Name: Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components (Video) | JoVE
Uploaded: 2023-06-02T15:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components at JoVE.com

For Figure 2, templates from Servier Medical Art (https://smart.servier.com/) were used. The FR lab is supported by the Association Fran&#231;aise contre les Myopathies - AFM&#160;via TRANSLAMUSCLE (grants 19507 and 22946), the Fondation pour la Recherche M&#233;dicale - FRM (EQU202003010217, ENV202004011730, ECO201806006793), the Agence Nationale pour la Recherche - ANR (ANR-21-CE13-0006-02, ANR-19-CE13-0010, ANR-10-LABX-73), and the La Ligue Contre le Cancer (IP/SC-17130). The above funders had no role in the design, collection, analysis, interpretation, or reporting of this study or the writing of this manuscript.

All experiments complied with French and EU animal regulations at the Institut Mondor de Recherche Biom&#233;dicale (INSERM U955), notably the directive 2010/63/UE. Animals were kept in a controlled and enriched environment at the animal facilities with certification numbers A94 028 379 and D94-028-028; they were handled only by authorized researchers and animal caretakers, and they were visually inspected by animal housing personnel for signs of discomfort during their lifetime. They were euthanized by&#160;cervical dis...

Harvesting Mouse Skeletal Muscle to Culture Stem Cells and Their Niche

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A., 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=An+absolute+requirement+for+Pax7-positive+satellite+cells+in+acute+injury-induced+skeletal+muscle+regeneration.">An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.</a> Development. 138 (17), 3639-3646 (2011).</li><li>McCarthy, J. J., 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=Effective+fiber+hypertrophy+in+satellite+cell-depleted+skeletal+muscle.">Effective fiber hypertrophy in satellite cell-depleted skeletal muscle.</a> Development. 138 (17), 3657-3666 (2011).</li><li>Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A., Kardon, G. <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,+connective+tissue+fibroblasts+and+their+interactions+are+crucial+for+muscle+regeneration.">Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration.</a> Development. 138 (17), 3625-3637 (2011).</li><li>Sambasivan, R., 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=Pax7-expressing+satellite+cells+are+indispensable+for+adult+skeletal+muscle+regeneration.">Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration.</a> Development. 138 (17), 3647-3656 (2011).</li><li>Hicks, M. R., Pyle, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+the+stem+cell+niche.">The emergence of the stem cell niche.</a> Trends in Cell Biology. 33 (22), 112-123 (2022).</li><li>Relaix, F., 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=Perspectives+on+skeletal+muscle+stem+cells.">Perspectives on skeletal muscle stem cells.</a> Nature Communications. 12 (1), 692 (2021).</li><li>Gama, J. F. G., 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=Role+of+regulatory+T+cells+in+skeletal+muscle+regeneration:+A+systematic+review.">Role of regulatory T cells in skeletal muscle regeneration: A systematic review.</a> Biomolecules. 12 (6), 817 (2022).</li><li>Loreti, M., Sacco, 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+jam+session+between+muscle+stem+cells+and+the+extracellular+matrix+in+the+tissue+microenvironment.">The jam session between muscle stem cells and the extracellular matrix in the tissue microenvironment.</a> NPJ Regenerative Medicine. 7 (1), 16 (2022).</li><li>Sambasivan, R., 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=Distinct+regulatory+cascades+govern+extraocular+and+pharyngeal+arch+muscle+progenitor+cell+fates.">Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates.