Cell sorting was performed at the FACS Core Facility, Aarhus University, Denmark. Figures were created using Biorender.com. We thank Dr. J. Farup for sharing the rabbit anti-PDGFRa antibody. This work was supported by an AUFF Starting Grant to E.P. and Start Package grants from&#160;NovoNordiskFonden to E.P. (0071113) and to A.D.M. (0071116).

<ol>
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The authors have no competing financial interests and no conflicts of interest.

Several steps are key in the execution of this protocol to achieve good yields. The individual muscles have a small volume compared to the amount of muscle used in bulk isolation protocols. This results in a risk of the muscle drying out during the dissection, which reduces yield. To prevent this, it is important to add medium to the muscles immediately after dissection. In addition, if dissection is taking longer, the skin can be removed from one limb at a time to reduce the time of exposure of the muscles to air. The smaller volume also results in an increased risk of over-digestion. To counter this, the present method requires lower amounts of collagenase II enzyme and reduced digestion times compared to bulk muscle protocols9,16. Enzymatic digestion also depends on the purity of the enzymes, and lower purity can negatively impact yield. In addition, mechanical digestion is critical. In case of insufficient cutting, the decreased surface area will hamper enzymatic digestion and lower the stem cell yield. In case of cutting too much, the increased surface area will cause over-digestion and lower the stem cell yield. The shaking water bath prevents precipitation of the digested muscle, improves the distribution of the enzyme, and aids in creating a homogeneous temperature, altogether enabling shorter incubation times. Therefore, the present method allows a significant reduction of the incubation time compared to other methods.
This protocol depends on cell dissociation and purification. These procedures mimic tissue injury, which activates the stem cells. Consistently, recent studies have revealed that MuSCs change their gene expression programs during the isolation procedure27,28,29,30. As a result, the purified stem cells are different from the cells in vivo in terms of gene expression patterns. A second limiting factor in the protocol is its dependence on FACS, which requires access to expensive equipment. FACS is the gold standard for isolating multiple cell populations simultaneously with high purity20. Recent advances using magnetic beads and microbubbles offer reductions in cost31,32, but whether they offer comparable yields to work on single muscles needs to be determined. Finally, the yield of the protocol is limited due to the small size of the muscles, thus posing restrictions on potential downstream assays.
Previous studies have relied on pooling different muscles when isolating MuSCs and/or FAPs to maximize cell yield. However, this averages out any tissue-specific differences in stem cell behavior and function between different muscles. The current protocol enables the isolation of MuSCs and FAPs from individual muscles for the downstream analyses of stem cell function. As an example of a downstream assay, stem cell activation was assayed by EdU incorporation, revealing that stem cells from different tissues display different activation kinetics. In previous works, the feasibility of using other downstream assays has been shown; these assays require smaller cell numbers, such as SmartSeq2 single-cell RNA sequencing, cell transplantation, microfluidic PCR, and clonal expansion assays22,33,34,35.
In conclusion, this protocol describes a method for the dissection of individual muscles to isolate and study MuSCs and FAPs. This strategy will enable experiments to gain a better understanding of stem cell function across different muscles in health and disease.

