Name: identification of skeletal muscle satellite cells by immunofluorescence with pax7 and laminin antibodies
Uploaded: 2018-04-19T13:00:00
Description: Watch this Scientific Journal Video about Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies at JoVE.com

We thank the NIAMS Light Imaging Section for providing the microscopes and technical help. The MF20 and Pax7 antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biological Sciences, The University of Iowa, Iowa City. This work was supported by the Intramural Research Program of NIAMS of the National Institutes of Health.

In this protocol, the anterior hind limb muscles of adult mice (2-6 months), tibialis anterior (TA) and extensor digitorum longus (EDL), were employed as an example to perform the immunofluorescence staining on their SCs. All the steps handling mice and muscle tissue dissections have been approved by the Animal Care and Use Committee (ACUC) of NIAMS/NIH.
1. Dissect the TA/EDL Muscle from the Mouse Hind Limb
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	<li>Euthanize the mouse in a CO2 filled euthanasia chamber under the...

<ol>
	<li>Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+and+cell+lineages+that+underlie+the+formation+of+skeletal+muscle.">Gene regulatory networks and cell lineages that underlie the formation of skeletal muscle.</a> Proc Natl Acad Sci U S A. 114 (23), 5830-5837 (2017).</li><li>Buckingham, M., Relaix, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX3+and+PAX7+as+upstream+regulators+of+myogenesis.">PAX3 and PAX7 as upstream regulators of myogenesis.</a> Semin Cell Dev Biol. 44, 115-125 (2015).</li><li>Relaix, F., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+insect+eye+to+vertebrate+muscle:+redeployment+of+a+regulatory+network.">From insect eye to vertebrate muscle: redeployment of a regulatory network.</a> Genes Dev. 13 (24), 3171-3178 (1999).</li><li>Chang, N. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+biology+of+satellite+cells+and+their+therapeutic+potential.">The emerging biology of satellite cells and their therapeutic potential.</a> Trends Mol Med. 14 (2), 82-91 (2008).</li><li>Wang, Y. X., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells,+the+engines+of+muscle+repair.">Satellite cells, the engines of muscle repair.</a> Nat Rev Mol Cell Biol. 13 (2), 127-133 (2011).</li><li>Rinaldi, F., Perlingeiro, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+for+skeletal+muscle+regeneration:+therapeutic+potential+and+roadblocks.">Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks.</a> Transl Res. 163 (4), 409-417 (2014).</li><li>Le Grand, F., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+satellite+cells+and+adult+myogenesis.">Skeletal muscle satellite cells and adult myogenesis.</a> Curr Opin Cell Biol. 19 (6), 628-633 (2007).</li><li>Giordani, L., Puri, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+control+of+skeletal+muscle+regeneration:+Integrating+genetic+determinants+and+environmental+changes.">Epigenetic control of skeletal muscle regeneration: Integrating genetic determinants and environmental changes.</a> FEBS J. 280 (17), 4014-4025 (2013).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nat Protoc. 10 (10), 1612-1624 (2015).</li><li>Feng, X., Adiarte, E. G., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+acts+directly+on+the+zebrafish+dermomyotome+to+promote+myogenic+differentiation.">Hedgehog acts directly on the zebrafish dermomyotome to promote myogenic differentiation.</a> Dev Biol. 300 (2), 736-746 (2006).</li><li>Juan, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+EZH2+controls+self-renewal+and+safeguards+the+transcriptional+identity+of+skeletal+muscle+stem+cells.">Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells.</a> Genes Dev. 25 (8), 789-794 (2011).</li><li>Jackson, K. A., Snyder, D. S., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+fiber-specific+green+autofluorescence:+potential+for+stem+cell+engraftment+artifacts.">Skeletal muscle fiber-specific green autofluorescence: potential for stem cell engraftment artifacts.</a> Stem Cells. 22 (2), 180-187 (2004).</li><li>Lepper, C., Conway, S. J., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+satellite+cells+and+embryonic+muscle+progenitors+have+distinct+genetic+requirements.">Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements.</a> Nature. 460 (7255), 627-631 (2009).</li></ol>

The above protocol was based on a method of Pax7/MF20 staining on zebrafish skeletal muscle12. The solutions used and blocking steps are identical or similar. The antibodies used are identical. The adjusted steps were based on the features of mouse muscle tissue and SCs. First, Laminin antibody was added in the mix to help visualize and confirm the position of SCs. It was particularly helpful to count the number of SCs under the microscope when using the dual filter cube of 488/555; this greatly e...