</a> Developmental Cell. 16 (6), 810-821 (2009).</li><li>Pereira, P. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+functions+of+platelet/endothelial+cell+adhesion+molecule-1+(CD31).">Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31).</a> Current Opinion in Hematology. 23 (3), 253-259 (2016).</li><li>Scholzen, T., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ki-67+protein:+From+the+known+and+the+unknown.">The Ki-67 protein: From the known and the unknown.</a> Journal of Cellular Physiology. 182 (3), 311-322 (2000).</li><li>Abou-Khalil, R., Le Grand, F., Chazaud, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+skeletal+muscle+reserve+cells.">Human and murine skeletal muscle reserve cells.</a> Stem Cell Niche. 1035, 165-177 (2013).</li><li>Pasut, A., Oleynik, P., 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=Isolation+of+muscle+stem+cells+by+fluorescence+activated+cell+sorting+cytometry.">Isolation of muscle stem cells by fluorescence activated cell sorting cytometry.</a> Methods in Molecular Biology. 798, 53-64 (2011).</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> Nature Protocols. 10 (10), 1612-1624 (2015).</li><li>Montarras, D., 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=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309 (5743), 2064-2067 (2005).</li><li>Qu, Y., Edwards, K., Barrow, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+use+of+primary+murine+myoblasts+in+small-molecule+screens.">Isolation, culture, and use of primary murine myoblasts in small-molecule screens.</a> STAR Protocols. 4 (2), 102149 (2023).</li><li>Danoviz, M. E., Yablonka-Reuveni, Z. <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:+Background+and+methods+for+isolation+and+analysis+in+a+primary+culture+system.">Skeletal muscle satellite cells: Background and methods for isolation and analysis in a primary culture system.</a> Methods in Molecular Biology. 798, 21-52 (2011).</li><li>Saclier, M., Theret, M., Mounier, R., Chazaud, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+macrophage+conditioned-medium+on+murine+and+human+muscle+cells:+analysis+of+proliferation,+differentiation,+and+fusion.">Effects of macrophage conditioned-medium on murine and human muscle cells: analysis of proliferation, differentiation, and fusion.</a> Methods in Molecular Biology. 1556, 317-327 (2017).</li><li>Giordani, L., 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=High-dimensional+single-cell+cartography+reveals+novel+skeletal+muscle-resident+cell+populations.">High-dimensional single-cell cartography reveals novel skeletal muscle-resident cell populations.</a> Molecular Cell. 74 (3), 609-621 (2019).</li><li>Tabula Muris Consortium 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=Single-cell+transcriptomics+of+20+mouse+organs+creates+a+Tabula+Muris.">Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.</a> Nature. 562 (7727), 367-372 (2018).</li><li>Brunetti, J., Koenig, S., Monnier, A., Frieden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopattern+surface+improves+cultured+human+myotube+maturation.">Nanopattern surface improves cultured human myotube maturation.</a> Skeletal Muscle. 11 (1), 12 (2021).</li><li>Denes, L. 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Adult skeletal muscle function is underpinned by a finely orchestrated set of cellular interactions and molecular signals. Here, a method is presented that allows for the study of these parameters in an ex vivo setting that closely resembles the physiological microenvironment.
Several groups have reported in vitro methods to culture myogenic cells. These methods aimed to isolate satellite cells to study their myogenic progenitor properties. Two main approaches are used to iso...

