 h; the day before dissection)
NOTE: The solutions, plates, and media are prepared under sterile conditions and filtered (0.45 µm) prior to use unless otherwise noted. Prepare stock solutions of dispase (11 U/mL in PBS) and collagenase II (1.000 U/mL in PBS) and store them at -20 °C (Table 1). The stocks are thawed and used for secondary digestion in step 4.2.6.
2. Preparation (0.5 h; the day of dissection):
3. Muscle dissection (20-30 min)
NOTE: This section of the protocol is carried out in a non-sterile environment. The procedure can be carried out using one or several mice. However, one mouse is sufficient to prepare both samples for sorting and controls for setting up compensation and FACS gates.
4. Muscle digestion to a single-cell suspension (~1 h 35 min)
NOTE: The following steps include non-sterile (steps 4.1-4.2) and sterile work environments (step 4.3).
5. Staining and sorting (~40 min + 30 min sort/sample)
NOTE: Work in a sterile environment on ice for the following steps.
6. EdU incorporation assay
NOTE: Work under sterile conditions and use the chemical fume hood when handling paraformaldehyde (PFA) for the following steps. EdU is a nucleotide analog incorporated into the DNA as the cells go through the S-phase of the cell cycle. It is mutagenic at high concentrations. Always wear gloves when handling EdU. Check the local guidelines for handling EdU waste.
7. Immunofluorescence staining
NOTE: This part of the protocol can be performed independently of section 6. When skipping section 6, please perform steps 6.3.1 and 6.3.2 to enable cell permeabilization before continuing with step 7.2 below.
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 an unstained sample to identify the cells and separate the singlets from doublets (Figure 3A). The subsequent gates were set using FMO controls to identify staining thresholds (Figure 3B). The stained sample was then gated for CD31/CD45-FITC and Sca1-PacBlue. The SCA1+/CD31–/CD45– population (FAPs) was sorted into a separate collection tube, while the double negative population was gated and plotted for VCAM1-PECy7 and forward scatter (FSC). The VCAM1+ population (MuSCs) was sorted into a separate collection tube. MuSCs and FAPs were quantified as the percentage of singlets (Table 3) and sorted into separate collection tubes containing 500 µL of the wash medium. The diaphragm and triceps muscle single-cell suspensions have a higher relative abundance of MuSCs than FAPs, whereas the other single-cell suspensions have a higher relative abundance of FAPs than MuSCs (Table 3).
Of the sorted cells, 1,000-3,000 cells were seeded and incubated for 24 h. After 24 h of incubation, the medium was removed and replaced with a fresh medium containing EdU. The cells were fixed at 48 h, stained for EdU, and subsequently with antibodies against Pax7 protein or PDGFRa protein, and imaged using an inverted epifluorescence microscope. Images were quantified using the Fiji plugin in ImageJ. Robust EdU staining was observed, though the fraction of EdU-positive cells was different for the two stem cell types and the different muscles (Figure 4A–C). MuSCs isolated from either the EDL or GA showed significantly lower EdU incorporation compared to MuSCs isolated from either the TA, diaphragm, gracilis, or triceps, whereas MuSCs isolated from the masseter and soleus fall in between and are not significantly different from either group (Figure 4A,B). This is consistent with our previous results22. Moreover, the tissues from which the MuSCs show high EdU incorporation levels are the same tissues from which the MuSCs were previously shown to express high levels of Pax3 protein22. FAPs isolated from the EDL showed a significantly lower EdU incorporation compared to FAPs isolated from the GA and soleus, while FAPs isolated from the TA showed a significantly lower EdU incorporation compared to FAPs isolated from the soleus (Figure 4A,C). This underscores the importance of analyzing stem cells from individual tissues rather than pooling muscles from different tissues for isolation. For all tissues, the mean fraction of EdU-positive MuSCs was higher compared to the mean fraction of EdU-positive FAPs, suggesting that MuSCs activate faster under the given conditions.
Finally, cell purity was confirmed with immunofluorescence staining (Figure 4D). On average, 97.71% (± 1.38%) of MuSCs stained positive for Pax7 protein, and 88.16% (±6.35%) of FAPs stained positive for PDGFRa protein, confirming the specificity of our stem cell isolation procedure (Figure 4D).
Figure 1: Schematic abstract of the protocol. Schematic showing the two main segments of the protocol, MuSC isolation (upper panel), quiescence assay (lower panel), and the key steps of the methodology used in each of them. Please click here to view a larger version of this figure.
Figure 2: Muscle location and isolation. (A) A scheme showing the location of each muscle. (B–S) Demonstration of muscle isolation for (B–D) gracilis, (E–G) TA/EDL, (H–J) triceps, (K–M) GA/soleus, (N–P) masseter, and (Q–S) diaphragm muscles. Please click here to view a larger version of this figure.
Figure 3: Gating strategy used to identify and sort MuSCs and FAPs in a diaphragm muscle sample. Cells are identified based on cell size (Forward Scatter (FSC)) and granularity (Side Scatter (SSC)). Singlets are selected based on FSC-A and FSC-W. Lineage– cells are gated for subsequent identification of MuSCs (Lineage–/VCAM1+), and FAP (Lineage–/SCA1+) cells are gated to identify FAPs. The same gating strategy was applied to (A) an unstained control, (B) FMO controls (FMO-SCA1PacBlue, FMO-CD31/45-FITC, and FMO-VCAM1-PECy7), and (C) a stained sample. Please click here to view a larger version of this figure.
