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>
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		<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>
		</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...

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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Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

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 ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

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 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 ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...