Name: isolation of quiescent stem cell populations from individual skeletal muscles
Uploaded: 2022-12-09T13:45:00
Description: Watch this Scientific Journal Video about Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles at JoVE.com

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).

The present protocol was performed in accordance with animal care guidelines at Aarhus University and local ethics regulations.
NOTE: Make sure to comply with the regulations of the local ethical committee for animal experimentation and handling of post-mortem rodent samples. Mice are a potential source of allergens; if available, turn on exhaust ventilation and place it over the workspace to avoid excessive exposure to allergens. Alternatively, wear a face mask if the experiment is carried ou...

<ol>
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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 s...

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.

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	<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 &#38; 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 &#38; 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&#160;</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&#160;&#160;</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 &#38; 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&#160;</td>
			
			<td>Bioreagent, Sigma Aldrich&#160;</td>
<td>Source: SLCK6209 (Cat# C8919)</td>			
<td></td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			
			<td>Worthington, Fisher Scientific&#160;</td>
<td>Lot: 40H20248 (cat# L5004177 )</td>			
<td>Collagenase</td>
		</tr>
		<tr>
			<td>Dispase</td>
			
			<td>Gibco, Fisher Scientific&#160;</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>
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			<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&#160;</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&#160;&#160;</td>
<td>K44104563 9Cat # 1000631000)</td>			
<td></td>
		</tr>
		<tr>
			<td>Head over head mini-tube rotator&#160;</td>
			
			<td>Fisher Scientific&#160;</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&#160;</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&#160;</td>
			
			<td>IKA&#160;</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&#160;</td>
<td>304827</td>			
<td>20G needle&#160;</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 &#181;M)</td>
			
			<td>Sarstedt AG &#38; Co. KG, Hounisen Laboratorieudstyr A/S</td>
<td>83.3945.040</td>			
<td>Cell strainer&#160;</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&#160;</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&#160;</td>
<td>ACTC-7712</td>			
<td>&#160;60 x 40cm, 8 layers</td>
		</tr>
		<tr>
			<td>Rainin, pipet-lite XLS</td>
			
			<td>Mettler Toledo, Thermo Scientific&#160;</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&#160;</td>
			
			<td>BD Emerald, Fisher Scientific</td>
<td>307731</td>			
<td>Syringe</td>
		</tr>
		<tr>
			<td>TC Dish 100, standard</td>
			
			<td>Sarstedt AG &#38; Co. KG, Hounisen Laboratorieudstyr A/S</td>
<td>83.3902</td>			
<td>Petri dish&#160;</td>
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		<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...

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 ...

