Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Summary

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Abstract

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Introduction

Fibro-adipogenic progenitor cells (FAPs) are a population of resident multipotent progenitor cells in skeletal muscle that play a critical role in muscle homeostasis, repair, and regeneration, and conversely, also mediate pathologic responses to muscle injury. As the name suggests, FAPs were originally identified as a progenitor population with the potential to differentiate into fibroblasts and adipocytes¹ and were purported to be the key mediators of fibro-fatty infiltration of skeletal muscle in chronic injury and disease. Further study revealed that FAPs are additionally capable of osteogenesis and chondrogenesis^2,3,4. Thus, they are more broadly notated in the literature as mesenchymal or stromal progenitors^3,5,6,7,8. In acute skeletal muscle injury, FAPs indirectly aid in regenerative myogenesis by transiently proliferating to provide a favorable environment for activated muscle satellite cells and their downstream myogenic progenitor (MPs) counterparts^1,9,10. In parallel with successful regeneration, FAPs undergo apoptosis, returning their numbers to baseline levels^1,9,10,11. In contrast, in chronic muscle injury, FAPs override pro-apoptotic signals, which results in their persistence^9,10,11 and abnormal muscle repair.

In vivo studies evaluating the cellular and molecular mechanisms by which FAPs mediate muscle responses have utilized murine animal models to date^{1,7,9,10,11,12,13,14}. While genetically engineered mice are powerful tools for use in these analyses, the small size of the animal limits tissue availability for study in long-term localized injury models where muscle atrophy can be profound, such as traumatic denervation. Furthermore, measurement of muscle strength and physical function requires ex vivo or in situ measurements that necessitate termination of the mouse, or in vivo methods that require surgery and/or a general anaesthetic to permit evaluation of muscle contractile performance^{15,16,17,18,19,20}. In rats, well validated and globally utilized muscle functional analyses, in addition to analyses for more complex motor behaviors such as gait analysis (e.g., Sciatic Function Index, CatWalk analysis) exist and are performed in awake and spontaneously moving animals^21,22,23,24. This additionally optimizes the principles of minimal morbidity in animal experimentation, and numbers of research animals used. The rat thereby provides the FAPs investigator the added flexibility of greater injured muscle volume for protein and cellular analyses and the ability to undertake serial assessments of muscle complex static and dynamic functional activity and behaviors, in the alert animal.

FAPs have primarily been identified and isolated from whole muscle samples using flow cytometry and Fluorescence-activated cell sorting (FACS) respectively. These are laser-based assays that are able to identify multiple specific cell populations based on characteristic features such as size, granularity, and a specific combination of cell surface or intracellular markers²⁵. This is highly advantageous in the study of an organ system such as skeletal muscle, as homeostasis and regeneration are complex, multifactorial processes coordinated by a plethora of cell types. A seminal study identified FAPs, as well as MPs, using flow cytometric methods in mouse skeletal muscle¹. They demonstrated that FAPs are mesenchymal in nature, as they lacked surface antigens specific to cells from endothelial (CD31), hematopoietic (CD45), or myogenic (Integrin-α7 [ITGA7]) origins, but expressed the mesenchymal stem cell marker Sca-1 (Stem cell antigen 1)¹ and differentiated into fibrogenic and adipogenic cells in culture. Other studies demonstrated successful isolation of mesenchymal progenitors in muscle based on the expression of an alternative stem cell marker, platelet-derived growth factor receptor alpha (PDGFRα)^2,7,8 and further analysis revealed these likely to be the same cell population as FAPs³. FAPs are now commonly identified in flow cytometry using either Sca-1 or PDGFRα as a positive selection marker^{1,9,10,11,12,13,14,26,27,28,29,30,31}. The use of PDGFRα is preferential for human tissue however, as a direct human homologue of murine Sca-1 has yet to be identified³². In addition, other cell surface proteins have been reported as markers of MPs (e.g., VCAM-1), providing a potential alternative to ITGA7 as an indicator of cells of myogenic lineage during FAPs isolation³³.

While flow cytometry/FACS is a powerful methodology for studying the role and pathogenic potential of FAPs in skeletal muscle^{1,9,10,11,13,29}, it is limited technically by the specificity and optimization of its required reagents. Since flow cytometric identification and isolation of FAPs has been developed and conducted in mouse animal models^1,9,10,11,29, this poses challenges for researchers who wish to study FAPs in other model organisms. Many factors - such as optimal tissue size to be processed, as well as reagent and/or antibody specificity and availability - differ depending on the species used.

In addition to the technical barriers to studying FAPs in a novel animal model, they have largely been studied in an acute, toxic setting - usually via intramuscular chemical injection or cardiotoxin. Evaluation of the long-term dynamics of FAPs is limited primarily to assessment in Duchenne's muscular dystrophy, using the mdx mouse model^9,10,11, and models of combination muscle injury such as massive rotator cuff tear where concurrent tendon transection and denervation is performed on shoulder musculature^26,27,28. The response of FAPs to the sole insult of chronic traumatic denervation, a common occurrence in work-place accidents in heavy industry, agriculture, and in birth traumas (brachial plexus injury)^34,35,36,37 with significant morbidity, has not been as well characterized, often limited to a short-term time frame^11,38.

We describe a method for identifying and isolating FAPs and MPs from healthy as well as severely atrophic and fibrotic skeletal muscle in the rat. First, identification of CD31-/CD45-/Sca-1+/VCAM-1- FAPs and CD31-/CD45-/Sca-1-/VCAM-1+ MPs using a tissue digestion and flow cytometry staining protocol is demonstrated and subsequent validation of our findings is performed through culture and immunocytochemical staining of FACS-isolated cells. Using this method, we also report a novel FAPs time-course in a long-term isolated denervation injury model in the rat.

Protocol

Investigators conducting this protocol must receive permission from their local animal ethics board/care committee. All animal work was approved by the St. Michael's Hospital Unity Health Toronto Animal Care Committee (ACC #918) and was conducted in accordance with the guidelines set forth by the Canadian Council on Animal Care (CCAC). A schematic of the flow cytometry protocol is shown in Figure 1. If the downstream application is FACS and subsequent cell culture, all steps should be completed with proper aseptic technique.

1. Muscle harvesting

1. Anesthetize rats using an appropriate anesthetic and sacrifice according to local vivarium and animal ethics board guidelines. This protocol harvests the gastrocnemius muscle from adult female Lewis rats (200-250 g), as an example. Rats were anesthetized using 2-3% Isoflurane and were sacrificed by intracardiac injection of T61.
2. Once the animal has been sacrificed, shave the whole hindlimb to facilitate the location of the muscle and minimize fur contamination of the harvested tissue.
3. Using a sterile scalpel, make two incisions in the skin: the first around the circumference of the ankle joint and the second up the midline of the medial aspect of the hindlimb from the ankle to the hip.
4. Peel back the skin and superficial muscle layers to reveal the underlying gastrocnemius, which originates at the medial and lateral condyles of the femur and inserts at the Achilles Tendon.
5. Use blunt dissection to separate the gastrocnemius from the surrounding tissue, handling the muscle only by the tendon to avoid crush injury.
6. Separate the gastrocnemius from its insertion by transecting the Achilles Tendon as distally as possible with sharp scissors. Once cut, grasp the Achilles tendon with forceps and gently peel the gastrocnemius off the underlaying bone. Still holding the muscle with forceps in one hand, locate the gastrocnemius' two origins and cut at the medial and lateral femoral condyles.
7. Blot the excised gastrocnemius gently against a sterile piece of gauze to remove as much blood as possible. Trim the muscle on a sterile surface and remove any excess connective tissue as well as the Achilles Tendon.
8. Place muscle in a weigh-boat and weigh using a precision scale. This protocol is optimized to digest muscle with a wet weight ranging from 200-600 mg. Operators may subdivide excess harvested tissue for other downstream assays, if desired.
9. Gently further divide harvested muscle to be used for flow cytometry into 3-4 smaller pieces (approximately 1-2 cm³) and submerge in ice-cold 1x PBS. Keep cold on ice until all samples have been harvested.

