This study was funded by a Mosaic grant (017.009.009) from The Netherlands Organization for Scientific Research (NWO) and a Start grant (S-13-167C) for young investigators from the AO Foundation. Z.Y.R is supported by the National Institutes of Health (grant # AG021566, AG035377, NS090051).

All experiments described herein were approved by the local Board for Animal Experiments from the Radboud University Nijmegen in accordance with Dutch laws and regulations (RU-DEC 2013-205).
1. Extracellular Matrix Gel Spots
<ol>
	<li>Perform the following steps one day before the isolation:
	<ol>
		<li>Thaw an aliquot extracellular matrix gel (100 &#181;l) at 4 &#176;C for at least 1.5 hr. Dilute 1:10 in Dulbecco's modified Eagle's medium; with 4,500 mg/L glucose, 4 mM L-glutamine, and 110 mg/ml sodium pyruvate (DMEM). Keep the extracellular matrix gel at 4 &#176;C at all times. Note: Abrupt temperature changes will result in uneven coating and crystal formation.</li>
		<li>Keep the diluted extracellular matrix gel solution on ice for 15 min.</li>
		<li>Pre-chill a 20 &#181;l micropipette for 10 min.</li>
		<li>Put 8-well chamber slides into a 100 mm Petri dish and transfer the dish onto a cold surface (e.g. a freezer pack) for 10 min.</li>
		<li>Use the pre-chilled micropipette to put a drop of 10 &#181;l extracellular matrix gel in each well. Keep the Petri dish on the cold surface for at least another 7 min (Figure 1A).</li>
		<li>Completely remove the remaining extracellular matrix gel (Figure 1B), and dry the wells at 37 &#176;C overnight.</li>
	</ol>
	</li>
</ol>
2. Dissection of Head Muscles (Masseter, Digastric, and Levator Veli Palatini)
<ol>
	<li>Before dissection, prepare 50 ml of phosphate-buffered saline (PBS) supplemented with 2% Penicillin-Streptomycin (P/S). Keep on ice.</li>
	<li>After euthanasia of one young adult rat (9 weeks) with CO2/O2, decapitate the head and remove the skin from the head. Transfer the head to ice-cold PBS supplemented with 2% P/S in a 50 ml tube.</li>
	<li>Masseter muscle (derived from the 1st branchial arch)
	<ol>
		<li>Place the head with one side up on a silicone pad and fix with hypodermic needles (Figure 2A).</li>
		<li>Identify the parotid gland and the facial nerve (Figure 2A). Expose the deep fascia covering the gland. Cut the fascia and remove the gland using dissection scissors. Identify the external auditory canal. Trace the facial nerve from the stylomastoid foramen and carefully remove the temporal, zygomatic, and buccal branches with a scalpel blade No. 15.</li>
		<li>Free the superficial head of the masseter muscle by removing the fascia. Identify both superficial and deep heads of the masseter muscle. Trace the superficial head until its thick tendinous aponeurosis inserted in the zygomatic process of the maxilla.</li>
		<li>Separate the tendon from its origin at the zygomatic process with a straight forceps. Cut it with a scalpel blade No. 15 or dissection scissors and carefully life it (Figure 2B).</li>
		<li>Dissect the superficial head of the masseter until its insertion at the angle and inferior half of the lateral surface of the ramus of the mandible with a scalpel blade No. 15 (Figure 2C). Now, completely remove the muscle.</li>
	</ol>
	</li>
	<li>Posterior belly of the digastric muscle (derived from the 2nd branchial arch)
	<ol>
		<li>Place the head in a supine position on the silicone pad and fix with hypodermic needles (Figure 3A).</li>
		<li>Remove the subcutaneous fat overlying both sublingual and submandibular glands. Next, remove the superficial fascia and glands using dissection scissors. Expose the digastric muscle (anterior and posterior belly).</li>
		<li>Hold the anterior tendon of the posterior belly with a straight forceps, cut it, and dissect it carefully until its origin in the tympanic bulla (Figure 3B). Do the same at the contralateral side.</li>
	</ol>
	</li>
	<li>Levator veli palatini muscle (derived from the 4th branchial arch)
	<ol>
		<li>After dissection of the posterior belly of the digastric muscle, localize the stylohyoid muscle, pull it laterally, and carefully remove it (Figure 4A).</li>
		<li>Localize the tendon of the levator veli palatini that inserts at the tympanic bulla (Figure 4A). Dissect it carefully and cut it on both sides.</li>
		<li>Look for the trachea and the esophagus that runs behind it. Lift the esophagus, and expose the pharynx and the larynx.</li>
		<li>Localize and dissect the area of the superior pharyngeal constrictor muscle. Identify the levator veli palatini and cut it at both sides (Figure 4B). 
Note: Directly after dissection, carefully remove tendon and connective tissue from each muscle under the stereo microscope. Submerge all specimens quickly in ethanol 70%, and transfer them to ice-cold PBS supplemented 2% P/S in a 15 ml tube.
</li></ol>
</li>
</ol>
3. Isolation of Satellite Cells
<ol>
	<li>Perform the following preparation steps for SC isolation from 3 groups of muscles:
	<ol>