Movement, breathing, metabolism, body posture, and body temperature maintenance all depend on skeletal muscle, and malfunctions in the skeletal muscle can, thus, cause debilitating pathologies (i.e., myopathies, muscular dystrophies, etc.)1. Given its essential functions and abundance, skeletal muscle has drawn the attention of research labs worldwide that strive to understand the key aspects that support normal muscle function and can serve as therapeutic targets. In addition, skeletal muscle is a widely used model to study regeneration and stem cell function, as healthy muscle can fully self-repair after complete injury and degeneration, mostly due to its resident stem cells2; these are also called satellite cells and are localized under the basal lamina in the periphery of the muscle fibers3.
The core cells of adult skeletal muscle are the myofibers (long syncytial multinuclear cells) and the satellite cells (stem cells with myogenic potential that are quiescent until an injury activates them). The latter cells are the central cells of muscle regeneration, and this process cannot occur in their absence4,5,6,7. In their immediate microenvironment, there are multiple cell types and molecular factors that signal to them. This niche is gradually established throughout development and until adulthood8. Adult muscle contains multiple cell types (endothelial cells, pericytes, macrophages, fibro-adipogenic progenitors-FAPs, regulatory T cells, etc.)9,10 and extracellular matrix components (laminins, collagens, fibronectin, fibrillins, periostin, etc.)11 that interact with each other and with the satellite cells in the context of health, disease, and regeneration.
Preserving this complex niche in experimental settings is fundamental but challenging. Equally difficult is to maintain or return to quiescence, a cell state that is critical for satellite cells9. Several methods have been introduced to partially tackle these challenges, each with its advantages and disadvantages (detailed in the discussion section). Here, a method is presented that can partially overcome these two barriers. Muscles are initially harvested and then broken down mechanically and enzymatically before the heterogenous cell mixture is put into culture. Over the course of the culture, many cell types of the niche are detected, and satellite cells that have returned to quiescence are observed. As a last step of the protocol, the immunofluorescence steps that allow for the detection of each cell type through the use of universally accepted markers, are presented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-CD31</td>
			<td>BD</td>
			<td>550274</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>anti-FOSB</td>
			<td>Santa Cruz</td>
			<td>sc-7203</td>
			<td>dilution 1:200</td>
		</tr>
		<tr>
			<td>anti-GFP</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>anti-Ki67</td>
			<td>Abcam</td>
			<td>ab16667</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>anti-MyHC</td>
			<td>DSHB</td>
			<td>MF20-c</td>
			<td>dilution 1:400</td>
		</tr>
		<tr>
			<td>anti-MYOD</td>
			<td>Active Motif</td>
			<td>39991</td>
			<td>dilution 1:200</td>
		</tr>
		<tr>
			<td>anti-MYOG</td>
			<td>Santa Cruz</td>
			<td>sc-576</td>
			<td>dilution 1:150</td>
		</tr>
		<tr>
			<td>anti-Pax7</td>
			<td>Santa Cruz</td>
			<td>sc-81648</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>anti-PDGFR&#945;</td>
			<td>Invitrogen</td>
			<td>PA5-16571</td>
			<td>dilution 1:50</td>
		</tr>
		<tr>
			<td>b-FGF</td>
			<td>Peprotech</td>
			<td>450-33</td>
			<td>concentration 4 ng/mL</td>
		</tr>
		<tr>
			<td>bovine serum albumin (BSA) &#8211; used for digestion&#160;</td>
			<td>Sigma Aldrich</td>
			<td>A7906-1006</td>
			<td>concentration 0.2%</td>
		</tr>
		<tr>
			<td>BSA IgG-free, protease-free &#8211; used for staining</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td>concentration 5%</td>
		</tr>
		<tr>
			<td>cell strainer 40 um</td>
			<td>Dominique Dutscher</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>cell strainer 70 um</td>
			<td>Dominique Dutscher</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>cell strainer 100 um</td>
			<td>Dominique Dutscher</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Roche</td>
			<td>10103586001</td>
			<td>concentration 0.5 U/mL</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Euromedex</td>
			<td>UD8050-05-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td>concentration 3 U/mL</td>
		</tr>
		<tr>
			<td>Dissection forceps size 5</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection forceps size 55</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors (big, straight)</td>
			<td>Fine Science Tools</td>
			<td>9146-11</td>
			<td>ideal for chopping</td>
		</tr>
		<tr>
			<td>Dissection scissors (small, curved)</td>
			<td>Fine Science Tools</td>
			<td>15017-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors (small, straight)</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>ThermoFisher</td>
			<td>41966-029</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU Click-iT kit</td>
			<td>ThermoFisher</td>
			<td>C10340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum &#8211; option 1</td>
			<td>Eurobio</td>
			<td>CVF00-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum &#8211; option 2</td>
			<td>Gibco</td>
			<td>10270-106&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning Life Sciences</td>
			<td>354234</td>
			<td>coating solution</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Dominique Dutscher</td>
			<td>090261</td>
			<td>flexible film</td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde &#8211; option 1</td>
			<td>PanReac AppliChem ITW Reagents</td>
			<td>211511.1209</td>
			<td>concentration 4%</td>
		</tr>
		<tr>
			<td>Paraformaldeyde &#8211; option 2</td>
			<td>ThermoFisher</td>
			<td>28908</td>
			<td>concentration 4%</td>
		</tr>
		<tr>
			<td>Shaking water bath</td>
			<td>ThermoFisher</td>
			<td>TSSWB27</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX100</td>
			<td>Sigma Aldrich</td>
			<td>T8532-500 ML</td>
			<td>concentration 0.5%</td>
		</tr>
		<tr>
			<td>Wild-type mice</td>
			<td>Janvier</td>
			<td>C57BL/6NRj</td>
			<td></td>
		</tr>
	</tbody>
</table>

unfractionated bulk culture of mouse skeletal muscle to recapitulate niche and stem cell quiescence

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

This protocol allows for muscle cell culture while preserving the satellite cells and most cells from their endogenous niche. Figure 2 summarizes the main steps of the protocol, while essential parts of the dissection and digestion are presented in Figure 1. Dissection of the hindlimb musculature is recommended (Figure 1A-C), as this group of muscles is well studied and shares a developmental origin...

Watch this Scientific Journal Video about Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components at JoVE.com

Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components (Video) | JoVE

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...