2. Muscle digestion

1. Remove muscle from PBS and place in a sterile 10 cm cell culture dish. Gently tear and mince tissue with forceps until pieces are approximately 3-4 mm³, removing as much connective tissue as possible. Once thoroughly minced, transfer to a sterile 50 mL conical tube containing 6 mL DMEM + 1% penicillin/streptomycin (P/S).
2. Add 10 µL of 300 mM CaCl₂ solution to 365 µL of Collagenase II solution (stock concentration 4800 U/mL) to activate the collagenase enzyme. Add the activated collagenase II solution to the 50 mL conical tube containing the tissue slurry. The final Collagenase II concentration is 250 U/mL.
3. Incubate tubes in a shaker for 1 h at 37 °C, 240 x g, making sure to manually swirl every 15 min to dislodge any tissue that has adhered to the side of the tube.
4. After 1 h, remove tubes from shaker and add the following per sample: 100 µL of Collagenase II (4,800 U/mL) and 50 µL of Dispase (4.8 U/mL).
5. Pipette samples using a serological pipette 15-20 times until the solution is homogenous. If processing multiple samples, use a separate sterile pipette for each sample to avoid sample cross-contamination.
6. Incubate again in a shaker for 30 min at 37 °C and 240 x g. After 15 min, shake samples by hand to dislodge adherent tissue off the side of the tube.

3. Generation of single cell suspension

1. Slowly shear samples through a 10 mL syringe with a 20 G needle for 10 cycles.
   NOTE: One cycle involves taking up muscle solution into syringe, and injecting it back into tube. Ensure to minimize any bubbles by completing shearing slowly, as excessive frothing can cause additional cell death³⁹.
2. Place a 40 µm cell strainer on a sterile 50 mL conical tube and wet it by pipetting 5 mL DMEM + 10% FBS & 1% P/S.
3. Pipette 1 mL of the sample at a time through the cell strainer.
4. Wash the cell strainer by pipetting DMEM with 10% FBS and 1% P/S through the strainer to bring the total volume in the tube to 25 mL.
5. Split 25 mL of the sample equally into two 15 mL conical tubes and centrifuge at 15 °C, 400 x g for 15 min.
   NOTE: Splitting the muscle solution into two 15 mL conical tubes ensures better cell recovery after centrifugation compared to a single tube.
6. Aspirate the supernatant and re-suspend the pellet in 1 mL 1x RBC Lysis buffer (see Supplementary File) at room temperature for 7 min to eliminate erythrocytes.
7. Bring up the volume to 10 mL with 9 mL of wash buffer (see Supplementary File) and spin tubes at 400 x g, 15 °C for 15 min.
8. Aspirate the supernatant and recombine pellets by re-suspending in 1 mL wash buffer.
9. Transfer an appropriate volume of cells to a separate 1.5 mL microcentrifuge tube and mix with trypan blue dye. Count live cells on a light microscope using a hemocytometer.

4. Antibody staining for flow cytometry

NOTE: The Sca-1 antibody must be conjugated to APC prior to flow cytometry/FACS experiments, as per the manufacturer's instructions. Performance must be validated for each batch of conjugates (Figure 2). Final conjugations can be stored in 20 µL aliquots at -20 °C and are stable for three weeks. Refer to the Supplementary File for full conjugation protocol.

1. For flow cytometry, transfer 1-2 x 10⁶ cells per experimental sample to a sterile 1.5 mL microcentrifuge tube. Bring volume up to 1 mL with wash buffer and place on ice.
2. For each experiment, set up the following required controls: i) unstained and ii) viability controls to accurately select for the live cell population; iii) fluorescence minus one (FMO) controls on single cell suspensions to set accurate gates for CD31-/CD45- fractions, FAPs, and MPs; and iv) single-stained compensation beads to correct for fluorescence spillover between channels.
   1. For all cell controls, aliquot 5 x 10⁵ - 1 x 10⁶ cells in 1 mL of wash buffer in a 1.5 mL microcentrifuge tube and place on ice.
   2. For bead controls, add 1 drop of positive compensation beads (~1.5 x 10⁵ beads per drop) to each labeled 1.5 mL microcentrifuge tube. The full complement of controls is listed in Table 1.
      NOTE: If the experiment is being performed for the first time, run single-stained controls for each conjugated antibody on single cell suspensions (in addition to unstained, viability, single-stained compensation bead and FMO controls) to assess the positive stained population in cells and validate staining observed on compensation beads. Validate every freshly-conjugated Sca-1::APC preparation by performing single-staining on compensation beads and single cell suspensions. Refer to Table 1 for a full list of staining controls.
3. To prepare the viability control, transfer half of the volume of cells from the "viability" tube to a new 1.5 mL microcentrifuge tube. Label this tube "Dead".
4. Incubate "Dead" tube at 65 °C for 2-3 min to kill the cells, then place on ice. After 2-3 min, re-combine dead cells with live cells remaining in the viability control tube. This population of cells will be used to set compensation values (if needed) and properly set gates for the viability dye.
5. Centrifuge the single cell suspensions (experimental samples and controls) at 500 x g, 4 °C for 5 min.
6. Aspirate the supernatant and re-suspend cell pellets in 100 µL wash buffer.
7. Add antibodies, depending on the experimental sample or control. Refer to the staining matrix (Table 2) for information on antibody combinations and amounts.
8. Gently flick each sample to ensure complete mixing and incubate on ice in the dark for 15 min. For compensation beads, incubate at room temperature in the dark for 15 min.
9. For single cell suspension experimental and control samples, bring up the volume to 1 mL by adding 900 µL wash buffer. For compensation bead controls, bring up the volume to 1 mL with 900 µL of 1x PBS.
10. Centrifuge single cell suspension samples at 500 x g, 4 °C for 5 min. Centrifuge compensation bead controls at 300 x g, 4 °C for 5 min.
11. For all single cell suspension samples, aspirate and discard supernatant and re-suspend cell pellet in 300 µL wash buffer. For compensation bead controls, aspirate and discard the supernatant, re-suspend the pellet in 300 µL of 1x PBS, then add 1 drop (~1.5 x 10⁵) of negative compensation beads.
12. Keep all single cell suspension samples on ice under aluminum foil and proceed to flow cytometric acquisition. Compensation bead controls should also be protected from light but can be kept at room temperature.
    NOTE: If experimental endpoint is FAPs identification by flow cytometry, please follow steps 5.1.1-5.1.11. If endpoint is cell isolation via FACS for culture and staining, please follow steps 5.2.1-5.2.9 and sections 6-7.

