Name: facs-isolation and culture of fibro-adipogenic progenitors and muscle stem cells from unperturbed and injured mouse skeletal muscle
Uploaded: 2022-06-08T14:30:00
Description: Watch this Scientific Journal Video about FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle at JoVE.com

We would like to thank Tom Cheung (The Hong Kong University of Science &#38; Technology) for advice on MuSC isolation. This work was funded by the NIAMS-IRP through NIH grants AR041126 and AR041164.

All animal experiments performed were conducted in compliance with institutional guidelines approved by the Animal Care and Use Committee (ACUC) of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS). Investigators performing this protocol must adhere to their local animal ethics guidelines.
NOTE: This protocol describes in detail how to isolate FAPs and MuSCs from hind limb and injured tibialis anterior (TA) muscles of adult male and female mice (3-6 months) and pr...

<ol>
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Establishing efficient and reproducible protocols for the identification and isolation of pure adult stem cell populations is the first and most critical step toward understanding their function. Isolated FAPs and MuSCs can be used to conduct multiomics analysis in transplantation experiments as a potential treatment for muscular diseases or can be genetically modified for disease modeling in stem cell therapy. 
The protocol described here provides standardized guidelines for the identificatio...

The skeletal muscle is the largest tissue in the body, accounting for ~40% of adult human weight, and is responsible for maintaining posture, generating movement, regulating basal energy metabolism, and body temperature1. Skeletal muscle is a highly dynamic tissue and possesses a remarkable ability to adapt to a variety of stimuli, such as mechanical stress, metabolic alterations, and daily environmental factors. In addition, skeletal muscle regenerates in response to acute injury, leading to complete restoration of its morphology and functions2. Skeletal muscle plasticity mainly relies upon a population of resident muscle stem cells (MuSCs), also termed satellite cells, which are located between the myofiber plasma membrane and the basal lamina2,3. Under normal conditions, MuSCs reside in the muscle niche in a quiescent state, with only a few divisions to compensate for cellular turnover and to replenish the stem cell pool4. In response to injury, MuSCs enter the cell cycle, proliferate, and either contribute to the formation of new muscle fibers or return to the niche in a self-renewal process2,3. In addition to MuSCs, homeostatic maintenance and regeneration of the skeletal muscle rely upon the support of a population of muscle resident cells named fibro-adipogenic progenitors (FAPs)5,6,7. FAPs are mesenchymal stromal cells embedded in the muscle connective tissue and capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage5,8,9,10. FAPs provide structural support for MuSCs as they are a source of extracellular matrix proteins in the muscle stem cell niche. FAPs also promote long-term maintenance of the skeletal muscle by secreting cytokines and growth factors that provide trophic support for myogenesis and muscle growth6,11. Upon acute muscle injury, FAPs rapidly proliferate to produce a transient niche that supports the structural integrity of the regenerating muscle and provides a favorable environment to sustain MuSCs proliferation and differentiation in a paracrine manner5. As regeneration proceeds, FAPs are cleared from the regenerative muscle by apoptosis, and their numbers gradually return to basal level12. However, in conditions favoring chronic muscle injury, FAPs override pro-apoptotic signaling and accumulate in the muscle niche, where they differentiate into collagen-producing fibroblasts and adipocytes, leading to ectopic fat infiltrates and fibrotic scar formation12,13.
Due to their multipotency and their regenerative abilities, FAPs and MuSCs have been identified as prospective targets in regenerative medicine for the treatment of skeletal muscle disorders. Therefore, to investigate their function and therapeutic potential, it is important to establish efficient and reproducible protocols for the isolation and culture of FAPs and MuSCs.
Fluorescence-activated cell sorting (FACS) can identify different cell populations based on morphological characteristics such as size and granularity, and permits cell-specific isolation based on the use of antibodies directed against cell surface markers. In adult mice, MuSCs express the vascular cell adhesion molecule 1 (VCAM-1, also known as CD106)14,15 and &#945;7-Integrin15, while FAPs express the platelet-derived growth factor receptor &#945; (PDGFR&#945;) and the stem cell antigen 1 (Sca1 or Ly6A/E)5,6,9,12,16,17. In the protocol described here, MuSCs were identified as CD31-/CD45-/Sca1-/VCAM-1+/&#945;7-Integrin+, while FAPs were identified as CD31-/CD45-/Sca1+/VCAM-1-/&#945;7-Integrin-. Alternatively, PDGFR&#945;EGFP mice were employed to isolate FAPs as CD31-/CD45-/PDGFR&#945;+/VCAM-1-/&#945;7-Integrin- events18,19. Furthermore, we compared the overlapping between the fluorescent signal of PDGFR&#945;-GFP+ cells to cells identified by the surface marker Sca1. Our analysis showed that all GFP-expressing cells were also positive for Sca1, indicating that either approach can be employed for the identification and isolation of FAPs. Finally, staining with specific marker antibodies confirmed the purity of each cell population.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL Polypropylene Round-Bottom Tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G BD Needle 1 in. single use, sterile</td>