		<li>Prepare 7.5 ml of 0.1% pronase in DMEM. Filter the solution through a 0.22 &#181;m filter. Pre-warm the solution at 37 &#176;C in a water bath for 10 min before isolation.</li>
		<li>Prepare 35 ml of DMEM supplemented with 10% Horse Serum (HS) and 1% P/S. Also pre-warm at 37 &#176;C in a water bath.</li>
		<li>Prepare 15 ml culture medium which consists of DMEM supplemented with 20% fetal bovine serum (FBS), 10% HS, 1% P/S and 1% chicken embryo extract (CEE). Pre-warm at 37 &#176;C in a water bath.</li>
		<li>Pre-coat six plastic pipettes (10 ml) with HS and dry for at least 10 min before use.</li>
	</ol>
	</li>
	<li>In the culture hood, transfer each muscle into a well of a 6-well plate. Using the dissection scissors, cut the muscle in small pieces of about 2 mm. Be careful not to mince the tissue too much.</li>
	<li>Carefully add 2.5 ml of 0.1% pronase solution to each well and incubate at 37 &#176;C for 60 min. Gently shake the plate after 20, 40, and 60 min. Note: The exact duration of the incubation depends on factors like age and strain of the animals.</li>
	<li>Monitor under the microscope. Check the muscle fragments and stop the enzymatic digestion when the fibers bundles get a loosened appearance (Figure 5).</li>
	<li>Add 2.5 ml of DMEM supplemented with 10% HS and 1% P/S. Transfer to a 15 ml tube and centrifuge the tubes at 400 x g for 5 min. Discard the supernatant by decantation.</li>
	<li>Add 5 ml DMEM supplemented with 10% HS and 1% P/S. Pipette the solution up and down with a 10 ml plastic pipette (trituration) for at least 20 times to homogenize the tissue.</li>
	<li>Centrifuge the tubes at 200 x g for 4 min. Collect the supernatant and transfer into a 15 ml tube.</li>
	<li>Add 5 ml DMEM supplemented with 10% HS and 1% P/S. Pipette again with a 10 ml plastic pipette until the tissue fragments passes easily through the pipette.</li>
	<li>Centrifuge the tubes at 200 x g for 4 min and collect the supernatant in a 15 ml tube.</li>
	<li>Put a cell strainer (40 &#181;m) onto a 50 ml tube and transfer the supernatant containing the dissociated cells onto the filter. Wash with 1 ml DMEM for maximal cell recovery.</li>
	<li>Centrifuge the tubes at 1,000 x g for 10 min and discard the supernatant with a pipet.</li>
	<li>Resuspend the pellet in 300 &#181;l culture medium and count the cells in a hemocytometer.</li>
</ol>
4. Differentiation of Satellite Cells on Extracellular Matrix Gel Spots
<ol>
	<li>Dilute the cell suspension to obtain 1.5 x 103 cells in 10 &#181;l of culture medium.</li>
	<li>Secure the covers of the chambers slides with tape and mark the spots with a black marker on the bottom side of the object glass.</li>
	<li>Using a micropipette, put a drop of 10 &#181;l cell suspension onto the extracellular matrix gel spot. Check under the microscope whether the drop of cell suspension has been placed correctly on the spot. Incubate for six hours at 37 &#176;C.</li>
	<li>Carefully add 400 &#181;l of culture medium (DMEM supplemented with 20% FBS, 10% HS, 1% P/S and 1% CEE) and incubate for three days at 37 &#176;C. 
	Note: At this point, freshly isolated SC are subjected to massive trauma (enzymatic digest and harsh trituration) and they need to recover. Do not disturb the cells during the first three days 37. Next, the culture medium can be changed depending on the type of experiment. 
	The extracellular matrix gel spots can be seeded with a high cell density (1.5-2.5 x 103/20 &#181;l) for differentiation assay. Culture medium (DMEM supplemented with 20% FBS, 10% HS, 1% P/S and 1% chicken embryo extract) can be replaced every third day.</li>
	<li>Alternatively, if expansion and passing is desired follow the next steps:
	<ol>
		<li>Thaw an aliquot extracellular matrix gel (500 &#181;l) at 4 &#176;C for at least 1.5 hr. Dilute 1:10 in DMEM and follow the recommendations in point 1.1.1.</li>
		<li>Pre-chill a 10 ml pipette for 10 min at 4 &#176;C.</li>
		<li>Transfer three T75 flasks onto a cold surface (e.g. a freezer pack) for 10 min.</li>
		<li>Use the pre-chilled pipette to put 1 ml extracellular matrix gel into each flask. Check that the surface is covered completely. Keep the flasks on the cold surface for at least another 7 min (Figure 1A).</li>
		<li>Completely remove the remaining extracellular matrix gel with a 10 ml pipette, and dry the wells at 37 &#176;C for 1 hr.</li>
		<li>After counting, resuspend the freshly isolated SCs in 10 ml of culture medium (DMEM supplemented with 20% FBS, 10% HS, 1% P/S and 1% chicken embryo extract) and seed in the pre-coated T75 flasks.</li>
		<li>After three days, change the medium (and every third day) until 80% confluence is reached. For passaging, wash the T75 flasks three times with PBS. Next add 1 ml 0.25% trypsin solution and incubate for three min at 37 &#176;C. Resuspend in 9 ml of culture medium (DMEM supplemented with 20% FBS, 10% HS, 1% P/S and 1% chicken embryo extract) and centrifuge at 200 x g for 5 min. Discard the supernatant. After counting, resuspend 1 x 106 cells in 1,000 &#181;l of culture medium and freeze the cells.</li>
	</ol>
	</li>
</ol>