5. Flow cytometry and fluorescence-activated cell sorting (FACS)

1. Flow cytometry
   NOTE: This protocol employs a benchtop flow cytometer equipped with 405 nm, 488 nm, and 640 nm lasers that are capable of simultaneously distinguishing 10 different colors. Bandpass filters and their associated fluorochromes used in this protocol are as follows: 450/50 (SYTOX Blue), 530/30 (FITC), 575/25 (PE), and 670/30 (APC). Voltages for each detector are as follows: FSC 700; SSC 475; FITC 360; PE 460; PE-Cy7 600; SYTOX Blue 360; APC 570. Ensure you are trained on the proper operation of the flow cytometer or cell sorter prior to use.
   1. Ensure the cytometer has been turned on for 10-20 min before use and has been primed by cleaning sequentially with clean, rinse, and sheath fluid solutions for 30-45 s each. Finish with a rinse with dH₂O. Ensure that an adequate volume of sheath fluid has been added to the storage container to maintain proper sample flow throughout acquisition.
   2. Set up the gating strategy to identify FAPs and MPs as delineated in Figure 3.
      NOTE: FAPs and MPs are identified by the following hierarchical gating strategy: i) SSC-A vs FSC-A (side cell scatter area versus forward cell scatter area to separate cells vs debris), ii) FSC-W vs FSC-H (forward cell scatter width versus forward cell scatter height to discriminate singlets from doublets in the FSC parameter), iii) SSC-W vs SSC-H (side cell scatter width versus side cell scatter height to discriminate singlets from doublets in the SSC parameter), iv) SSC-A vs SYTOX Blue (to distinguish live versus dead singlets), v) SSC-A vs CD31/45::FITC (to exclude CD31+ and CD45+ cells from further analysis), and vi) Sca-1::APC vs VCAM-1::PE from the CD31-/CD45- (Lineage; Lin-) population (identification of FAPs and MPs). FAPs are identified as CD31-/CD45-/Sca-1+/VCAM-1- events and MPs are identified as CD31-/CD45-/Sca-1-/VCAM-1+ events.
   3. First run each single-stained compensation bead control through the cytometer on low speed to generate compensation values used to correct for any fluorescence spillover between channels. Assess compensation by comparing fluorescent signal of each control in its own detector (e.g., SSC-A vs APC for Sca-1::APC single-stained beads) as well as all other detectors. There should be two distinct populations (one with negative and one with positive signal) in the appropriate detector and only a negative population in all other detectors. Set the stopping gate to 10,000 compensation bead events and record the data.
      NOTE: In between acquisition of each sample, make sure to run dH₂O through the cytometer for 10-20 sec to avoid sample-to-sample contamination.
   4. Next process the unstained and viability control samples to properly gate on live single cells. Set the stopping gate to 10,000 singlet events and record data.
      NOTE: Approximately 5 min before acquisition of each single cell suspension sample with the exception of the unstained sample, add 1 µL of SYTOX Blue viability dye (300 µM working concentration diluted from 1 mM stock solution) to each sample and flick gently to mix (final concentration 1 µM).
   5. Then acquire the remaining single cell suspension control samples. Assess each FMO control with its appropriate plot in the gating strategy (Figure 3). For example, assess the FITC signal of the CD31+CD45 FMO to ensure an accurate CD31-/CD45- gate. An optimal example is shown in Figure 3G. If the protocol is being performed for the first time, single-stained controls on cells should be run before the acquisition of FMO controls.
   6. Assess the fluorescent signal of each single-stained cell sample in its appropriate detector as well as in all other detectors to validate proper compensation. Set the stopping gate to 10,000 live singlet events and record on the software.
   7. Once all controls (single cell suspensions and beads) have been processed, prepare all experimental samples by first measuring and recording the volume of each sample. These measurements will be used to accurately quantify FAPs and MPs, as described in step 5.1.11. Then, add 50 µL of precision counting beads and gently mix by pipetting up and down 2-3 times.
   8. Briefly run the first experimental sample to validate identification of the counting bead population. This population appears as a small distinct cluster separate from the general cell population on the FSC-A vs SSC-A plot (Figure 3A, red box). Create a gate around the counting bead population. Then acquire data for each experimental sample by processing through the cytometer on low speed. Set the stopping gate to 10,000 counting bead events and record.
      NOTE: Investigators may alternatively identify counting beads by setting up an additional plot assessing SSC-A versus any of the detectors, as the counting beads are fluorescent in all detectors.
   9. After all samples have been processed, clean the cytometer using the appropriate protocols. Export all data for analysis.
   10. Open all data files on an appropriate flow cytometry analysis software. Set the gating strategy as used for data acquisition as described in step 5.1.2. Examine controls in the same order as in data acquisition (e.g., unstained, viability, single stain, then FMO controls) to re-validate the gating strategy. Once accurate gates have been set using FMO controls, apply the gates to all experimental samples. Export raw data as a spreadsheet for quantification.
   11. Calculate the number of FAPs and MPs in each experimental sample using the counting beads:
       
       where, Acquired Cell Count is the number of recorded events of pertinent cell population (e.g. FAPs or MPs) on the acquisition software; Acquired Bead Count is the number of recorded events of counting beads on the acquisition software; Counting Beads Volume is the volume of counting bead solution added in step 5.1.7; Sample Volume is the volume of each stained experimental sample prior to addition of counting beads.; Bead Concentration is the number of beads per µL solution; this value is found on the product datasheet.
2. FACS - sorting for cell culture
   NOTE: This protocol performs FACS on a cell sorter equipped with 4 lasers (UV, Violet, Blue, Red) that is capable of simultaneously distinguishing 11-14 colors. Follow the experimental sample staining (section 4) and flow cytometry protocol, with the exceptions of steps 1 to 3 delineated below, to optimize the FACS workflow:
   1. Increase the concentration of cells in the experimental samples to be sorted to 7 x 10⁶ cells/mL to generate robust yields of FAPs and MPs.
   2. To account for this significant increase in the cell concentration, double all antibody concentrations in the experimental samples to be sorted.
   3. Process the final stained cell samples through a 40 µM cell strainer cap affixed to a 5 mL polystyrene tube immediately prior to sorting to reduce cell clumping and increase sort yields.
   4. Collect single, live rat FAPs and MPs directly from the cell sorter into a 5 mL polypropylene collection tube containing 1 mL of sterile, 100% Fetal Bovine Serum (FBS). Keep cells on ice until sorting is complete.
      NOTE: If conducting FACS at an off-site location, transfer all sorted cells on ice and in a secured, covered container.
   5. Working in a sterile biosafety cabinet (BSC), bring volume of sorted cells up to 7 mL with appropriate growth media (e.g., FAP growth media (FAP GM) for sorted FAPs, and MP growth media (MP GM) for sorted MPs; see Supplementary File for recipes) and centrifuge at 500 x g, 4 °C for 7 min to remove as much residual wash buffer as possible.
   6. Resuspend pellets in 1 mL of appropriate growth media and plate into a 12-well plate containing a sterile, collagen-coated 12 mm glass coverslip/well for subsequent immunostaining (see section 6).
      NOTE: If immunocytochemistry staining for collagen, plate sorted cells into a 12 well plate containing a sterile, laminin-coated 12 mm glass coverslip/well, instead of collagen-coated. If immunocytochemistry experiments of immediately isolated progenitors are required, seed FAPs and MPs at a density of 15,000 cells per cm² and proceed directly to step 6.1. For long-term cultures to induce progenitor differentiation, seed FAPs at a density of 5,000 cells per cm², and MPs at a density of 7,500 cells per cm².
   7. Incubate cells at 37 °C and 5% CO₂ in a cell culture incubator. After 72 h in culture, change half of the media. Change media fully every 2-4 d after.
   8. To induce myocyte development, switch MPs cultures to MP differentiation (MD) medium on Day 9 of culture. To induce adipocytes, switch FAPs cultures to FAP adipogenic differentiation (AD) medium on Day 10 of culture.
   9. To induce fibrogenesis, FAPs may be switched to fibrogenic differentiation (FD) media at variable times during culture, or alternatively, may be seeded directly into FD media following isolation (step 5.2.6) (See Supplementary File for all media recipes).

6. Immunocytochemistry of cultured FAPs and MPs

1. To validate cell sorting and demonstrate purity of FAPs and MPs cultures, immunostain with cell-type specific markers including PDGFRα (FAPs marker), Pax-7 (muscle stem [satellite] cell marker), Fibroblast-specific protein (FSP-1, fibroblast marker), Perilipin-1 (Plin-1, adipocyte marker), Collagen type 1 (Col1a1, indicator of fibrosis), Myosin Heavy Chain (MHC, mature myocyte marker).
   1. For immunostaining of freshly sorted cells, centrifuge the 12-well plate at 200 x g for 3 min at room temperature to facilitate adherence of cells to the coverslip/well. This step is not necessary for long-term cultures. Remove culture media.
   2. For immunostaining with FSP-1, fix cells with 1 mL 100% methanol (MeOH) for 2 min at 4 °C. If immunostaining for PDGFRα, Plin-1, Pax7 or Col1a1, fix cell cultures with 1 mL 4% PFA in 1x PBS for 15 min at room temperature. MHC immunostaining tolerates either fixative.
      NOTE: For methanol-fixed cells, skip step 6.2 and proceed to step 6.3.
2. Aspirate 4% PFA and quickly wash cell cultures 3-4 times with 1x PBS. Add 1 mL of 100 mM Glycine in 1x PBS and incubate for 10 min at room temperature to inactivate residual PFA. Aspirate and wash 1-2 times with 1x PBS.
   NOTE: Cells can be left at this stage in 2 mL of 1x PBS, wrapped in cling film and stored at 4 °C for 7-10 days maximum.
3. After washing, add 1 mL of 0.1% Triton-X in 1x PBS and incubate for 20 min to permeabilize cell membranes.
4. Wash wells 2-3 times with 1-2 mL of 1x PBS then block cells with 1 mL of 1x PBS + 3% BSA per well for 1 h at room temperature.
5. Pipette 80 µL of primary antibody diluted in 1x PBS + 3% BSA (PDGFRα 1:100, Pax7 neat, FSP-1 1:50, Plin-1 1:400, Col1a1 1:250, MHC 3 µg/mL) onto a piece of parafilm taped to a mobile container. Using sterile fine forceps, carefully lift the coverslip out of the well and invert onto the drop of antibody solution. Incubate coverslip with two wet pieces of paper towel and cover container in plastic film to avoid evaporation of the antibody solution. Incubate overnight at 4 °C.
   NOTE: Staining coverslips out of the well utilizes less antibody (~80 µL) than staining inside the well (500 µL minimum).
6. On Day 2, leave coverslips at room temperature for 30 min to warm. Using forceps carefully right and transfer coverslips back to their respective wells (cells facing up) and wash 2-3 times with 1-2 mL of 1x PBS for 2 min each to remove as much primary antibody as possible.
7. Using the same staining technique as with primary antibody staining, stain cells with goat anti-rabbit Alexa Fluor 488 secondary antibody (1:400) to detect FSP-1, Plin-1, Col1a1, or PDGFRα and goat anti-mouse Alexa Fluor 555 secondary antibody (1:300) to detect MHC or Pax-7. Incubate cells for 1 h at room temperature and keep cells protected from light.
8. Return cells to the well and incubate cells with Hoechst (1:10,000) for 2-4 min at room temperature. Wash cells another 2-3 times with 1x PBS for 2 min each to remove excess Hoechst.
9. Mount coverslips onto glass slides using an anti-fade fluorescent mounting medium and leave slides to dry overnight in the dark at room temperature. Store mounted coverslips at 4 °C in the dark.