Skeletal muscle is composed of multinucleated muscle cells, called myotubes, organized in myofibers, which generate force and movements through contraction. Most skeletal muscles, except for some craniofacial muscles, are derived from a temporary embryonic structure called a somite1. Myogenic precursor cells delaminate from the epithelial somite to become myoblasts. Myoblasts further differentiate into myocytes that fuse to become myotubes to form multi-nucleated myofibers. The above process is called myogenesis and is characterized by temporally-regulated control of gene expression. Myogenic precursors express Pax3 and Pax7, whereas myoblasts express MyoD and/or Myf5 and myocytes express myogenin and myosins2,3. Muscle growth is a process in which myofibers become larger by incorporating more myonuclei into existing fibers (hyperplasia) and by an increase in muscle fiber size (hypertrophy)4. During muscle growth, there is a sustainable source of myogenic cells that have stem cell properties in that they can differentiate and self-renew. These cells are termed satellite cells based on their physical location between the sarcolemma (cell membrane of the myofiber) and the basal lamina5. SCs vigorously contribute to muscle growth in the juvenile stage (the first 2-3 weeks of postnatal mice), but become quiescent in resting adult muscle6. Remarkably, they can be re-activated in response to muscle injures and differentiate into new muscle cells to repair the damaged muscle7.
The stem cell properties make the study of SCs relevant for both basic muscle biology and therapies of muscle diseases8. As a result, it has been an area of intense investigation in the past decades. A tremendous progress has been made in dissecting the genetics and epigenetics of SCs9,10. Techniques involved in isolating and identifying SCs in situ were developed and optimized along the way11. Immunofluorescent staining allows the identification of SCs through the use of specific antibodies, including that for Pax7. However, the scarcity and small size of the SCs combined with a strong auto-fluorescence of adult skeletal muscle tissue render the visualization challenging. Here, we describe an immunofluorescent staining protocol optimized for mouse muscle tissue for Pax7 and based on an existing method for zebrafish muscle12. In addition, a Laminin antibody labeled with a distinct fluorophore is employed to identify the basal lamina under which the SCs are located. This protocol consistently allows the visualization of Pax7-positive SCs and myogenic precursors under all tested physiological conditions and developmental stages.

<table>
	<tbody>
		<tr>
			<td>methylbutane</td>
			<td>Sigma-Aldrich</td>
			<td>M32631</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature (O.C.T.) compound</td>
			<td>Electron Microscopy Sciences</td>
			<td>62550-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Gibco, Themo Fisher</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>16% PFA</td>
			<td>TED PELLA</td>
			<td>50-00-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Thermo Fisher</td>
			<td>0 1-6201</td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td>115-007-003</td>
			<td></td>
		</tr>
		<tr>
			<td>20x Citrate Buffer</td>
			<td>Thermo Fisher</td>
			<td>00 500</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>Laminin polyclonal rabbit antibody</td>
			<td>Sigma-Aldrich</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr>
			<td>MF20 mono-clonal mouse antibody (IgG2b) (supernatant)</td>
			<td>Developmental Study Hybridoma Bank</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A-21121</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG2b cross-absorbed secondary antibody, Alexa Fluor 647</td>
			<td>Thermo Fisher</td>
			<td>A-21242</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555</td>
			<td>Thermo Fisher</td>
			<td>A32732</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1860 cryostat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DM6000 wide-field fluorescent microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMR wide-field fluorescent microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM510 confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM780 confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cuisinart electronic pressure cooker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Following the above steps, SCs can be successfully visualized in adult resting muscle sections under a fluorescent microscope (Figure 1). Although the adult muscle tissue has strong auto-fluorescence in certain type of fibers, the bright Alexa series dyes can overcome the background noise and the signal stands out (Figure 1A, B; arrow heads). The two-photon confocal microscopy captures a relatively cleaner image ...

Watch this Scientific Journal Video about Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies at JoVE.com

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

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, ...