			<td>BD Biosciences&#160;</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Alpha 7 Integrin PE (clone:R2F2) (RatIgG2b)</td>
			<td>The University of British Columbia</td>
			<td>53-0010-01</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD31 Antibody</td>
			<td>BioLegend</td>
			<td>102510</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD45 Antibody</td>
			<td>BioLegend</td>
			<td>103112</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSMelody Cell Sorter</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer-Lok tip control syringe, 10-mL</td>
			<td>BD Biosciences&#160;</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse CD106 Antibody</td>
			<td>BioLegend</td>
			<td>105703</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J&#160; mouse (Female and Male)</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.129S4-Pdgfratm11(EGFP)Sor/J mouse</td>
			<td>The Jackson Laboratory</td>
			<td>007669</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning BioCoat Collagen I 6-well Clear Flat Bottom TC-treated Multiwell Plate</td>
			<td>Corning</td>
			<td>356400</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning BioCoat Collagen I 12-well Clear Flat Bottom TC-treated Multiwell Plate</td>
			<td>Corning</td>
			<td>356500</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning BioCoat Collagen I 24-well Clear Flat Bottom TC-treated Multiwell Plate</td>
			<td>Corning</td>
			<td>356408</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Solution (1 mg/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>62248</td>
		</tr>
		<tr>
			<td>Disposable Aspirating Pipets, Polystyrene, Sterile</td>
			<td>VWR</td>
			<td>414004-265</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 40 &#181;m Cell Strainer, Blue, Sterile</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 60 mm TC-treated Cell Culture Dish, Sterile</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 15-mL</td>
			<td>VWR</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge Tubes, Polypropylene, Sterile, Corning, 50-mL</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Tubes, Polypropylene, Corning</td>
			<td>VWR</td>
			<td>60819-728</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Tubes, Polystyrene, with 35um Cell Strainer Cap Corning</td>
			<td>VWR</td>
			<td>21008-948</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor, Basic, Human, Recombinant (rhFGF, Basic)</td>
			<td>Promega</td>
			<td>G5071</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo 10.8.1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Collagenase, Type II, powder</td>
			<td>ThermoFisher Scientific</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Dispase, powder</td>
			<td>ThermoFisher Scientific</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco DMEM, high glucose, HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>12430054</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum, certified, United States</td>
			<td>ThermoFisher Scientific</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Ham&#39;s F-10 Nutrient Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>11550043</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Horse Serum, New Zealand origin</td>
			<td>ThermoFisher Scientific</td>
			<td>16050122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco PBS, pH 7.4</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco PBS (10x), pH 7.4</td>
			<td>ThermoFisher Scientific</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin-Glutamine (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 555</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21127</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened Fine Scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen 7-AAD (7-Aminoactinomycin D)</td>
			<td>ThermoFisher Scientific</td>
			<td>A1310</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse PDGF&#160;R alpha Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1062</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Fisher Scientific</td>
			<td>NC9624464</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>31872</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue anti-mouse Ly-6A/E (Sca 1) Antibody</td>
			<td>BioLegend</td>
			<td>108120</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16%</td>
			<td>Fisher Scientific</td>
			<td>NCC0528893</td>
			<td></td>
		</tr>
		<tr>
			<td>Pax7 mono-clonal mouse antibody (IgG1) (supernatant)</td>
			<td>Developmental Study Hybridoma Bank</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine7 Streptavidin</td>
			<td>BioLegend</td>
			<td>405206</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Dumont #5 Forceps</td>
			<td>Fine Science Tools Inc</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
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facs-isolation and culture of fibro-adipogenic progenitors and muscle stem cells from unperturbed and injured mouse skeletal muscle