7. Oil Red O (ORO) staining of cultured FAPs and MPs

1. Perform ORO staining on non-permeabilized cells, as permeabilization of the cell membrane can result in non-specific/undesired staining of non-adipogenic cell types. Prior to commencing staining, prepare an ORO working stock (See Supplementary File for the recipe) and incubate at room temperature for 20 min.
2. After 20 min, filter the solution using a 0.2 µm filter in order to remove any undissolved aggregates.
3. Aspirate media from well and add 1 mL of 10% Neutral Buffered Formalin (10% NBF). Incubate for 5 min at room temperature.
   NOTE: Cell confluency can result in lifting from the well/coverslip. Take care when aspirating/adding solutions.
4. Aspirate and add 1 mL of fresh 10% NBF and incubate for at least 1 h at room temperature.
   NOTE: The protocol can be stopped at this point, as cells can be left in 10% NBF overnight.
5. Quickly wash the wells once with 1 mL of 60% isopropanol, then aspirate and allow the wells to dry completely (approximately 2 min).
6. Add 400 µL Oil Red O working stock per well and incubate for 10 min at room temperature, making sure to avoid pipetting any ORO on the walls of the plate.
7. Remove all of the Oil Red O and quickly wash the well 4 times with dH₂O.
   NOTE: If stained wells contain coverslips, mount using the same technique as described in step 6.9.
8. Image either mounted coverslips or the stained well using a brightfield microscope.

8. Tissue staining of contralateral and denervated rat gastrocnemius sections

1. Picrosirius Red (PSR)
   1. Perform PSR staining on 5 µm-thick, formalin-fixed paraffin embedded (FFPE) rat gastrocnemius histologic sections as previously described⁴⁰.
2. Oil Red O (ORO)
   1. Fix 5 µm-thick isopentane-frozen rat gastrocnemius histologic sections in 4% PFA for 10 min, incubate in 60% isopropyl alcohol for 1 min.
   2. Stain with ORO working stock for 12 min. Incubate in 60% isopropyl alcohol for 1 min, wash for 10 min in dH₂O. Mount on coverslips using a water-soluble mounting media.
3. Sca-1 and laminin tissue fluorescent immunohistochemistry (IHC)
   1. Perform fluorescent IHC on 5 µm-thick isopentane-frozen rat gastrocnemius histologic sections.
   2. Hydrate samples in 1x PBS for 5 min, fix in 4% PFA for 10 min then incubate samples in tissue IF blocking solution (see Supplementary File) for 90 min.
   3. Incubate with anti-Sca-1 primary antibody (1:500) diluted in 1x PBS + 0.05% Tween at 4 °C overnight.
   4. On Day 2, wash three times in 1x PBS + 0.05% Tween for 5 min each, then incubate in goat anti-rabbit Alexa Fluor 555 (1:500) for 1 h.
   5. Wash again (as before), incubate with blocking solution for 1 h, then add anti-laminin primary antibody (1:500) diluted in 1x PBS + 0.05% Tween for 1 h.
   6. Wash again (as before), then incubate in goat anti-rabbit Alexa Fluor 488 (1:500) for 1 h (for laminin).
   7. Wash again (as before) then incubate in DAPI (1:10,000) for 4 min. Wash and mount on coverslips using anti-fade mounting medium.

Representative Results

Identifying FAPs and MPs via flow cytometry using a novel antibody panel including Sca-1 and VCAM-1
The gating strategy for identifying FAPs in rat muscle is based upon flow cytometry protocols in the mouse²⁹, which gate on CD31 (endothelial) and CD45 (hematopoietic) positive cells (termed the lineage [Lin]) and examines the fluorescent profile of FAPs marker Sca-1 and MPs marker ITGA7 from the linage-negative (Lin-) population. In the absence of commercially available, fluorophore-conjugated, and flow cytometry-validated antibodies for rat Sca-1 and ITGA7, we self-conjugated a rat Sca-1::APC antibody, and undertook an alternative strategy to identify the MPs. As VCAM-1 was recently shown to be an efficient single positive selection marker for the isolation of rat MPs³³ and a rat-specific, conjugated, flow cytometry-validated antibody targeting VCAM-1 is available, we utilized VCAM-1 to identify MPs, as opposed to ITGA7.

We confirmed successful conjugation and performance of the Sca-1:APC antibody with single staining of compensation beads and cell suspensions generated from healthy rat gastrocnemius (Figure 2A,B). A five-point titration of Sca-1::APC (Figure 2C) was performed in addition to all other antibodies (CD31::FITC, CD45::FITC, VCAM-1:PE) used in the protocol (Supplementary Figure 1), to identify the optimum concentrations. Using this novel panel design, putative FAPs and MPs were simultaneously identified from healthy gastrocnemius (Figure 3), whereby cells single-positive for Sca-1 (Lin-/Sca-1+/VCAM-1-) were designated FAPs (red box) and cells single-positive for VCAM-1 (Lin-/Sca-1-/VCAM-1+) were designated MPs (blue box). We also identified a population of cells double positive for Sca-1 and VCAM-1 (Lin-/Sca-1+/VCAM-1+) (Figure 3F; upper right quadrant).

Validation of identification of FAPs and MPs by FACS and cell culture
To validate the protocol presented for flow cytometric identification of FAPs and MPs in rat skeletal muscle, we sought to isolate live cells for culture in vitro. Using FACS, viable FAPs and MPs were isolated using the same gating strategy as in the flow cytometric analysis. Approximately 20,000-40,000 FAPs and 30,000-50,000 MPs were collected for cell culture from a single gastrocnemius muscle from a 230 g rat.

To confirm the purity of each population, we co-immunostained freshly sorted FAPs and MPs for PDGFRα and Pax7. PDGFRα is the second mesenchymal progenitor marker, besides Sca-1, commonly used to identify FAPs^2,7,8. Pax-7 is a well-recognized and widely used marker of muscle satellite (stem) cells⁴¹. The sorted population of FAPs displayed positive staining for PDGFRα with no contamination by Pax7 positive cells (Figure 4A; top row). Conversely, the sorted population of MPs stained positive for Pax7 with an absence of PDGFRα positive cells (Figure 4A; bottom row), validating the ability of Lin-/Sca-1+/VCAM-1- and Lin-/Sca-1-/VCAM-1+ cell surface antigen profiles to isolate pure populations of FAPs and MPs respectively.

We next cultured sorted FAPs and MPs over a time course of 10-12 days in conditions to induce adipogenic, fibrogenic, and myogenic differentiation. By day 12, FAPs cultures subjected to adipogenic conditions contained cells with either a fibroblast-like morphology or a multilocular morphology similar to that of white pre-adipocytes with fat droplets (data not shown). Immunostaining for fibroblast specific protein-1 (FSP-1) confirmed fibroblast differentiation, while staining for Plin-1 (Perilipin-1; an adipocyte marker)⁴² and Oil red O confirmed the differentiation of adipocytes and the presence of neutral triglycerides and lipids, respectively (Figure 4B). The absence of contaminating cells from a myogenic lineage in the FAPs cultures was confirmed by co-immunostaining for myosin heavy chain (MHC), a marker of differentiated myocytes. FAPs cultures subjected to fibrogenic differentiation (FD) were assessed for Collagen type 1 (Col1a1) expression, one of the main FAPs-derived collagens⁸. Co-immunostaining for Col1a1 and MHC at Day 11 revealed robust Collagen type 1 expression with no contaminating MHC positive cells (Figure 4B). Final confirmation of purity of the FAPs population was undertaken by culturing FAPs in myogenic media, to encourage the outgrowth of any contaminating MPs. No MHC positive cells were observed (Supplementary Figure 2A).