Figure 4: Quantification of cell activation by EdU staining. (A) Representative images of EdU staining of MuSCs (top panels) and FAPs (bottom panels) from a diaphragm sample. Shown are merged images of EdU and Hoechst (left panels), and the single channels in green (EdU, middle panels) and blue (Hoechst, right panels). (B,C) Bar graph showing the percentage of EdU-positive (B) MuSCs or (C) FAPs for indicated muscles. Plotted is the mean ± SEM. Each dot represents a mouse. Statistical analysis was performed in GraphPad using two-tailed Student's t-tests, with significance set to *p < 0.05 and **p < 0.01. (D) Immunofluorescence staining of MuSCs (top panels) and FAPs (bottom panels) with antibodies against the MuSC marker Pax7 (left side) and the FAP marker PDGFRa (right side). Shown are merged images of Pax7 with Hoechst, followed by the single channels, and merged images of PDGFRa with Hoechst, followed by the single channels. N = 3. Please click here to view a larger version of this figure.
Solutions | Reagents | Amount |
Wash medium | F-10 Nutrient mixture (Ham) (1x), +L-glutamine | 445 mL |
Horse serum | 50 mL | |
Pen/strep | 5 mL | |
Dissociation buffer (1st digestion) | Collagenase type II | 650 U/mL |
Wash medium | 100 mL | |
Dispase stock (2nd digestion) | Dispase in PBS | 11 U/mL |
Collagenase stock (2nd digestion) | Collagenase type II in PBS | 1000 U/mL |
PBS 1x | PBS 10x powder concentrate | 9.89 g/L |
Autoclaved/sterile water | 1 L | |
Acidic water | Glacial acetic acid (100%) Anhydrous for analysis | 5.15 mL |
Autoclaved/sterile water | 895 mL | |
Collagen solution (0.002%) | Collagen from calf-skin | 20 mL |
Acidic water | 800 mL | |
Triton X-100 | Triton X-100 | 0.5% (v/v) |
PBS 1x | 99.5% | |
Blocking buffer | PBS 1x | 18 mL |
Donkey serum | 2 mL |
Table 1: Table of recipes.
Sample no. | Name |
1 | Unstained |
2 | Fluorescence minus VCAM1-PeCy7 |
3 | Fluorescence minus SCA1-PacificBlue |
4 | Fluorescence minus CD31/45-FITC |
5 | Experimental stain (all four antibodies) |
Table 2: Overview of staining, controls, and samples prepared for each tissue prior to sorting.
Tissue | Antibody mix | Cells | Singlets | Lin neg | FAPs | MuSCs |
Diaphragm | unstained | 100% | 82% | 55% | 0.5% | 0.0% |
FMO Sca1-PacBlue | 100% | 83% | 19% | 0.3% | 4.3% | |
FMO CD31/45-488 | 100% | 84% | 40% | 9.4% | 5.6% | |
FMO Vcam1-PeCy7 | 100% | 78% | 20% | 5.6% | 0.0% | |
stained | 100% | 77% | 19% | 2.4% | 3.8% | |
Gracilis | stained | 100% | 96% | 11% | 2.6% | 1.4% |
TA | stained | 100% | 88% | 16% | 2.8% | 2.2% |
EDL | stained | 100% | 86% | 26% | 19.2% | 0.8% |
Soleus | stained | 100% | 91% | 41% | 13.3% | 1.1% |
GA | stained | 100% | 94% | 51% | 6.1% | 1.5% |
Triceps | stained | 100% | 92% | 30% | 2.6% | 4.5% |
Masseter | stained | 100% | 85% | 27% | 19.3% | 2.6% |
Table 3: Overview of relative cell type abundance in the FACS data.
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 smaller volume also results in an increased risk of over-digestion. To counter this, the present method requires lower amounts of collagenase II enzyme and reduced digestion times compared to bulk muscle protocols9,16. Enzymatic digestion also depends on the purity of the enzymes, and lower purity can negatively impact yield. In addition, mechanical digestion is critical. In case of insufficient cutting, the decreased surface area will hamper enzymatic digestion and lower the stem cell yield. In case of cutting too much, the increased surface area will cause over-digestion and lower the stem cell yield. The shaking water bath prevents precipitation of the digested muscle, improves the distribution of the enzyme, and aids in creating a homogeneous temperature, altogether enabling shorter incubation times. Therefore, the present method allows a significant reduction of the incubation time compared to other methods.
This protocol depends on cell dissociation and purification. These procedures mimic tissue injury, which activates the stem cells. Consistently, recent studies have revealed that MuSCs change their gene expression programs during the isolation procedure27,28,29,30. As a result, the purified stem cells are different from the cells in vivo in terms of gene expression patterns. A second limiting factor in the protocol is its dependence on FACS, which requires access to expensive equipment. FACS is the gold standard for isolating multiple cell populations simultaneously with high purity20. Recent advances using magnetic beads and microbubbles offer reductions in cost31,32, but whether they offer comparable yields to work on single muscles needs to be determined. Finally, the yield of the protocol is limited due to the small size of the muscles, thus posing restrictions on potential downstream assays.
Previous studies have relied on pooling different muscles when isolating MuSCs and/or FAPs to maximize cell yield. However, this averages out any tissue-specific differences in stem cell behavior and function between different muscles. The current protocol enables the isolation of MuSCs and FAPs from individual muscles for the downstream analyses of stem cell function. As an example of a downstream assay, stem cell activation was assayed by EdU incorporation, revealing that stem cells from different tissues display different activation kinetics. In previous works, the feasibility of using other downstream assays has been shown; these assays require smaller cell numbers, such as SmartSeq2 single-cell RNA sequencing, cell transplantation, microfluidic PCR, and clonal expansion assays22,33,34,35.
In conclusion, this protocol describes a method for the dissection of individual muscles to isolate and study MuSCs and FAPs. This strategy will enable experiments to gain a better understanding of stem cell function across different muscles in health and disease.
The authors have nothing to disclose.
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).
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) |