Skeletal muscle has a high capacity for regeneration due to the presence of muscle stem cells (MuSCs). MuSCs are located on the myofibers, underneath the basal lamina, and reside in a quiescent state of prolonged, reversible cell cycle exit1,2,3,4. Upon injury, MuSCs activate and enter the cell cycle to give rise to amplifying progenitors that can differentiate and fuse to form new myofibers2,5. Previous work has showed that MuSCs are absolutely essential for muscle regeneration6,7,8. Moreover, a single MuSC can engraft and generate both new stem cells and new myofibers9. Skeletal muscle also harbors a population of mesenchymal stromal cells called fibro-adipogenic progenitors (FAPs), which play a crucial role in supporting MuSC function during muscle regeneration6,10,11,12.
Due to their potential to coordinate muscle regeneration, there has been tremendous interest in understanding how MuSCs and FAPs work. Quiescent MuSCs are marked by expression of the transcription factors Pax7 and Sprouty1, and the cell surface protein calcitonin receptor, whereas quiescent FAPs are marked by the cell surface protein platelet-derived growth factor receptor alpha (PDGFRa)10,12,13,14,15. Previous studies have showed that MuSCs and FAPs could be purified from skeletal muscles using cell surface markers and fluorescence-activated cell sorting (FACS)9,15,16,17,18,19,20,21. While these protocols have greatly advanced the ability to study MuSCs and FAPs, one drawback is that most of these protocols require the isolation of MuSCs from a pool of different muscle tissues. Recent work from us and others have revealed differences in cell phenotype and gene expression levels between MuSCs isolated from different tissues22,23. MuSCs from the diaphragm, triceps, and gracilis show faster activation than MuSCs from lower hindlimb muscles22, while MuSCs from extraocular muscle show faster differentiation than MuSCs from the diaphragm and lower hindlimb muscles23.
This protocol describes the isolation of MuSCs and FAPs from individual skeletal muscles (Figure 1). This includes the dissection of the diaphragm, triceps, gracilis, tibialis anterior (TA), soleus, extensor digitorum longus (EDL), gastrocnemius (GA), and masseter muscles. Dissected muscles are subsequently dissociated by enzymatic digestion using collagenase II (a protease that specifically targets the Pro-X-Gly-Pro amino sequence in collagen, enabling the degradation of connective tissue and tissue dissociation24) and dispase (a protease that cleaves fibronectin and collagen IV, enabling further cell dissociation25). MuSCs and FAPs are isolated from single-cell suspensions by FACS. As examples of downstream assays for cell analysis, stem cell activation is determined by assaying 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation, while cell purity is determined by immunofluorescence staining for the cell-type specific markers Pax7 and PDGFRa.