MP cultures subjected to myogenic conditions demonstrated MHC-expressing mature myocytes and multi-nucleated myotubes 12 days post plating (Figure 4C). Co-immunostaining of the MP cultures with FSP-1, and Plin-1 demonstrated the absence of contaminating fibroblasts or adipocytes, respectively. Similarly, ORO staining did not demonstrate lipid contamination (Fig. 4C). Col1a1, which is highly expressed by fibroblasts, has also been reported to be produced to a lesser extent by myogenic precursors and myoblasts, although there are contradictory data in the literature in this regard^8,43,44. While co-immunostaining of MPs with Col1a1 and MHC revealed myocyte differentiation of our MP culture, no evidence of Col1a1 immunopositivity was found (Figure 4C). To further confirm the purity of the population, MP cultures were subjected to adipogenic or fibrogenic conditions to encourage the growth of adipocytes and fibroblasts (Supplementary Figure 2B). No contaminating cells from the FAPs lineage were observed.

While we had demonstrated the ability to identify and sort pure populations of FAPs and MPs from rat muscle, we next sought to determine the identity of the Lin-/Sca-1+/VCAM-1+ (double positive) cells. Since Sca-1 expression has been reported on a very small proportion of MPs (approximately 3%) in healthy muscle⁴⁵ and similarly few FAPs (approx. 4%) have been reported to express VCAM-1 in healthy muscle¹⁰, immunostaining of freshly sorted Lin-/Sca-1+/VCAM-1+ cells was performed for PDGFRα and Pax7 along with culturing them in myogenic, adipogenic, and fibrogenic conditions to induce myogenesis, adipogenesis and fibrogenesis respectively. It was found that the double-positive cells were a mixed population of MPs and FAPs, with freshly sorted cells immunostaining positive for either PDGFRα or Pax7 (Supplementary Figure 3A) and cultured cells differentiating into mature myocytes, adipocytes or fibroblasts (Supplementary Figure 3B,C).

ITGA7 vs VCAM-1 to identify MPs during flow cytometric FAPs identification
While a successful protocol for flow cytometric identification of rat FAPs and MPs using Sca-1 and VCAM-1 respectively was established, we sought to determine if a self-conjugated ITGA7 antibody could similarly be used to identify MPs instead of VCAM-1, as is standard in the mouse. A rat-specific ITGA7 antibody was conjugated to PE-Cy7 (see Supplementary File) and the antibody's performance was validated on commercial compensation beads and single cell suspensions generated from rat gastrocnemius (Supplementary Figure 4A-C). The ITGA7::PE-Cy7 antibody performed adequately on single staining and FMO experiments (Supplementary Figure 4D). However, when full staining gastrocnemius cell suspensions with CD31::FITC, CD45::FITC, Sca-1::APC, and ITGA7::PE-Cy7, an interaction became evident between the Sca-1::APC and ITGA7::PE-Cy-7 antibodies. Subsequent culture of both the FACS sorted Lin-/Sca-1+/ITGA7- cells (purported FAPs) and Lin-/Sca-1-/IGTA7+ cells (purported MPs) (Supplementary Figure 4E) yielded predominantly FAPs and with few MPs (Supplementary Figure 4F), indicating the interaction between Sca-1::APC and ITGA7::PE-Cy-7 antibodies negatively impacted the specificity of cell identification.

A novel FAPs time-course in long-term denervated skeletal muscle
As FAPs dynamics have been assessed in the context of short-term traumatic denervation, in murine models^{11, 38}, we sought to validate the performance of our method for FAPs isolation from both healthy and severely atrophic, fibrotic muscle. Rats were subjected to traumatic long-term denervation injury using the well-validated unilateral tibial nerve transection model⁴⁶ with gastrocnemius muscle harvested at four serial timepoints over 14 weeks post-denervation from the denervated limb and contralateral innervated limb (to serve as an internal control). As has been previously reported, denervated muscle demonstrated progressive atrophy (Figure 5A,B), with increasing fibrosis and fat deposition (Figure 5C-F) over time⁴⁷.

Our protocol generated an adequate number of cells for flow cytometric analysis, even though 12 and 14 week-denervated gastrocnemius weighed approximately 0.2-0.3 g (15-20% of respective contralateral control muscle) (Figure 5B). We observed up-regulation of FAPs in the denervated gastrocnemius muscle compared to the innervated contralateral control muscle, maintained for the 14 week duration of the experiment (Figure 6A,B), concordant with the progressive muscle fibrosis and fat deposition. Sca-1 immunostaining of muscle histologic cross sections confirmed localization of Sca-1 expressing cells to areas of fibro-fatty change in 14 week-denervated muscle (Figure 5G), in addition to baseline expression in the interstitium between myofibers in healthy muscle. A distinct subset of cells emerged in the FAPs population over time post-denervation characterized by a robust increase in Sca-1 signal (Sca-1 high; Figure 6A, red box) on flow cytometric analysis, compared to the FAPs basal Sca-1 expression (Sca-1 Med/Low; Figure 6A, green box). Quantification of these subpopulations revealed differential dynamics over time; Sca-1 High FAPs increased significantly at 12- and 14-weeks post-denervation (Figure 6B), comprising approximately half of the total FAPs population (Figure 6C). In contrast, Sca-1 Med/Low FAPs were the dominant subpopulation at 2- and 5-weeks post-denervation (Figure 6C) and showed only a transient increase in levels early post-denervation (Figure 6B).

In contrast to the sustained presence of FAPs in long-term denervated muscle, MPs demonstrated an expected biphasic response to denervation. Initially the MPs increased in denervated muscle above that seen in the control limb, but at 5 weeks post-denervation the population began to decline (Figure 6D). In keeping with the well-known exhaustion of the muscle satellite cell population in long-term denervated muscle⁴⁷, by 12 weeks MPs were either at or below baseline levels post tibial nerve transection.

We also analyzed the dynamics of the Lin-/Sca-1+/VCAM-1+ population (Supplementary Figure 3D-E). While the frequency of double positive cells relative to the Lin- population progressively decreased in denervated muscle over time, when the absolute cell count was determined per gram of muscle a temporary increase in the double positive population was observed, prior to falling to or below baseline levels by 14 weeks post-denervation.

Figure 1: Graphical schematic for FAPs and MPs identification, isolation, and culture: Graphical schematic depicting FAPs and MPs identification from rat gastrocnemius. Samples are harvested and mechanically minced before undergoing two sequential enzymatic digestions. Tissue preparations are then processed and filtered to generate a single cell suspension. A cocktail of fluorescently-conjugated antibodies is added to each sample which is then run through a flow cytometer or cell sorter to respectively identify or isolate FAPs and MPs. Please click here to view a larger version of this figure.

Figure 2: Sca-1::APC self-conjugated antibody validation and titration. Validation of Sca-1::APC antibody conjugation by single-staining on commercial compensation beads (A) and single cell suspensions generated from healthy rat gastrocnemius muscle (B). Distinct populations of both Sca-1 labeled beads and cells are evident (black boxes). Antibody titration was performed by testing five different concentrations of Sca-1::APC on single cell suspensions (C); the optimal concentration was chosen based on greatest fluorescence intensity with minimal background staining and found to be batch dependent. A representative titration is shown with the batch specific optimal concentration indicated by the red box. Please click here to view a larger version of this figure.

Figure 3: Flow cytometry identification of FAPs and MPs in rat gastrocnemius. (A-F) Gating strategy for flow cytometric analysis of FAPs and MPs. (A) Samples are first gated to exclude debris (black box) as well as to gate for counting beads (red box). Cells are then gated to exclude doublets both by front scatter (FSC) (B) and side scatter (SSC) characteristics (C). The viability of resulting single cells is assessed by staining with SYTOX Blue (D). SYTOX Blue negative (live) singlets are then assessed for FITC signal identifying CD31 and CD45 (Lineage; Lin) in order to exclude Lin+ fractions from further analysis (black box Lin- cells) (E). The Lin- population is then assessed for Sca-1::APC versus VCAM-1::PE signal (F). FAPs are identified as Lin-/Sca-1+/VCAM-1- (F; red box) while MPs are identified as Lin-/Sca-1-/VCAM-1+ events (F; blue box). A Lin-/Sca-1+/VCAM-1+ double positive population is also evident (upper right quadrant). (G) FMO (Fluorescence Minus One) controls for CD31::FITC and CD45::FITC demonstrate proper compensation and gating for Lin- cells. (H-I) Sca-1::APC versus VCAM-1::PE plots of FMO controls demonstrate proper compensation and gating for FAPs (Sca-1 FMO) and MPs (VCAM-1 FMO). Please click here to view a larger version of this figure.