This protocol is significant because it enables the study of stem cell function across different skeletal muscles, deepening our understanding of how they contribute to homeostasis, regeneration, and disease progression. Using this protocol, we can isolate two populations of live stem cells from specific muscles and study how they work. To begin muscle isolation, spray down the euthanized mouse with ethanol and place it with the abdomen facing up.Using scissors, make a 0.5 centimeter long horizontal incision into the abdominal skin. Using both hands, pull the skin to expose the torso as well as the lower extremities up to the hind limbs. Locate the gracilis or the inner thigh muscle and using a pair of curved forceps grab onto it and slightly lift the muscle.Using scissors, make a 0.5 centimeter incision to cut out the gracilis muscle. Repeat the procedure on the other leg. Using a scalpel, cut the fascia by making a 0.5 centimeter incision along the lateral side of the tibia.Then grab the fascia using curved forceps and remove it by pulling, exposing the tendons at the distal end of the hind limb. Next, insert a pair of straight forceps with super fine tips in between the distal tendon of the TA and the EDL muscle. Separate the muscles by sliding the forceps toward the proximal end of the muscles.Bring the forceps back to the distal end and cut the distal tendon. Gently grab the distal tendon of the TA with curved forceps and lift the muscle up and over its proximal attachment. To carefully cut the proximal tendon, cut the other end at the closest possible to its attachment and transfer the TA to a Petri dish.Then use the straight forceps with super fine tips to go underneath the distal tendon of the EDL and slide the forceps toward the proximal end of the muscle to separate the muscles from each other. Bring the forceps back to the distal end and cut the distal tendon without damaging the muscle. By gently grabbing the distal tendon of EDL with curved forceps, lift the muscle up and over its proximal attachment to carefully cut the proximal tendon as close to its attachment as possible.Cut the other end and transfer the EDL muscle to a Petri dish. Repeat the procedure for the other hind limb. Use straight forceps with super fine tips to reach in between the achilles tendon and the lower hind limb bones.Slide the forceps towards the proximal end of the muscle to separate the muscle from the bones. Bring the forceps back to the distal end and cut the distal tendon. Pull the gastrocnemius or GA muscle up and over the fibula.To locate the proximal soleus tendon. Insert the straight forceps in between the soleus and GA muscles and move the forceps toward the distal end of the muscles to separate the soleus from the GA.Now cut the proximal soleus tendon. Grab it with curved forceps and carefully lift the sous to access its distal tendon.Cut the distal tendon to isolate the soleus from the GA and place the soleus in the Petri dish containing the wash medium. Then cut the GA and place it in the Petri dish. To isolate the triceps muscle, use straight forceps having super fine tips to reach in between the triceps and the humerus bone and slide the forceps toward the proximal end of the muscle to separate the muscle from the bone.Next, cut the distal end of the triceps muscle and using curved forceps pull it up and over the elbow to access the proximal tendon. Cut the proximal tendon of the triceps and transfer the muscle into a Petri dish. Repeat the procedure for the other four limb.To remove the fur and the skin from the jaw, use scissors to make a 0.5 centimeter incision below the eye, cutting in the caudal direction. Then using the thumbs and the index fingers of both hands, pinch onto each side of the incision and remove the skin by pulling it upward and downward. Locate the major tendon of the masseter in the caudal side below the eye and insert the flat scalpel blade in between the bone and the muscle to cut the tendon.Grab the major masseter tendon with curved forceps and cut it with a scalpel blade or scissors in the rostral direction to separate the masseter muscle from the jawbone. Place the isolated masseter muscle in the Petri dish and repeat the procedure for the second masseter muscle. Use scissors to make a thoracotomy in the middle of the sternum and cut through the sternum.Expose the diaphragm by cutting 360 degrees through the rib cage. Then to separate the upper body from the abdomen use scissors to cut through the trachea, esophagus, vena cava, and abdominal aorta. Next, using scissors, preform a laparotomy one centimeter below the sternum and make a cut around 360 degrees.Place the closed scissors between the rib cage and the abdominal organs and press down. Gently pull on the rib cage to separate it from the abdominal organs. To separate the diaphragm from the rib cage, loosely hold the diaphragm between two fingers and cut through the rib cage with scissors.Cut the diaphragm as close to the ribs as possible around 360 degrees and place the isolated diaphragm in a Petri dish. Mince the isolated muscles one by one by cutting them into pieces of roughly one millimeter. Transfer the minced muscles to a 15 liter conical tube containing five liters of dissociation buffer, and incubate the tube at 37 degrees Celsius for 35 minutes in a shaking water bath at a speed of 60 rotations per minute.After enzymatic digestion of the minced muscles, transfer the suspension from the 15 milliliter tube to a 50 milliliter tube. Using a 10 milliliter syringe fitted with a 20 gauge needle, re-suspend the sample by pulling it up and down through the needle five times. Draw the cell suspension into the syringe and strain the sample into a new 50 milliliter conical tube fitted with a cell strainer on top.To retrieve all mono nucleated cells, wash the empty conical tube with 20 milliliters of wash media and pour it through the strainer to combine with the strained sample. Retrieve the remaining volume under the cell strainer with a P1000 pipette. Following tissue dissociation and antibody staining, the skeletal muscle stem cells or MuSCs and fibro adipogenic progenitors or FAPs from individual muscles were purified by fluorescence activated cell sorting.After initial gating to identify the cells and separate the singlets from doublets, subsequent gates were set using FMO controls to identify staining thresholds. The stain sample was gated and the SCA1 positive CD31 negative and CD45 negative population corresponding to FAPs was sorted into a separate collection tube. The double negative population was further gated to sort the VCA positive population corresponding to the MuSCs.The diaphragm and triceps muscle single cell suspensions, unlike the others, had a higher relative abundance of MuSCs than FAPs. EDU staining, though robust, indicated a difference in fractions of EDU positive cells for the two stem cell types and across the different muscles. For all tissues, however, the mean fraction of EDU positive MuSCs was higher than that of EDU positive FAPs.MuSCs isolated from EDL and GA showed significantly lower EDU incorporation compared to those from TA, diaphragm, gracilis or triceps. Similarly, significant variation in EDU incorporation was also observed in FAPs isolated from different muscles. Finally, cell purity was confirmed with immunofluorescent staining, indicating the specificity of the stem cell isolation protocol.The most critical step is the mechanical digestion of the tissue. When the tissue is cut too much, the viability declines. When the tissue is cut too little, the EEL declines.The protocol can be combined with assays that measure cell behavior, such as engraftment following transplantation to better understand stem cell functions in vivo. This technique may answer whether differences in stem cell behavior within individual muscles can contribute to disease phenotypes and diseases characterized by unique patterns of affected muscles.

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