<table><tbody><tr><td>1.5 mL tube( PCR performance tested, PP, 30,000 xg, DNA/DNase-/RNase-free, Low DNA binding, Sterile )</td><td>Sarstedt AG &amp;amp; Co. KG, Hounisen Laboratorieudstyr A/S</td><td>72.706.700</td><td>1.5 mL tube</td></tr><tr><td>15 mL tube (PP/HD-PE, 20,000 xg, IVD/CE, IATA, DNA/DNase-/RNase-free, Non-cytotoxic, pyrogen free, Sterile)</td><td>Sarstedt AG &amp;amp; Co. KG, Hounisen Laboratorieudstyr A/S</td><td>62.554.502</td><td>15 mL tube</td></tr><tr><td>5 mL polystyrene round-bottom tube</td><td>Falcon, Fisher Scientific&amp;nbsp;</td><td>352054</td><td>FACS tube without strainer cap</td></tr><tr><td>5 mL polystyrene Round-bottom tube with cell-strainer cap</td><td>Falcon, Fisher Scientific&amp;nbsp;&amp;nbsp;</td><td>352235</td><td>FACS tube with strainer cap</td></tr><tr><td>5 mL tube (PP, non sterile autoclavable)</td><td>VWR collection</td><td>525.0946</td><td>5 mL tube</td></tr><tr><td>50 mL tube( PP/HD-PE, 20,000 xg, IVD/CE, ADR, DNA/DNase-/RNase-free, non-cytotoxic, pyrogen free, Sterile)</td><td>Sarstedt AG &amp;amp; Co. KG, Hounisen Laboratorieudstyr A/S</td><td>62.547.254</td><td>50 mL tube</td></tr><tr><td>Alexa Fluor 555 Donkey anti-rabbit IgG (H+L)</td><td>Invitrogen, Thermo Fisher</td><td>Lot: 2387458 (Cat # A31572)</td><td></td></tr><tr><td>Alexa Fluor 647 donkey-anti mouse IgG (H+L)</td><td>Invitrogen, Thermo Fisher</td><td>Lot: 2420713 (Cat#A31571)</td><td></td></tr><tr><td>ARIA 3</td><td>BD</td><td></td><td>FACS, Core facility Aarhus University</td></tr><tr><td>Centrifuge 5810</td><td>eppendorf</td><td>EP022628188</td><td>Centrifuge</td></tr><tr><td>Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye</td><td>Invitrogen, Thermo Fisher</td><td>Lot: 2387287 (Cat# C10337)</td><td>Cell Proliferation Kit</td></tr><tr><td>Collagen from calf-skin&amp;nbsp;</td><td>Bioreagent, Sigma Aldrich&amp;nbsp;</td><td>Source: SLCK6209 (Cat# C8919)</td><td></td></tr><tr><td>Collagenase type II</td><td>Worthington, Fisher Scientific&amp;nbsp;</td><td>Lot: 40H20248 (cat# L5004177 )</td><td>Collagenase</td></tr><tr><td>Dispase</td><td>Gibco, Fisher Scientific&amp;nbsp;</td><td>Lot: 2309415 (cat# 17105-041 )</td><td>Dispase</td></tr><tr><td>Donkey serum (non-sterile)</td><td>Sigma Aldrich, Merck</td><td>Lot: 2826455 (Cat# S30-100mL)</td><td></td></tr><tr><td>Dumont nr. 5, 110 mm</td><td>Dumont, Hounisen Laboratorieudstyr A/S</td><td>1606.327</td><td>Straight forceps with fine tips</td></tr><tr><td>Dumont nr. 7, 115 mm</td><td>Dumont, Hounisen Laboratorieudstyr A/S</td><td>1606.335</td><td>Curved forceps</td></tr><tr><td>F-10 Nutrient mixture (Ham) (1x), +L-glutamine</td><td>Gibco, Fisher Scientific&amp;nbsp;</td><td>Lot. 2453614 (cat# 31550-023)</td><td></td></tr><tr><td>FITC anti-mouse CD31</td><td>BioLegend, NordicBioSite</td><td>MEC13.3 (Cat # 102506)</td><td></td></tr><tr><td>FITC Anti-mouse CD45</td><td>BioLegend, NordicBioSite</td><td>30-F11 (Cat# 103108)</td><td></td></tr><tr><td>Glacial acetic acid (100%)</td><td>EMSURE, Merck&amp;nbsp;&amp;nbsp;</td><td>K44104563 9Cat # 1000631000)</td><td></td></tr><tr><td>Head over head mini-tube rotator&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>15534080 (Model no. 88861052)</td><td>Head over head mini-tube rotator</td></tr><tr><td>Horse serum</td><td>Gibco, Fisher Scientific&amp;nbsp;</td><td>Lot. 2482639 (cat# 10368902 )</td><td></td></tr><tr><td>Isotemp SWB 15</td><td>FisherBrand, Fisher Scientific</td><td>15325887</td><td>Shaking water bath</td></tr><tr><td>MS2 mini-shaker&amp;nbsp;</td><td>IKA&amp;nbsp;</td><td></td><td>Vortex