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

This protocol allows the isolation of approximately one million FAPs and up to 350,000 MuSCs from uninjured hind limbs of wild-type adult mice (3-6 months), corresponding to a yield of 8% for FAPs and 3% for MuSCs of total events. When sorting cells from damaged TA 7 days post-injury, two to three TA muscles are pooled to obtain up to 300,000 FAPs and 120,000 MuSCs, which correspond to a yield of 11% and 4%, respectively. Post-sort purity values are usually above 95% for FAPs and MuSCs.
The ga...

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

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

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

This protocol describes a method for the simultaneous isolation of fibro adipogenic, progenitors, and muscle stem cells using fluorescence activators of sorting. Pure populations of these cells are a prerequisite for understanding their biological role in physiological and pathological conditions. Using this technique, Forbes and muscle stem cells were identified and isolated from either armpit or injured muscle.The high filled impunity of Forbes and muscle stem cells have allowed us to use this cells for different downstream techniques such as saccharine transplantation and mudial mix analysis. To begin, place the euthanized mouse supine on a dissection pad and spray it with 70%ethanol To avoid contamination use forceps to pinch the center of the mouse belly skin and cut approximately one centimeter opening horizontally. Grasp the wound edges and disk in the mouse by pulling the opening in the opposite directions to uncover the muscles underneath, expose one side of the hind limb at the time.Before collecting muscles, Remove intermuscular fat between the hamstrings in the proximal end of the quadriceps to improve the isolation of fibro adipogenic progenitor cells and muscle stem cells. Next, without damaging the tissue use sharp forceps to break and peel off the fascia to expose the underneath tibialis anterior or TA and extensor digitorum longus or EDL muscles. Run the sharp tip of the forceps underneath the TA EDL muscles to detach the muscles from the tibia.Cut off the tendons, attaching the TA EDL to the ankle and while holding it with forceps, trim the muscles with scissors along the longitudinal line of the TA EDL. Cut the tendons around the knee to detach the whole TA EDL muscles. Place the muscles in a six centimeter dish kept on ice.To isolate gastrocnemius muscles cut all tendons at the ankle and detach the muscles from the tibia and fibula. Cut around the knee and transfer the muscles to the dish. Peel off the fascia around the quadriceps and separate it from the femur by running the sharp tip of the forceps between the muscle and bone.Cut the tendons around the knee and trim the rest of the muscle by cutting along the femur while holding the quadriceps with forceps at the distal end. Place the muscles in the six centimeter dish. Detach the hamstrings and remaining muscles around the femur and transfer those to the dish.Collect the hind limb muscles in a dish kept on ice. Aspirate wash medium from the dish and add one to two milliliters of muscle dissociation buffer to keep the muscles moist. Next, mince muscles thoroughly Use a scalpel to tear and slice the muscle and then use scissors to keep cutting the muscle into small pieces for one to two minutes until obtaining a slurry paste of well minced tissue.Then transfer the minced muscles to the conical tube containing muscle dissociation Buffer. Seal the tubes with laboratory film and incubate in a 37 degree Celsius water bath with agitation for one hour. After one hour, remove the tube from the shaker and fill it to 50 milliliters With wash medium, gently invert the tube twice to ensure mixing.Centrifuge the cells in a swinging bucket Rotor at 250 times G for five minutes at four degree Celsius. Transfer 42 milliliters of supernatant into two new tubes and leave eight milliliters in the original tube. Fill the new tubes containing 21 milliliters of the supernatant up to 50 milliliters with wash medium.Then centrifuge the cells at 350 times G for eight minutes at four degrees Celsius. Aspirate the supernatant and keep the pellets on ice. Next, add one milliliter of collagenases two stock and one milliliter of dis based stock in the original tube containing eight milliliters of muscle dissociation buffer.Using a five milliliter serological pipette resuspend the pellet 10 times without clogging. Eject the solution toward the wall of the tube, avoiding the formation bubbles. Seal the tubes with laboratory film and incubate in a 37 degrees Celsius water bath with agitation for 30 minutes.After 30 minutes, remove the tube from the shaker aspirate and eject the muscle suspension 10 times through a 10 milliliter syringe with a 20 gauge needle, fill each tube up to 