The authors have no conflicting interests to disclose.

SCs from different branchiomeric head muscles were isolated from one 9-week-old Wistar rat and cultured directly on extracellular matrix gel spots without prior expansion and passaging. After isolation, the cells were counted and seeded at the same cell density. For the parallel isolation of three different muscles, this method takes about 4 hr. To avoid culture contamination, a critical step is the rapid washing in alcohol 70% after dissection of the muscles.
During SC isolation it is important to cut the muscle tissue into small pieces (about 2 mm) but avoid too much mincing as this will result in a small cell yield because of cell damage. Also, the duration of the enzymatic digestion must be checked carefully under the microscope to avoid further damage. The aim of the digestion is to dissociate the myofibers. Since more than 90% of the isolated cells express Pax7, no further purification is required (Figures 6-8). This avoids extra purification steps in other methods such as pre-plating on uncoated dishes14,37,38, fractionation on Percoll39,40, or fluorescent- or magnetic cell sorting41,43. For trituration it is essential to induce shear between the tissue fragments and the opening of the pipette tip as this allows the mechanical release of the SCs. If the trituration with a 10 ml pipette (inside diameter tip: 1 mm) is difficult, a 5 ml (inside diameter tip: 2 mm) pipette can be used first. Alternatively, glass Pasteur pipettes can be cut at the desired diameter and be used. This method is simple, efficient and allows the simultaneous isolation of SC from different muscle samples.
The culture plates for SCs can also be coated with gelatin or collagen, but our previous studies show that extracellular matrix gel is far better for the maintenance of the myogenic potential than collagen38. The extracellular matrix gel spots of millimeter size (10 &#181;l/&#216; 2 mm or 20 &#181;l/&#216; 4 mm) allows the study of proliferation and differentiation of SCs with limited numbers of cells. For the differentiation assay about 8 to 20 times fewer cells are required compared to a 24-well plate (&#216; 15.6 mm), and about 80 to 200 times fewer compared to 35 mm Petri dishes (&#216; 35 mm)14,38.
Since extracellular matrix gel is expensive, this method is also more cost-efficient. In addition, the chamber slides can be replaced by plastic cover slips to further reduce the costs. For the preparation of the extracellular matrix gel spots overnight drying of the chamber slides is essential. As the extracellular matrix gel spots are transparent, it is necessary to mark the spots at the bottom side using back lighting. The chambers slides are fixed in a Petri dish for easy manipulation. Further cell culture expansion is not necessary, which offers the possibility to study the SCs of smaller muscles or small muscle samples. Alternatively, e.g. for PCR or muscle constructs if more cells are needed, the freshly isolated SCs can first be expanded in T75 flasks as indicated above.
SCs isolated using this protocol are not suitable for further purification with flow cytometry immediately after isolation. The digestion with pronase causes extensive digestion of the surface antigens14. Horse serum and fetal bovine serum that are used for cell culture must first be properly characterized before isolation, as different lot numbers differentially affected myoblasts proliferation and differentiation.
In recent years, there is a growing interest in the muscles derived from the branchial arches and the head mesoderm (e.g. the extraocular muscles)24. It has been clearly demonstrated that head and limb muscles possess highly different properties. Masseter muscle from old animals seems to retain their regenerative capacity in comparison with limb muscles25,26. SCs from the extraocular muscles possess a robust proliferation and differentiation capacity comparable to SCs from head muscles, and show a larger engraftment potential than limb muscle SCs24.
The fiber type distribution and myosin composition varies among muscle groups and also between species. Muscles originating from the first branchial arch in humans contain both slow and fast fibers (subtypes IIA and IIX), neonatal myosins and myosins typical for developing cardiac muscle. In rodents these muscles contain about 95% fast fibers myosin IIA and IIb)44-46. Studies on avian muscles show that SCs from different muscle fiber types vary in differentiation capacity. SCs from fast fibers only differentiate into fast muscle fibers, while SCs from slow fibers can differentiate into both fiber types47. In addition, the percentage of SCs in fast muscle fibers is lower than in slow muscle fibers48,49. This indicates that the fiber type distribution must be taken into account for studies on muscles in the craniofacial area. Similar to cleft palate muscles, the LVP in rodents contains almost exclusively fast fibers50. For that reason, SCs from the LVP are suitable for pre-clinical studies in the field of cleft palate.
This protocol offers new possibilities to study SCs derived from branchiomeric head muscles or other smaller muscles or smaller muscles samples. This will facilitate the development of new therapies to improve the regeneration of muscles in the maxillofacial area in conditions such as cleft palate but also in other conditions affecting smaller muscles.

An erratum was issue for <a href="http://www.jove.com/video/52802">Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles</a>. The fourth figure was updated to explain the isolation of the LVP better.