unit</td></tr><tr><td>Needle 20 G (0.9 mm x 25 mm)</td><td>BD microlance, Fisher Scientific&amp;nbsp;</td><td>304827</td><td>20G needle&amp;nbsp;</td></tr><tr><td>Neutral formalin buffer 10%</td><td>CellPath, Hounisen Laboratorieudstyr A/S</td><td>Lot: 03822014 (Cat # HOU/1000.1002)</td><td></td></tr><tr><td>Non-pyrogenic cell strainer (40 &amp;micro;M)</td><td>Sarstedt AG &amp;amp; Co. KG, Hounisen Laboratorieudstyr A/S</td><td>83.3945.040</td><td>Cell strainer&amp;nbsp;</td></tr><tr><td>Pacific Blue anti-mouse Ly-6A/E (Sca-1)</td><td>BioLegend, NordicBioSite</td><td>D7 (Cat# 108120)</td><td></td></tr><tr><td>Pax7 primary antibody</td><td>DSHB</td><td>Lot: 2/3/22-282ug/mL (Cat# AB 528428)</td><td></td></tr><tr><td>PBS 10x powder concentrate</td><td>Fisher BioReagents, Fisher Scientific</td><td>BP665-1</td><td></td></tr><tr><td>PE/Cy7 anti-mouse CD106 (VCAM1)</td><td>BioLegend, NordicBioSite</td><td>429 (MVCAM.A) (Cat # 105720)</td><td></td></tr><tr><td>Pen/strep</td><td>Gibco, Fisher Scientific&amp;nbsp;</td><td>Lot. 163589 (cat# 11548876 )</td><td></td></tr><tr><td>Pipette tips p10</td><td>Art tips, self sealing barrier, Thermo Scientific</td><td>2140-05</td><td>Low retention, pre-sterilized, filter tips</td></tr><tr><td>Pipette tips p1000</td><td>Art tips, self sealing barrier, Thermo Scientific</td><td>2279-05</td><td>Low retention, pre-sterilized, filter tips</td></tr><tr><td>Pipette tips p20</td><td>Art tips, self sealing barrier, Thermo Scientific</td><td>2149P-05</td><td>Low retention, pre-sterilized, filter tips</td></tr><tr><td>Pipette tips p200</td><td>Art tips, self sealing barrier, Thermo Scientific</td><td>2069-05</td><td>Low retention, pre-sterilized, filter tips</td></tr><tr><td>Protective underpad</td><td>Abena&amp;nbsp;</td><td>ACTC-7712</td><td>&amp;nbsp;60 x 40cm, 8 layers</td></tr><tr><td>Rainin, pipet-lite XLS</td><td>Mettler Toledo, Thermo Scientific&amp;nbsp;</td><td>2140-05, 2149P-05, 2279-05, 2069-05</td><td>Pipettes (P10, P20, P200, P1000)</td></tr><tr><td>Recombinant anti-PDGFR-alpha</td><td>RabMAb, abcam</td><td>AB134123</td><td></td></tr><tr><td>Scalpel (shaft no. 3)</td><td>Hounisen, Hounisen Laboratorieudstyr A/S</td><td>1902.502</td><td>Scalpel</td></tr><tr><td>Scalpel blade no. 11</td><td>Heinz Herenz, Hounisen Laboratorieudstyr A/S</td><td>1902.0911</td><td>Scalpel</td></tr><tr><td>Scanlaf mars</td><td>Labogene</td><td>class 2 cabinet: Mars</td><td>Flow bench</td></tr><tr><td>ScanR</td><td>Olympus</td><td></td><td>Microscope, Core facility Aarhus University</td></tr><tr><td>Scissors</td><td>FST</td><td>14568-09</td><td></td></tr><tr><td>Series 8000 DH</td><td>Thermo Scientific</td><td>3540-MAR</td><td>Incubator</td></tr><tr><td>Serological pipette 10 mL</td><td>VWR</td><td>612-3700</td><td>Sterile, non-pyrogenic</td></tr><tr><td>Serological pipette 5 mL</td><td>VWR, Avantor delivered by VWR</td><td>612-3702</td><td>Sterile, non-pyrogenic</td></tr><tr><td>Syringe 5 mL, Luer tip (6%), sterile&amp;nbsp;</td><td>BD Emerald, Fisher Scientific</td><td>307731</td><td>Syringe</td></tr><tr><td>TC Dish 100, standard</td><td>Sarstedt AG &amp;amp; Co. KG, Hounisen Laboratorieudstyr A/S</td><td>83.3902</td><td>Petri dish&amp;nbsp;</td></tr><tr><td>Tissue Culture (TC)-treated surface, black polystyrene, flat bottom, sterile, lid, pack of 20</td><td>Corning, Sigma Aldrich</td><td>3764</td><td>96-well Half bottom plate</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich, Merck</td><td>Source: SLCJ6163 (Cat # T8787)</td><td></td></tr></tbody></table>