Figure 4: Sorted FAPs and MPs cell culture and immunostaining. (A) Co-immunostaining for PDGFRα (green) and Pax7 (red) on freshly sorted FAPs (top row) and MPs (bottom row). Nuclei stain blue with DAPI. FAPs solely express PDGFRα and no Pax7 positive cells are evident, while MPs exclusively express Pax7 with no PDGFRα positive cell contamination, demonstrating the purity of both sorted populations. (B) FAPs at Day 12 in adipogenic differentiation media (AD) stain positive for Fibroblast-specific Protein 1 (FSP-1; green) and Perilipin-1 (Plin-1; green), demonstrating differentiated fibroblasts and adipocytes, respectively. Nuclei are stained with DAPI (blue). Oil Red O (ORO) staining (red) indicates the presence of neutral triglycerides and lipids arising from mature adipocytes by Day 12. FAPs subjected to fibrogenic differentiation media (FD) demonstrate expression of FAPs-derived Collagen type 1 (Col1a1; green). FAPs cultured in either adipogenic or fibrogenic media do not co-immunostain for myosin heavy chain (MHC, red), indicating an absence of contaminating myocytes. (C) MPs grown in myogenic differentiation media (MD) on Day 12 display MHC positive (red) staining demonstrating the presence of mature myocytes and fused multinucleated myotubes. Nuclei are stained with DAPI (blue). MP cultures were clear of fibroblast and adipocyte contamination as indicated by the absence of co-immunostaining for FSP-1 (green), Plin-1 (green) and ORO staining. MPs grown on laminin to accommodate Co1a1 immunostaining do not display the same degree of myotube fusion by Day 12 as occurs on collagen. Col1a1 (green) is absent from MP cultures. Please click here to view a larger version of this figure.

Figure 5: Atrophy, fibrosis, and fatty infiltration of long-term denervated muscle. (A) Representative harvested gastrocnemius muscles at four serial timepoints post-denervation. Gastrocnemius denoted CONTRA is a representative contralateral limb sample from a two-week denervated animal. (B) Quantification of muscle atrophy post-denervation expressed as the ratio of denervated (DEN) gastrocnemius wet weight to that of the contralateral (CONTRA) limb. (C) Picrosirius Red (PSR) stained histologic cross-sections of gastrocnemius harvested 2 or 14 weeks post-denervation demonstrate progressive fibrosis. Muscle stains yellow, collagen stains red. (D) Fibrosis quantified by determining area of PSR positive tissue relative to total tissue area. (E) Oil Red O (ORO) stained cross-sections of gastrocnemius harvested 2- or 14-weeks post-denervation, counter-stained with hematoxylin. Lipids stain red. (F) Fatty infiltration quantified by determining area of ORO stained tissue relative to total tissue area. (G) Gastrocnemius histologic cross-sections harvested 2- or 14-weeks post-denervation, immunostained for laminin (green) and Sca-1 (red). Laminin positively stains the basal lamina that surrounds individual myofibers. Sca-1 identifies multiple progenitor cell types. Nuclei are stained with DAPI (blue). Inset shows localization of Sca-1+ cells outside the basal lamina in healthy muscle; yellow arrows denote presence of Sca-1+ cells within fibrotic areas. (Data are mean +/- S.D; n=3 for 2 and 14 week timepoints. Data in panel B analyzed by one-way ANOVA, data in panels D and F analyzed by two-way ANOVA. Post-hoc Sidak's test performed to correct for multiple comparisons. * = P<0.05 between CONTRA and DEN; # = P<0.05 between samples) Please click here to view a larger version of this figure.

Figure 6: FAPs and MPs dynamics in long-term denervated rat gastrocnemius. (A) Representative Sca-1::APC versus VCAM-1::PE panels of the Lin- (CD31-/CD45-) population from muscle samples harvested 2-, 5-, 12-, or 14-weeks post-denervation. Top row shows plots from the denervated (DEN) gastrocnemius, while the bottom row shows those from the contralateral, unoperated (CONTRA) gastrocnemius. Lin-/Sca-1+/VCAM-1- FAPs (Total) are subdivided into Sca-1 High (red box) and Sca-1 Med/Low (green box) fractions, while Lin-/Sca-1-/VCAM-1+ MPs are shown in the blue box. (B) Quantification of FAPs (Total, Sca-1 High, Sca-1 Med/Low) at each timepoint post-denervation, reported as the frequency (%) of Lin- cells (top row) and the number of cells per gram muscle (bottom row) normalized to the contralateral control gastrocnemius. (C) Relative proportions of Sca-1 High and Sca-1 Med/Low subpopulations of the total FAPs population. (D) Quantification of MPs at each timepoint post-denervation reported as the frequency (%) of Lin- cells (left graph) and as the number of cells per gram muscle (right graph) normalized to the contralateral control. (Data are mean +/- S.D; n=4 for 2 and 12 week timepoints; n=5 for 5 week timepoint; n=2 for 14 week timepoint. Data analyzed by one-way ANOVA; Post-hoc Sidak's test to correct for multiple comparisons. # = P<0.05.) Please click here to view a larger version of this figure.

Supplementary Figure 1: Antibody validation and titration. Validation of CD31::FITC (A-C), CD45::FITC (D-F), and VCAM-1::PE (G-I) commercially-conjugated antibodies on compensation beads (A,D,G) and single-cell suspensions from healthy rat gastrocnemius muscle (B,E,H). Each antibody was titrated at 5 different concentrations (C,F,I). The optimal concentration was chosen based on greatest fluorescence intensity with minimal background staining (red boxes). Please click here to download this File.

Supplementary Figure 2: Sorted FAPs and MPs in reverse differentiation culture conditions. (A) FAPs cultured in myogenic differentiation media (MD) at Day 12 co-immunostained for FSP-1 (green), Plin-1 (green) or Col1a1 (green) and MHC (red) do not demonstrate myocyte contamination. Positive FSP-1, and Col1a1 staining reveal FAPs differentiation to fibroblasts. ORO staining (red) reveals lipid production although Plin-1 positive adipocytes are not readily visible. Nuclei stained with DAPI (blue). (B) MPs subjected to adipogenic differentiation media died. MPs cultured in FAP Growth Media (FAP GM) for 12 days and co-immunostained for FSP-1 (green) or Plin-1 (green) and MHC (red) demonstrate differentiated myocytes and myotubes with an absence of FSP-1 or Plin-1 positive cells. The absence of ORO staining further confirms the lack of adipogenic cells in culture. MPs grown in fibrogenic differentiation media (FD) and co-immunostained for Col1a1 and MHC show mature myocytes and an absence of Col1a1 positive cells. MPs grown on laminin to accommodate Col1a1 immunostaining do not display the same degree of fusion to myotubes at 12 days in culture as occurs on collagen. Please click here to download this File.

Supplementary Figure 3: Lin-/Sca-1+/VCAM-1+ cells are a mixed population of FAPs and MPs. (A) PDGFRα and Pax7 co-immunostaining of freshly isolated Lin-/Sca-1+/VCAM-1+ cells show a mixed population of PDGFRα single positive (white arrows) and Pax7 single positive (yellow arrow) cells identifying FAPs and MPs, respectively. (B) Lin-/Sca-1+/VCAM-1+ cultures on Day 12 grown in myogenic differentiation media (MD) contain both FSP-1 single positive (green) and MHC single positive (red) cells identifying fibroblasts and myocytes/tubes, respectively. An absence of Plin-1 (green) and ORO (red) staining indicate the absence of mature adipocytes. Co-immunostaining for Col1a1 (green) and MHC (red) show mature myocytes and fibroblasts. (C) Lin-/Sca-1+/VCAM-1+ cells on Day 12 grown in adipogenic differentiation media (AD) similarly show a mix of FSP-1 (green) single positive and MHC (red) single positive cells, but also with Plin-1 (green) single positive cells and ORO staining present, confirming the differentiation of fibroblasts, myocytes and adipocytes. Cultures grown in fibrogenic differentiation media (FD) show mature myocytes and Col1a1 single positive cells (fibroblasts). (D) Representative Sca-1::APC versus VCAM-1::PE panels of the Lin- (CD31-/CD45-) population from denervated muscle samples harvested 2-, 5-, 12-, or 14-weeks post-denervation; Lin-/Sca-1+/VCAM-1+ population are indicated in the purple box. (E) Quantification of Lin-/Sca-1+/VCAM-1+ cells across four serial time-points post-denervation, reported as the frequency of Lin- cells (left graph) and cells per gram muscle (right graph) normalized to the contralateral gastrocnemius. (Data are mean +/- S.D; n=4 for 2 and 12 week timepoints; n=5 for 5 week timepoint; n=2 for 14 week timepoint. Data were analyzed by one-way ANOVA; post-hoc Sidak's test to correct for multiple comparisons; # = P<0.05.) Please click here to download this File.