Erratum: Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

About 1:500 to 1:1,000 newborns exhibit a cleft involving the lip and/or palate (CLP); thus this is the most common congenital malformation in humans1. The muscles of the soft palate are critical for the functioning of the soft palate during speech, swallowing, and sucking. If a cleft of the soft palate is present, these muscles are abnormally inserted into the posterior end of the palatal bone.
The soft palate moves up and down during speech, preventing air to escape through the nose. Children with a cleft in the palate do not have this control function resulting in a phenomenon known as velopharyngeal dysfunction2,3. Although the treatment protocols are variable, surgical repair of the soft palate takes place in early childhood (6-36 months of age)4. The abnormally inserted muscles of the soft palate can be surgically corrected5-7, however, velopharyngeal dysfunction persists in 7% to 30% of the patients2,3,8-10.
The ability of skeletal muscle to regenerate through the action of satellite cells (SCs) is well established11,12. Upon muscle injury, SCs are activated and migrate to the site of injury. They then proliferate, differentiate, and fuse to form new myofibers or repair damaged ones13. Quiescent SCs express the transcription factor Pax714,15, while their progeny, the proliferating myoblasts, additionally express the myogenic determination factor 1 (MyoD)16. Differentiating myoblasts start to express myogenin (MyoG)17. The terminal differentiation of myoblasts is marked by the formation of myofibers, and the expression of muscle-specific proteins such as myosin heavy chain (MyHC)16,18.
Recently, several strategies have been used in regenerative medicine to improve muscle regeneration of limb muscles19-23. Specific studies on branchiomeric head muscles are also important because it was recently demonstrated that they differ from other muscles in several aspects24. In contrast with limb muscles, it has been suggested that branchiomeric head muscles contain less SCs25, regenerate slower, and more fibrous connective tissue is formed after injury26 In addition, proliferating SCs from branchiomeric head muscles also express other transcription factors. For instance, Tcf21, a transcription factor for craniofacial muscle formation is strongly expressed in regenerating head muscles but hardly in regenerating limb muscles25. The muscles in the soft palate of CLP patients are usually smaller and less well-organized compared to normal palatal muscles27,28. Slow and fast fibers are both present in the soft palate muscles but the slow fibers are more abundant. In contrast, cleft muscles contain a higher proportion of fast fibers and also a reduced capillary supply compared with normal soft palate muscles29-31. Fast fibers are more prone to contraction-induced injury31-33. The accompanying poor capillary supply may also promote fibrosis34,35. All these aspects may contribute to the poor regeneration of soft palate muscles after surgical cleft closure36. In view of this, a protocol for the isolation and characterization of branchiomeric head muscle SCs is crucial. This provides the possibility to study SC biology of branchiomeric head muscles. In addition, new therapies based on tissue engineering can be developed to promote muscle regeneration after surgery in CLP and other conditions compromising the craniofacial area.
In general, SCs can be obtained after dissociation of muscle tissue14. Mincing, enzymatic digestion, and trituration are generally required to release SCs from their niche. SCs can be purified by pre-plating on uncoated dishes14,37,38, fractionation on Percoll39,40, or fluorescent- or magnetic cell sorting41-43. Here we present a new economic and rapid protocol for the isolation of satellite cells from branchiomeric head muscles of young adult rats. This protocol is based on a previous manuscript14 and specifically adapted for small tissue samples. The isolation of SCs from representative muscles originating from the 1st, 2nd, and 4th branchial arches are described. After isolation, low numbers of satellite cells are cultured on extracellular matrix gel spots of millimeter size to study their differentiation. This approach avoids the requirement for the expansion and passaging of SCs.

<table><tbody><tr><td>Hypodermic Needle 25&amp;nbsp;G 0.5&amp;nbsp;x&amp;nbsp;25&amp;nbsp;m</td><td>BD Microlance</td><td>300400</td><td></td></tr><tr><td>Dissecting scissors</td><td>Braun</td><td>BC154R</td><td></td></tr><tr><td>Micro forceps straight</td><td>Braun</td><td>BD330R</td><td></td></tr><tr><td>Surgical Scalpel Blade No.&amp;nbsp;15</td><td>Swann-Morton</td><td>0205</td><td></td></tr><tr><td>Alcohol 70%</td><td>Denteck</td><td>2,010,005</td><td></td></tr><tr><td>Permanox Slide, 8 Chamber</td><td>Thermo Scientific</td><td>177445</td><td></td></tr><tr><td>6 well cell culture plate</td><td>Greiner bio-one</td><td>657160</td><td></td></tr><tr><td>Cell Culture Dishes (100 x 20 mm)</td><td>Greiner bio-one</td><td>664160</td><td></td></tr><tr><td>15 ml sterile conical centrifuge tube</td><td>BD Biosciences</td><td>352097</td><td></td></tr><tr><td>50 ml sterile conical centrifuge tube</td><td>BD Biosciences</td><td>352098</td><td></td></tr><tr><td>Cell strainer (40 &amp;mu;m)</td><td>Gibco</td><td>431750</td><td></td></tr><tr><td>10 ml serological pipette</td><td>Greiner bio-one</td><td>607180</td><td></td></tr><tr><td>20&amp;nbsp;&amp;micro;l FT20</td><td>Greiner bio-one</td><td>774288</td><td></td></tr><tr><td>Matrigel, Phenol-Red Free</td><td>BD Biosciences</td><td>356237</td><td>10 ml</td></tr><tr><td>Pronase</td><td>Calbiochem</td><td>53702</td><td>10&amp;nbsp;KU</td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco</td><td>14190-144</td><td>500 ml</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium, high glucose, GlutaMAX Supplement, pyruvate&amp;nbsp;</td><td>Gibco</td><td>10569-010</td><td>500 ml</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific</td><td>3600511</td><td>500 ml</td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>26050088</td><td>500 ml</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco</td><td>15140-122</td><td>100 ml</td></tr><tr><td>Chicken Embryo Extract</td><td>MP Biomedicals</td><td>2850145</td><td>20 ml</td></tr></tbody></table>

isolation and characterization of satellite cells from rat head branchiomeric muscles