isolation of quiescent stem cell populations from individual skeletal muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation assay.

Following the protocol for individual skeletal muscle isolation (Figure 2), the gracilis, TA, EDL, GA, soleus, triceps, masseter, and diaphragm muscles were isolated from three Swiss male outbred mice that had been discontinued from a local breeding program (Figure 2). Following tissue dissociation and antibody staining, MuSCs and FAPs from the individual muscles were purified by FACS (Figure 3). The initial gating was obtained with an unstained sample to identify the cells and separate the singlets from doublets (Figure 3A). The subsequent gates were set using FMO controls to identify staining thresholds (Figure 3B). The stained sample was then gated for CD31/CD45-FITC and Sca1-PacBlue. The SCA1+/CD31-/CD45- population (FAPs) was sorted into a separate collection tube, while the double negative population was gated and plotted for VCAM1-PECy7 and forward scatter (FSC). The VCAM1+ population (MuSCs) was sorted into a separate collection tube. MuSCs and FAPs were quantified as the percentage of singlets (Table 3) and sorted into separate collection tubes containing 500 &#181;L of the wash medium. The diaphragm and triceps muscle single-cell suspensions have a higher relative abundance of MuSCs than FAPs, whereas the other single-cell suspensions have a higher relative abundance of FAPs than MuSCs (Table 3).
Of the sorted cells, 1,000-3,000 cells were seeded and incubated for 24 h. After 24 h of incubation, the medium was removed and replaced with a fresh medium containing EdU. The cells were fixed at 48 h, stained for EdU, and subsequently with antibodies against Pax7 protein or PDGFRa protein, and imaged using an inverted epifluorescence microscope. Images were quantified using the Fiji plugin in ImageJ. Robust EdU staining was observed, though the fraction of EdU-positive cells was different for the two stem cell types and the different muscles (Figure 4A-C). MuSCs isolated from either the EDL or GA showed significantly lower EdU incorporation compared to MuSCs isolated from either the TA, diaphragm, gracilis, or triceps, whereas MuSCs isolated from the masseter and soleus fall in between and are not significantly different from either group (Figure 4A,B). This is consistent with our previous results22. Moreover, the tissues from which the MuSCs show high EdU incorporation levels are the same tissues from which the MuSCs were previously shown to express high levels of Pax3 protein22. FAPs isolated from the EDL showed a significantly lower EdU incorporation compared to FAPs isolated from the GA and soleus, while FAPs isolated from the TA showed a significantly lower EdU incorporation compared to FAPs isolated from the soleus (Figure 4A,C). This underscores the importance of analyzing stem cells from individual tissues rather than pooling muscles from different tissues for isolation. For all tissues, the mean fraction of EdU-positive MuSCs was higher compared to the mean fraction of EdU-positive FAPs, suggesting that MuSCs activate faster under the given conditions.
Finally, cell purity was confirmed with immunofluorescence staining (Figure 4D). On average, 97.71% (&#177; 1.38%) of MuSCs stained positive for Pax7 protein, and 88.16% (&#177;6.35%) of FAPs stained positive for PDGFRa protein, confirming the specificity of our stem cell isolation procedure (Figure 4D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64557/64557fig1v2.jpg" /> 
Figure 1: Schematic abstract of the protocol. Schematic showing the two main segments of the protocol, MuSC isolation (upper panel), quiescence assay (lower panel), and the key steps of the methodology used in each of them. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64557/64557fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64557/64557fig2v2.jpg" /> 