The overall goal of this protocol is to identify adult skeletal muscle satellite cells via immunofluorescence. This method can answer key questions related to the number and distribution of muscle stem cells in both physiological as well as pathological conditions. The main advantage of the technique is that one can identify muscle stem cells in their physiological environment with only limited manipulation.Demonstrating the procedure will be Faiza Naz, a postdoc from my laboratory. To begin, prepare a liquid nitrogen bath by pouring liquid nitrogen into a Dewar flask. Next, pour 10 milliliters of methylbutane into a 20-milliliter plastic beaker and transfer the beaker to the liquid nitrogen bath.Lay the dissected tibialis anterior muscle onto the flat surface of a small plastic syringe plunger. Next, cover the dissected muscle with a few drops of Optimal Cutting Temperature compound or O.C.T.Remove the beaker of methylbutane from the liquid nitrogen bath, then thaw the surface using the handles of a pair of scissors. Quickly dip the O.C.T.covered tissue into the melted methylbutane to snapfreeze it.Next, use forceps to detach the embedded tissue from the plunger and transfer it to a 1.5-milliliter microcentrifuge tube. Then, store the microcentrifuge tube in the liquid nitrogen. To start cryostat sectioning, add a drop of O.C.T.compound onto the center of the specimen holder of a cryostat and allow it to partially solidify.Then, stick the embedded tissue into the O.C.T.compound with the ankle side down and knee side up. Next, mount the specimen holder onto the cryostat and cut the tissue in 10-micrometer intervals. Collect four to eight cryosections on each frost-coated slide.Dry the sections at room temperature for three hours, then collect the slides in a slide box and store them at minus 80 degrees Celsius. Next, remove a slide from the slide box and warm it to room temperature. Then, use a liquid blocker pen to mark an area around the cryosections to prevent liquids from flowing off the side.In a fume hood, add 300 to 500 microliters of 4%paraformaldehyde to the marked area. Incubate the slide at room temperature for 10 minutes to fix the sections. Next, rinse the paraformaldehyde from the slide with PBS in a slide container.To begin antigen retrieval, fill a slide container with 200 milliliters of citrate buffer and transfer the slide to the container. Then, transfer the slide container to a pressure cooker at high pressure for 10 minutes. The antigen retrieval step is optional if the sections are dried at room temperature for 30 minutes and proceed immediately.Then the antigen retrieval step is not necessary. But if the sections are dried at room temperature for more than three hours then stored at minus 80 degrees Celsius, the antigen retrieval step is required. Next, cool the slide container under running water for 10 minutes.Then transfer the slide to a new container with 200 milliliters of PBST and rinse with PBST. Add water-soaked lab wipes to the tray, then remark the rinsed slide with a PAP pen and place it in the tray. Add 200 microliters of blocking solution to the slide.Then cover the tray and incubate at room temperature for 30 minutes. The blocking step is very important because the Pax7 antibody being used is a mouse mono-clonal antibody. The mouse-on-mouse blocking step reduces the unspecific bindings.Then, rinse the slide with PBST in the slide container and return it to the tray. Add 200 microliters of Primary Antibody mix to the slide and incubate at room temperature for one hour. Next, rinse the slide with PBST, then add 200 microliters of Secondary Antibody mix and incubate in the dark at room temperature for one hour.After the incubation period, rinse the slide with PBST. Dilute DAPI in a slide container and incubate the slide in the container for three minutes. Then, rinse the DAPI with PBST for five minutes.Finally, mount the slide with mounting media and apply a coverslip. In this study, satellite cells are successfully visualized in adult resting muscle sections under a fluorescent microscope. The background noise usually seen with adult muscle tissue and certain types of fibers is reduced by using mouse-on-mouse blocking.The specific immunostain signals are enhanced by using secondary antibodies conjugated with bright Alexa dyes. A two-photon confocal microscope captures a sharper image compared to the wide-field fluorescent microscope. The same protocol can also be applied to muscle tissue from juvenile mice, mouse embryos, and injured adult muscle tissues.Both juvenile and embryonic muscle tissues depict more activated satellite cells or Pax7 myogenic precursors with relatively larger nuclei. Thus it becomes easier to visualize the satellite cells of younger mice than of adult mice. In the injured muscle tissue, more activated Pax7-positive cells are visible.Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it is important to remember that noise coming from autofluorescence and unspecific mouse-on-mouse immuno-binding cannot be totally eliminated, but with this protocol the noise can be largely reduced. After watching this video, you should have a good understanding of how to identify satellite cells in muscle cryosections.

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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 ...

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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 ...

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 ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

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 ...