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

Using this protocol, the masseter muscle (one side) yields 0.8-1 x 106 cells, the digastric muscle (posterior belly) yields 1.5-2 x 105 cells, and levator veli palatini muscle yields 1-1.5 x 105 cells. Cell yields depend on the muscle type, strain, and age of the animal. For comparison between the three muscle groups, freshly isolated SCs were seeded at the same cell density (1.5 x 103/10 &#181;l). Directly after isolation, more than 90% of the freshly isolated cells express Pax7 (Figure 6).
Day 4, 7 and 10 cultures were stained with antibodies against Pax7, MyoD, MyoG and MyHC immunostaining. Five arbitrary fields were counted per culture using a 20X objective. At day 4 Pax 7 and Myo D is expressed in all muscle groups (Figures 6 and 7 and 8), however the progeny of SatCs from the masseter and digastric muscles start expressing myogenin earlier than the levator veli palatini muscle (Figure 9). At day 10, the expression of MyoG is strongly reduced in all groups (Figure 9). A few days after seeding on the extracellular matrix gel spots, the proliferating cells begin to fuse and form multi-nucleated myotubes, which express myosin heavy chain. Small myotubes are clearly visible at day 7 (Figure 10). At day 10, twitching of the myotubes can be observed (Video 1).
<img alt="Figure 1" src="/files/ftp_upload/52802/52802fig1.jpg" /> 
Figure 1: Extracellular matrix gel spots in a chamber slide. (A) For easy manipulation, place the 8-well chamber slide into a 100 mm Petri dish. Pipet 10 &#181;l extracellular matrix gel in each chamber and put it on a cold surface (7 min). (B) Chamber slide after the excess extracellular matrix gel is removed.
<img alt="Figure 2" src="/files/ftp_upload/52802/52802fig2.jpg" /> 
Figure 2: Dissection of the masseter muscle. (A) Head of the animal in a lateral view. Ear (E), Parotid gland (P) and facial nerve (VII). (B) Tendinous aponeurosis (Te) of the superficial head of the masseter muscle (Ms) and temporal muscle (T). Separate the tendon from its insertion with a forceps. (C) Carefully dissect the muscle until its insertion at the ramus of the mandible. E: ear, P: parotid gland, VII: facial nerve, T: Temporal muscle, Ms: superficial head of the masseter muscle, Te: tendon, Mp: deep head of the masseter muscle.
<img alt="Figure 3" src="/files/ftp_upload/52802/52802fig3.jpg" /> 
Figure 3: Dissection of the posterior belly of the digastric muscle. (A) Head of the animal in a supine position. Localize the submandibular gland (Sg), masseter muscle (M), facial nerve (VII) and sternocleidomastoid muscle (SCM). Remove the submandibular gland. (B) Localize the digastric muscle anterior (AD) and posterior belly (PD). With a straight forceps, take the anterior tendon of the posterior belly, cut it and dissect it carefully until its origin in the tympanic bulla (ty). E: ear, Sg: submandibular gland, VII: facial nerve, M: masseter muscle, SMC: sternocleidomastoid muscle, AD: anterior belly digastric muscle, PD: posterior belly digastric muscle, Ty: Tympanic bulla.
<img src="/files/ftp_upload/52802/52802fig4new.jpg" align="Figure 4" /> Figure 4: Dissection of the levator veli palatini muscle. (A) General view after dissection of the digastric muscle (posterior belly). Stylohyoid muscle (St) and tendon of the levator veli palatini can be localized. Note the trachea (T) and esophagus (Es) running behind it. (B) After lifting the trachea and the esophagus the pharynx (P) is exposed. The levator veli palatini that runs laterally towards the soft palate is now visible. The arrow indicates the dissected superior pharyngeal constrictor muscle; note the levator veli palatini muscles at both sides. E: ear, St: stylohyoid muscle, VII: facial nerve, M: masseter muscle, AD: anterior belly digastric muscle, PD: posterior belly digastric muscle, T: trachea, Es: esophagus, P: Pharynx, *levator veli palatini muscle.