Figure 2: Muscle location and isolation. (A) A scheme showing the location of each muscle. (B-S) Demonstration of muscle isolation for (B-D) gracilis, (E-G) TA/EDL, (H-J) triceps, (K-M) GA/soleus, (N-P) masseter, and (Q-S) diaphragm muscles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64557/64557fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64557/64557fig3v2.jpg" /> 
Figure 3: Gating strategy used to identify and sort MuSCs and FAPs in a diaphragm muscle sample. Cells are identified based on cell size (Forward Scatter (FSC)) and granularity (Side Scatter (SSC)). Singlets are selected based on FSC-A and FSC-W. Lineage- cells are gated for subsequent identification of MuSCs (Lineage-/VCAM1+), and FAP (Lineage-/SCA1+) cells are gated to identify FAPs. The same gating strategy was applied to (A) an unstained control, (B) FMO controls (FMO-SCA1&#173;PacBlue, FMO-CD31/45-FITC, and FMO-VCAM1-PECy7), and (C) a stained sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64557/64557fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64557/64557fig4v2.jpg" /> 
Figure 4: Quantification of cell activation by EdU staining. (A) Representative images of EdU staining of MuSCs (top panels) and FAPs (bottom panels) from a diaphragm sample. Shown are merged images of EdU and Hoechst (left panels), and the single channels in green (EdU, middle panels) and blue (Hoechst, right panels). (B,C) Bar graph showing the percentage of EdU-positive (B) MuSCs or (C) FAPs for indicated muscles. Plotted is the mean &#177; SEM. Each dot represents a mouse. Statistical analysis was performed in GraphPad using two-tailed Student&#39;s t-tests, with significance set to *p &#60; 0.05 and **p &#60; 0.01. (D) Immunofluorescence staining of MuSCs (top panels) and FAPs (bottom panels) with antibodies against the MuSC marker Pax7 (left side) and the FAP marker PDGFRa (right side). Shown are merged images of Pax7 with Hoechst, followed by the single channels, and merged images of PDGFRa with Hoechst, followed by the single channels. N = 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64557/64557fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solutions</td>
			<td>Reagents</td>
			<td>Amount&#160;</td>
		</tr>
		<tr>
			<td rowspan="3">Wash medium</td>
			<td>F-10 Nutrient mixture (Ham) (1x), +L-glutamine</td>
			<td>445 mL</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td rowspan="2">Dissociation buffer (1st digestion)</td>
			<td>Collagenase type II&#160;</td>
			<td>650 U/mL</td>
		</tr>
		<tr>
			<td>Wash medium</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Dispase stock (2nd digestion)</td>
			<td>Dispase in PBS</td>
			<td>&#160;11 U/mL</td>
		</tr>
		<tr>
			<td>Collagenase stock (2nd digestion)</td>
			<td>Collagenase type II in PBS</td>
			<td>1000 U/mL</td>
		</tr>
		<tr>
			<td rowspan="2">PBS 1x</td>
			<td>PBS 10x powder concentrate</td>
			<td>9.89 g/L</td>
		</tr>
		<tr>
			<td>Autoclaved/sterile water</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td rowspan="2">Acidic water</td>
			<td>Glacial acetic acid (100%) Anhydrous for analysis</td>
			<td>5.15 mL</td>
		</tr>
		<tr>
			<td>Autoclaved/sterile water</td>
			<td>895 mL</td>
		</tr>
		<tr>
			<td rowspan="2">Collagen solution (0.002%)</td>
			<td>Collagen from calf-skin&#160;</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>Acidic water</td>
			<td>800 mL</td>
		</tr>
		<tr>
			<td rowspan="2">Triton X-100</td>
			<td>Triton X-100</td>
			<td>0.5% (v/v)</td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>99.5%</td>
		</tr>
		<tr>
			<td rowspan="2">Blocking buffer</td>
			<td>PBS 1x</td>
			<td>18 mL</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>2 mL</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Table of recipes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample no.</td>