Supplementary Figure 4: ITGA7 as a marker of myogenic cells in flow cytometry of rat skeletal muscle. Validation of ITGA7::PE-Cy7 antibody conjugation by single-staining on commercial compensation beads (A) and single cell suspensions generated from healthy rat gastrocnemius muscle (B). Populations of both ITGA7 labeled beads and cells are evident (black boxes) (C) Antibody titration was performed by testing five different concentrations on single cell suspensions. Red box indicates ideal concentration. (D) Plots of Fluorescence Minus One (FMO) controls demonstrate the absence of fluorescence spillover across the antibody conjugates, but full stain of cell suspensions reveals a non-specific interaction between Sca-1::APC and ITGA7::PE-Cy7 (green box). Gating (E) to separate Lin-/Sca-1+/ITGA7- cells (purported "FAPs") and Lin-/Sca-1-/IGTA7+ cells (purported "MPs"). (F) Co-immunostaining of FACS-sorted cell cultures at 12 days post plating with Perilipin-1 (green) and MHC (red) demonstrates both groups of sorted cells matured predominantly into adipocytes with few myocytes present. Please click here to download this File.

1. Unstained

2. Viability Control

3. CD31 + CD45 FMO

4. Sca-1 FMO

5. VCAM-1 FMO

6. CD31 + CD45 Single Stain (Beads)

7. CD31 + CD45 Single Stain (Cells)

8. Sca-1 Single Stain (Beads)

9. Sca-1 Single Stain (Cells)

10. VCAM-1 Single Stain (Beads)

11. VCAM-1 Single Stain (Cells)

Table 1: Flow cytometry antibody staining controls. Full complement of antibody staining controls. If the experiment is being performed for the first time, single stained controls should be run on both compensation beads and single cell suspensions. For all subsequent experiments, single stained controls only need to be run on compensation beads.

	CD31::FITC (µg)	CD45::FITC (µg)	Sca-1::APC (µg)*	VCAM-1::PE (µg)	SYTOX Blue (µM; Final Concentration)
Unstained Control
	--	--	--	--	--
Single-Stained Controls
SYTOX Blue (Viability Control)	--	--	--	--	1
CD31 & CD45	0.5	0.25	--	--	1
Sca-1	--	--	0.25	--	1
VCAM-1	--	--	--	0.25	1
Fluorescence Minus One (FMO)
CD31 & CD45	--	--	0.25	0.25	1
Sca-1	0.5	0.25	--	0.25	1
VCAM-1	0.5	0.25	0.25	--	1
Experimental Samples
Full Stain	0.5	0.25	0.25	0.25	1

Table 2: Flow cytometry antibody staining matrix. The antibody amount for staining of experimental and control samples for flow cytometry is shown. All staining is performed in a total volume of 100 µL of wash buffer. *The optimal amount of self-conjugated Sca-1::APC antibody may vary depending on batch used. All freshly conjugated Sca-1::APC batches should be first validated by single-staining both compensation beads and single cell suspensions.

Supplementary file: Reagent recipes. Please click here to download this File.

Discussion

An optimized, validated FAPs isolation protocol for rat muscle is essential for researchers who wish to study injury models that are not feasible in the mouse for biologic or technical reasons. For example, mice are not an optimal animal model in which to study chronic local, or neurodegenerative injuries such as long-term denervation. Biologically, the short lifespan and rapid aging of mice make it difficult to accurately delineate the muscle sequalae due to denervation from the confounding factor of aging. From a technical standpoint, the drastically reduced muscle mass due to severe atrophy would be insufficient for effective flow cytometric analysis. In fact, mouse FAPs isolation protocols suggest grouping healthy muscles together to better increase yield of sorted FAPs²⁹. While this solves the technical barrier of obtaining adequate muscle tissue for analysis, it simultaneously limits researchers from assessing FAPs on a muscle-specific level. Since specific muscle groups vary inherently in their fiber type composition, degree of vascularization, and mitochondrial numbers⁴⁷ for example, combining multiple different muscles for flow cytometric analysis prevents muscle-specific FAPs characterization. In contrast, we show that both healthy and long-term denervated, atrophic and fibrotic rat gastrocnemius provide an adequate amount of starting material for effective flow cytometry. Moreover, in healthy samples, the surplus of muscle can be further subdivided for multiple downstream assays, allowing researchers to concomitantly analyze the cellular, molecular, and histological features of the same tissue. Finally, for experiments manipulating FAPs in injury models with therapeutics, well validated analyses of physical function and gait in alert and active rats are available^21,22,23,24, enabling serial assessment of muscle function without the need for termination of the animal.

A key obstacle in creating an optimized FAPs isolation protocol for the rat was antibody availability. Our initial approach sought to copy the antibody panel and gating strategy protocols widely employed in the mouse^{1,7,8,9,10,11,12,13,14}. While commercially available, conjugated, flow cytometry-tested, and validated antibodies that recognize rat were available for the lineage markers CD31 and CD45, none existed for positive FAPs identifying markers Sca-1 or PDGFRα, as well as for the negative selection marker ITGA7. Unconjugated, primary antibodies specific to these markers and purported to be effective in flow cytometry were available, but of the FAPs markers Sca-1 and PDGFRα, only the Sca-1 antibody had been validated in the published literature in flow cytometric analysis of rat cells⁴⁸. We, therefore, selected Sca-1 as the positive selection marker for FAPs. The prospect of using secondary antibodies to delineate Sca-1 and ITGA7 markers was not feasible for several reasons, but primarily because the only rat-specific antibodies for Sca-1 and ITGA7 validated for flow cytometry were generated in the same host species. Therefore, the remaining option was self-conjugation of both antibodies using commercially-available kits.

The process of choosing an optimal kit for antibody conjugation was extensive, as many kits are completely or partially intolerant to various buffer diluents (e.g., Glycine, Glycerol, BSA, Sodium Azide) present in primary antibodies. In addition, the fluorophore provided must be compatible with the lasers equipped within the flow cytometer/cell sorter available to the investigator, and not emit at a similar wavelength to other fluorophores used to identify other cell types in the sample. The presence of diluent components in the rat-specific PDGFRα primary antibodies that were poorly compatible with the conjugation kits, was an additional consideration in the choice of Sca-1 as the positive FAPs selection marker in this protocol. While these results demonstrate that both Sca-1::APC and ITGA7::PE-Cy7 conjugations resulted in identification of distinct positive-stained populations on both compensation beads and cells, the former were found to exhibit decay in fluorescence sooner than was advertised by the manufacturer. It is highly recommended that researchers conjugate Sca-1::APC according to the manufacturer's instructions, immediately prior to the first time use (e.g., the day before the experiment) to ensure robust signal, plan experiments accordingly such that a single batch of conjugated antibody can be used within the antibody's window of effectiveness, and validate each batch by single-staining compensation beads and titrating on single cell suspensions. More importantly, however, the critical interaction noted between Sca-1::APC and ITGA7::PE-Cy7 prevented the accurate delineation of populations of FAPs vs MPs, necessitating an alternative strategy be employed.

Boscolo Sesillo et al. recently demonstrated the successful flow cytometric isolation of rat muscle stem cells using VCAM-1 as a single positive selection marker³³ in CD31, CD45 and CD11b negative cells. With VCAM-1 as the MP selection marker, we utilized a novel antibody panel to identify FAPs (Lin-/Sca-1+/VCAM-1-) and MPs (Lin-/Sca-1-/VCAM-1+) and validated the approach with in vitro culture of FACS sorted cells from healthy gastrocnemius muscle. Freshly isolated FAPs and MPs immunostained solely for their respective alternative markers PDGFRα and Pax7. Cultured FAPs differentiated into populations of mature fibroblasts and adipocytes, and MPs into mature myocytes/myotubes. No cross contamination of FAPs and MPs within the cultures occurred. Immunostaining of histologic cross-sections confirmed infiltration of areas of fibro-fatty degradation in denervated muscle with Sca-1 positive cells.