<img alt="Figure 5" src="/files/ftp_upload/52802/52802fig5.jpg" /> 
Figure 5: Appearance of the muscle tissue (A) before and (B) after enzymatic digestion with pronase. Note that muscle bundles appear to be loosened after enzymatic digestion.
<img alt="Figure 6" src="/files/ftp_upload/52802/52802fig6highres.jpg" width="350" /> 
Figure 6: Pax 7 immunostaining. Freshly isolated SCs, applied to extracellular matrix gel at the end of isolation (about 6 hours after initial tissue digestion). Five arbitrary fields were counted using a 10X objective with an average of 210 cells per field. Approximately 90% of the cells are Pax 7 positive. DAPI: blue, Pax7: red. Scale bar, 100 &#181;m.
<img alt="Figure 7" src="/files/ftp_upload/52802/52802fig7highres.jpg" width="700" /> 
Figure 7: Pax 7, MyoD immunostaining. Day 4, 7 and 10 cultures were stained with antibodies against Pax7, and MyoD immunostaining. (A&#8211;C) and (D&#8211;F) Representative photomicrographs of day 4 and 7 cultures from the masseter muscle. (G and H) The number of Pax7+ and MyoD+ nuclei per microscopic field was counted and expressed as a percentage of the total number of nuclei (DAPI). DAPI: blue, Pax7: red, and MyoD: green. Scales bar, 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52802/52802fig7highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/52802/52802fig8highres.jpg" width="700" /> 
Figure 8: Distribution of Pax7&#177;/MyoD&#177; in cultures from mononucleated cells in cultures from masseter, digastric and levator veli palatine muscle. (A&#8211;C) Day 4, 7 and 10 cultures were stained with antibodies against Pax7, and MyoD immunostaining. The total number of cells is based on of the total number of nuclei (DAPI). (D) Data quantification of Pax7&#177;/MyoD&#177; cells. <a href="https://www.jove.com/files/ftp_upload/52802/52802fig8highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/52802/52802fig9highres.jpg" width="700" /> 
Figure 9: Myogenin immunostaining. Day 4, 7 and 10 cultures were stained with antibodies against Myogenin. (A&#8211;D) Representative photomicrographs of day 4 and 7 cultures from the levator veli palatine muscle. (E) The number of MyoG+ nuclei per microscopic field was counted and expressed as a percentage of the total number of nuclei (DAPI). (F) Data quantification of MyoG+ cells. DAPI: blue, Myogenin: green. Scales bar, 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52802/52802fig9highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/52802/52802fig10.jpg" /> 
Figure 10: Myosin Heavy Chain immunostaining. Day 4, 7 and 10 cultures were stained with antibodies against myosin heavy chain (MyHC). Representative photomicrographs of day 4, 7 and 10 cultures from the digastric (DIG) muscle. At day 7, small myotubes are present while at day 10 long and well-organized myotubes are evident. Scales bar, 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52802/52802fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Myotube twitching. Examples of two representative fields with twitching myotubes are shown for day 10 cultures from digastric muscle. <a href="https://www.jove.com/files/ftp_upload/52802/Video1.mpg" target="_blank">Please click here to view this video.</a>