			<td>Name&#160;</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Unstained</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Fluorescence minus VCAM1-PeCy7</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Fluorescence minus SCA1-PacificBlue</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Fluorescence minus CD31/45-FITC</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Experimental stain (all four antibodies)</td>
		</tr>
	</tbody>
</table>
Table 2: Overview of staining, controls, and samples prepared for each tissue prior to sorting.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tissue</td>
			<td>Antibody mix</td>
			<td>Cells</td>
			<td>Singlets</td>
			<td>Lin neg</td>
			<td>FAPs</td>
			<td>MuSCs</td>
		</tr>
		<tr>
			<td rowspan="5">Diaphragm</td>
			<td>unstained</td>
			<td>100%</td>
			<td>82%</td>
			<td>55%</td>
			<td>0.5%</td>
			<td>0.0%</td>
		</tr>
		<tr>
			<td>FMO Sca1-PacBlue</td>
			<td>100%</td>
			<td>83%</td>
			<td>19%</td>
			<td>0.3%</td>
			<td>4.3%</td>
		</tr>
		<tr>
			<td>FMO CD31/45-488</td>
			<td>100%</td>
			<td>84%</td>
			<td>40%</td>
			<td>9.4%</td>
			<td>5.6%</td>
		</tr>
		<tr>
			<td>FMO Vcam1-PeCy7</td>
			<td>100%</td>
			<td>78%</td>
			<td>20%</td>
			<td>5.6%</td>
			<td>0.0%</td>
		</tr>
		<tr>
			<td>stained</td>
			<td>100%</td>
			<td>77%</td>
			<td>19%</td>
			<td>2.4%</td>
			<td>3.8%</td>
		</tr>
		<tr>
			<td>Gracilis</td>
			<td>stained</td>
			<td>100%</td>
			<td>96%</td>
			<td>11%</td>
			<td>2.6%</td>
			<td>1.4%</td>
		</tr>
		<tr>
			<td>TA</td>
			<td>stained</td>
			<td>100%</td>
			<td>88%</td>
			<td>16%</td>
			<td>2.8%</td>
			<td>2.2%</td>
		</tr>
		<tr>
			<td>EDL</td>
			<td>stained</td>
			<td>100%</td>
			<td>86%</td>
			<td>26%</td>
			<td>19.2%</td>
			<td>0.8%</td>
		</tr>
		<tr>
			<td>Soleus</td>
			<td>stained</td>
			<td>100%</td>
			<td>91%</td>
			<td>41%</td>
			<td>13.3%</td>
			<td>1.1%</td>
		</tr>
		<tr>
			<td>GA</td>
			<td>stained</td>
			<td>100%</td>
			<td>94%</td>
			<td>51%</td>
			<td>6.1%</td>
			<td>1.5%</td>
		</tr>
		<tr>
			<td>Triceps</td>
			<td>stained</td>
			<td>100%</td>
			<td>92%</td>
			<td>30%</td>
			<td>2.6%</td>
			<td>4.5%</td>
		</tr>
		<tr>
			<td>Masseter</td>
			<td>stained</td>
			<td>100%</td>
			<td>85%</td>
			<td>27%</td>
			<td>19.3%</td>
			<td>2.6%</td>
		</tr>
	</tbody>
</table>
Table 3: Overview of relative cell type abundance in the FACS data.

Watch this Scientific Journal Video about Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles at JoVE.com

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2&#180;-deoxyuridine incorporation assay.

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...

isolation-quiescent-stem-cell-populations-from-individual-skeletal

Nanyang Technological University

Research

JoVE Journal

Biology

3.2K Views.  Aarhus University. Dit protocol is belangrijk omdat het de studie van de stamcelfunctie in verschillende skeletspieren mogelijk maakt, waardoor ons begrip van hoe ze bijdragen aan homeostase, regeneratie en ziekteprogressie wordt verdiept. Met behulp van dit protocol kunnen we twee populaties van levende stamcellen isoleren van specifieke spieren en bestuderen hoe ze werken. Om spierisolatie te beginnen, spuit u de geëuthanaseerde muis met ethanol en plaatst u deze met de buik naar boven gericht.Maak me...

Video: Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

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 ...

Developmental Biology

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 ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Medicine

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

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