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>
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		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>91500-09</td>
			<td></td>
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			<td>Student Dumont #5 Forceps</td>
			<td>Fine Science Tools Inc</td>
			<td>91150-20</td>
			<td></td>
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			<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.

facs-isolation-culture-fibro-adipogenic-progenitors-muscle-stem-cells

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.