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Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

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

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

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.9K Views.  Radboud University Medical Center. This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Video: Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric 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 ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

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

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

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 ...

Biology

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 ...

Functional Isolation of Single Motor Units of Rat Medial Gastrocnemius Muscle

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in hindlimb muscles (such as the medial gastrocnemius, soleus, or plantaris muscle) in experimental rats. A crucial element of the method is the application of electrical stimuli delivered to a motor axon isolated from the ventral root. The stimuli may be delivered at constant or variable inter-pulse intervals. This method is suitable for experiments on animals at varying stages of maturity (young, adult or old). Moreover, this protocol can be used in experiments studying variability and plasticity of motor units evoked by a large spectrum of interventions. The results of these experiments may both augment basic knowledge in muscle physiology and be translated into practical applications. This procedure focuses on the surgical preparation for the recording and stimulation of MUs, with an emphasis on the necessary steps to achieve preparation stability and reproducibility of results.

This method allows the recording of the force of twitch and tetanic contractions and action potentials in three types of motor units in the rat medial gastrocnemius muscle. The functional isolation of a single motor unit is induced by electrical stimulation of the axon.

This work outlines functional isolation of motor units (MUs), a standard electrophysiological method for determining characteristics of motor units in ...

Neuroscience

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

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 ...

Medicine

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.

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 ...