50 milliliters with wash medium and invert the tube. Then centrifuge at 250 times G for five minutes at four degree Celsius and transfer the supernatant into a new tube. Leaving eight milliliters in the original tube.Fill the new tube up to 50 milliliters with wash medium, again centrifuge the cells at 350 times G for eight minutes at four degree Celsius. Remove the supernatant and keep the pellet on ice. Place a 40 micron nylon cell strainer in the original 50 milliliter conical tube containing eight milliliters of wash medium and pre-wet the strainer with one to two milliliters of wash medium.While holding the strainer, Use a 10 milliliter pipette to resuspend the pellet gently. Filter the suspension through the cell strainer back into the same tube to minimize cell loss. Filter the other tube pellets back into the original tube by adding four to five milliliters of wash medium to each tube to resuspend the pellets and transfer the solution to the self strainer positioned in the original tube.After collecting all the pellets into the original 50 milliliter tube rinse the self strainer with another four to five milliliters of wash medium. Use a 1000 microliter pipette to collect all the liquid from the underside of the self strainer. Fill each tube with wash medium up to 50 milliliters and invert the tube.Centrifuge the tube at 250 times G for five minutes at four degrees Celsius. Remove the supernatant immediately without disturbing the pellet and resuspend the pellet in 600 microliters of wash medium. Transfer the suspension to a two milliliter micro centrifuge tube.Add antibodies CD 31 APC CD 45 APC SCA one Pacific Blue VCAM one biotin and alpha seven Integrin PE into a two millimeter tube containing cell suspension. Gently mix and incubate the cells in a rotating shaker at four degree Celsius for 45 minutes protecting them from light. After incubation, fill all the tubes up to two milliliters with wash medium and centrifuge at 250 times G for five minutes at four degree Celsius.Remove the supernatant and resuspend the pellet in 300 microliters of wash medium. Next, add streptavidin antibody into tubes and incubate the cells in a rotating shaker at four degree Celsius for 20 minutes. After incubation, fill tubes containing streptavidin antibody to two milliliters with wash medium.Then, centrifuge the tubes and aspirate the supernatant completely. After the second wash, resuspend the cells in the experimental tube in 500 microliters of wash medium. Pre-wet a cell strainer cap of a five milliliter polystyrene round bottom tube with 200 microliters of wash medium.Transfer the cell suspension from the experimental tube into the five milliliter polystyrene round bottom tube and allow it to filter by gravity. Print the two milliliter tube containing the experimental sample suspension with 300 microliters of wash medium and pass it through the same strainer cap. Use a 200 microliter pipette to collect all the liquid from the underside of the cell strainer.Seal with a cap, leave tubes on ice and protect them from light. The gating strategy adopted for the isolation of fibro adipogenic progenitor and muscle stem cells is shown here. The PD GFR alpha E G F P knock in reporter mice expresses the H two B E G F P fusion gene from the endogenous PD G FFR alpha locus, allowing efficient and specific labeling of the PD G GFR alpha lineage.The purity of the isolated cell populations was confirmed by immuno staining of co-culture GFP positive fibro adipogenic progenitor and muscle stem cells with PAC-seven antibodies. GFP positive fibro adipogenic progenitor did not express the PAC-seven marker, while muscle stem cells reacted with this antibody. All GFP expressing cells were positive for SCA one and negative for CD 31, CD 45 VCAM one and alpha seven integrin.The fibro adipogenic progenitor and muscle stem cells isolated from mice injured with intramuscular injections of no toxin seven days post-injury are shown. As muscle stem cells are activated an increase in cellular size. It is important to adjust the F SCA and SS CA parameters and expand the gate to include those cells in the center.Quantification of fibro adipogenic progenitor and muscle stem cells in uninjured and seven days post-injury TA muscles are shown here. Although the peak and proliferation was observed between days two and three a low rate of proliferating cells was still appreciable seven days after injury. Performing proper muscle mincing is of critical importance to release a single cells.Furthermore, mononucleotide cell isolation is achieved by running the suspension to a 10 milliliter syringe with a 20 gauged needle. This technique will help scientists to study the biological transcriptional and epigenomic profile of Forbes and muscle stem cells in normal condition and during the deeper stages of muscle regeneration.

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