While we undertook flow cytometric analysis of long-term denervated muscle to validate our protocol in severely atrophic, fibrotic and fat-infiltrated muscle, we incidentally observed the emergence of a FAPs sub-population with increased Sca-1 signal (denoted Sca-1 High) as compared to the baseline Sca-1 expression (denoted Sca-1 Med/Low) over time. Sca-1 High FAPs increased significantly late post-denervation (12 weeks plus), while Sca-1 Med/Low FAPs peaked early at 5 weeks before declining back to baseline levels. Heterogeneity in the FAPs phenotype has been reported^10,30. FAPs with higher Sca-1 expression were shown to differentiate more readily into adipocytes, and when exposed to fibrogenic stimulation increased expression of Col1a1^30,49. The dynamics of the FAPs sub-populations observed here appear to be in-line with the time-course of denervation-induced sequelae and muscle's regenerative capacity⁴⁷ and thus may characterize FAPs sub-populations with differing cellular programs. Sca-1 High FAPs become up-regulated at late time-points concomitant with fibro/fatty infiltration and a decline in regenerative potential, while Sca-1 Med/Low FAPs become up-regulated during muscle's regenerative window and may aid in effective regenerative myogenesis. The FAPs isolation protocol presented here provides investigators the ability to identify and isolate these various populations for future study.

We identified a population of cells staining positive for both Sca-1 and VCAM-1 (Lin-/Sca-1+/VCAM-1+) and ascertained this double positive population to be a mixture of FAPs and MPs. Separation of this population using a third marker - ITGA7 - was not possible due to the interaction between Sca-1 and ITGA7 primary antibodies. Malecova and colleagues reported a sub-population of VCAM-1 expressing FAPs that is nearly absent in healthy muscle, transiently increased with acute inflammation, and their persistence in mdx mice (model of muscular dystrophy) was associated with chronic muscle inflammation and fibrosis¹⁰. In contrast, and although not a pure FAPs population, we found that the Lin-/Sca-1+/VCAM-1+ population decreased to or below baseline levels with chronic muscle fibrosis at 12- and 14-weeks post-denervation. Denervation does not induce the same inflammatory reaction and cycling regeneration attempts experienced by mdx mice, which may explain the differences in our results. Our identification of MPs in the Lin-/Sca-1+/ VCAM-1+ cell population is in keeping with the report of Sca-1 expression on a very small proportion of MPs in healthy muscle, with a transient increase following injury during myoblast proliferation and subsequent withdrawal from the cell cycle⁴⁵. Thus, while our antibody labeling and FACS gating strategy successfully isolates pure populations of FAPs and MPs for study, experiments requiring flow cytometry-based quantification of these populations may slightly underestimate their numbers due to the small percentage of cells in both progenitor lines that co-express Sca-1 and VCAM-1. Regardless, the current protocol clearly demonstrates the classic biphasic response of MPs to denervation injury, with short-term upregulation and subsequent depletion of the population in long-term denervated muscle. Similarly, the increase in FAPs reported in isolated shorter-term denervation injury is recapitulated here, and shown to be further sustained using the long-term tibial nerve transection model.

In summary, this protocol provides researchers with a previously unexplored animal, the rat, in which to use flow cytometric methods to simultaneously study FAPs and MPs. Experiments in mice limited by quantity of skeletal muscle, and the frequent need to undertake terminal experiments to assess muscle strength and function, can be readily conducted in the larger rat and with a more extensive range of non-lethal strength and functional assessment methods. Overall, FAPs studies in the rat may uncover novel roles of this key progenitor population in acute and chronic muscle and peripheral nerve trauma and disease, subsequently increasing the potential for developing cell-specific therapies.

Materials

Name	Company	Catalog Number	Comments
5 mL Polypropylene Round-Bottom Tube	Falcon	352063
5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap	Falcon	352235
10 cm cell culture dishes	Sarstedt	83.3902
12-well cell culture plate	ThermoFisher	130185
12 mm glass coverslips, No.2	VWR	89015-724
10 mL Syringe	Beckton Dickenson	302995
15 mL centrifuge tubes	FroggaBio	91014
20 gauge needle	Beckton Dickenson	305176
25mL Serological pipette	Sarstedt	86.1685.001
40µm cell strainer	Fisher Scientific	22363547
50mL centrifuge tubes	FroggaBio	TB50
AbC Total Antibody Compensation Beads	ThermoFisher	A10497
Ammonium Chloride, Reagent Grade	Bioshop	AMC303.500
APC Conjugation Kit, 50-100µg	Biotium	92307
Aquatex Aqueous Mounting Medium	Merck	108562
Biolaminin 411 LN	Biolamina	LN411
Bovine Serum Albumin (BSA)	Bioshop	ALB001
Calcium Chloride	Bioshop	CCL444.500
Collagenase Type II	Gibco	17101015
CountBright Plus Absolute Counting Beads	ThermoFisher	C36995
Dexamethasone	Millipore Sigma	D4902
Dispase	Gibco	17105041
Dulbecco’s Modified Eagle Medium (DMEM) (1X)	Gibco	11995-065	(+)4.5 g/L D-Glucose (+)L-Glutamine (+)110 mg/L Sodium Pyruvate
EDTA	FisherScientific	S311
FACSClean Solution	Beckton Dickenson	340345
FACSDiva Software	Beckton Dickenson	--
FACSRinse Solution	Beckton Dickenson	340346
Fetal Bovine Serum	Sigma	F1051
Flow Cytometry Sheath Fluid	Beckton Dickenson	342003
FlowJo Software	Beckton Dickenson	--
Fluorescent Mounting Medium	Dako	S302380-2
Goat anti-mouse Alexa Fluor 555 secondary antibody	Invitrogen	A21424
Goat anti-rabbit Alexa Fluor 488 secondary antibody	Invitrogen	A11008
Goat anti-rabbit Alexa Fluor 555 secondary antibody	Invitrogen	A21429
Goat Serum	Gibco	16210-064
Ham's F10 Media	ThermoFisher	11550043	(+) Phenol Red (+) L-Glutamine (-) HEPES
Hank’s Balanced Salt Solution (HBSS) (1X)	Multicell	311-513-CL
Heat Inactivated Horse Serum	Gibco	26050-088
Hemocytometer	Reichert	N/A
HEPES, minimum 99.5% titration	Sigma	H3375
Horse Serum	ThermoFisher	16050130
Human Transforming Growth Factor β1 (hTGF-β1)	Cell Signaling	8915LF
Humulin R	Lilly	HI0210
IBMX	Millipore Sigma	I5879	Also known as 3-Isobutyl-1-methylxanthine
Isopropanol	Sigma	I9516	Also known as 2-propanol
Lewis Rat, Female	Charles River Kingston	004 (Strain Code)	200-250 grams used
LSRFortessa X-20 Benchtop Cytometer	Beckton Dickenson	--
Microcentrifuge	Eppendorf	EP-5417R
MoFlo XDP Cell Sorter	Beckman Coulter	--
Mouse Anti-CD31::FITC Antibody	Abcam	ab33858	Clone TLD-3A12
Mouse Anti-CD45::FITC Antibody	Biolegend	202205	Clone OX-1
Mouse Anti-CD106::PE Antibody	Biolegend	200403	Also known as VCAM-1
Mouse Anti-MHC Antibody	Developmental Studies Hybridoma Bank (DSHB)	N/A	Also known as MF20
Mouse Anti-Pax7 Antibody	Developmental Studies Hybridoma Bank (DSHB)	N/A
Neutral Buffered Formalin, 10 %	Sigma	HT501128
Oil Red O	Millipore Sigma	O0625
PE-Cy7 Conjugation Kit	Abcam	ab102903
Penicillin-Streptomycin	Sigma	P4333
Phosphate Buffered Saline, pH 7.4 (1X)	Gibco	10010-023	(-)Calcium Chloride (-)Magnesium Chloride
Potassium Bicarbonate, Reagent Grade	Bioshop	PBC401.250
Rabbit Anti-Fibroblast Specific Protein 1 (FSP-1) Antibody	Invitrogen	MA5-32347	FSP-1 also known as S100A4
Rabbit Anti-Integrin-a7 Antibody	Abcam	ab203254
Rabbit Anti-Laminin Antibody	Sigma	L9393
Rabbit Anti-Perilipin-1 Antibody	Abcam	ab3526
Rabbit Anti-Sca-1 Antibody	Millipore Sigma	AB4336
Rabbit Recombinant Anti-Collagen Type I Antibody	Abcam	ab260043	Also known as Col1a1
Rabbit Recombinant Anti-PDGFR Alpha Antibody	Abcam	ab203491
Recombinant Human FGF-basic	Gibco	PHG0266
Sodium Azide	Sigma	S2002
Triton-X-100	Fisher Scientific	BP151
Troglitazone	Millipore Sigma	T2573
Tween-20	Bioshop	TWN510