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

All experiments complied with French and EU animal regulations at the Institut Mondor de Recherche Biom&#233;dicale (INSERM U955), notably the directive 2010/63/UE. Animals were kept in a controlled and enriched environment at the animal facilities with certification numbers A94 028 379 and D94-028-028; they were handled only by authorized researchers and animal caretakers, and they were visually inspected by animal housing personnel for signs of discomfort during their lifetime. They were euthanized by&#160;cervical dislocation prior to dissection. No interventional procedures were performed during the animals&#39; lifetimes; thus, acquiring approval for the procedure from an Ethics Committee and the French Ministry of Higher Education, Research and Innovation was not necessary. Indeed, no ethics clearance is required for euthanization and post-mortem dissection according to the directive 2010/63/UE. The results presented in this manuscript are from the wild-type C57BL/6NRj line (see Table of Materials) and the transgenic Tg:Pax7-nGFP line12(bred by our team). The protocol was applied to male and female mice aged 8-12 weeks of age.
1. Reagent and equipment preparation pre-digestion
<ol>
	<li>Spray the dissection tools (straight and curved scissors, forceps, see the Table of Materials) with 70% ethanol, and dry them with paper. Coat a cork plate with aluminum foil, and keep 10 cm Petri dishes (one per animal) nearby. Have paper and 70% ethanol in reach. 
	NOTE: At the end of the dissection, rinse the dissection tools with water, then spray them with 70% ethanol, and dry them with paper.</li>
	<li>Set a rotating water bath to 37 &#176;C, and prepare the digestion mix (20 mL/animal) by combining DMEM with 1% penicillin-streptomycin, 0.5 U/mL collagenase, 3 U/mL dispase (see the Table of Materials), and 0.2% BSA in 50 mL tube(s).</li>
	<li>Pass the digestion mix through a 0.22 &#181;m filter in a cell culture hood. 
	NOTE: It is recommended to prepare the digestion mix fresh every time.</li>
</ol>
2. Reagent and equipment preparation post-digestion
<ol>
	<li>Post-digestion, the mix can be frozen or cultured. For freezing, prepare 10% DMSO:90% fetal bovine serum (FBS), as well as a set of cryotubes (1 mL of cell suspension per 2 mL cryotube). For the culture, prepare culture medium (DMEM supplemented with 1% penicillin-streptomycin, 4 ng/mL bFGF, and 20% FBS) and a set of 8-well plates. The plates must be coated before plating the cells (details are provided in step 7.1).</li>
	<li>For the staining, prepare 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) (0.15 mL/well of the 8-well plate) and blocking solution (5% IgG-free bovine serum albumin [BSA] in PBS; 0.15 mL/well of the 8-well plate). 
	CAUTION: Do not breathe in the PFA powder; prepare and handle it under a chemical hood.</li>
</ol>
3. Dissection
<ol>
	<li>Spray the euthanized animal with 70% ethanol. Make a horizontal incision (left side of the body to the right side) with big scissors at the level of the abdomen, and cut around the waist. Pull the skin off of the hindlimbs to reveal the muscles (Figure 1A).</li>
	<li>Place the animal on the aluminum foil-covered cork plate, and pin down the opposite forelimb and hindlimb. Quickly remove all the hindlimb muscles (front and back) into a 10 cm Petri dish placed on ice (Figure 1B,C). Take special care to remove the adipose tissue from the areas around the quadriceps and the posterior muscles. Fascia, nerves, and tendons can also be removed at this point if this does not compromise the overall time spent on dissection. 
	NOTE: An optimal dissection time for both the hindlimbs should be around 15-20 min. It is advised that the dissection time does not exceed 30 min.</li>
	<li>Add drops of DMEM to the muscles occasionally to keep them moist, but not too much, as this will render chopping difficult. Repeat for the other hindlimb. Once all the muscles of one animal are in the Petri dish (Figure 1D), chop them finely with scissors for 7-10 min to obtain a smooth homogenate (Figure 1E). 
	NOTE: In this protocol, DMEM supplemented with L-glutamine, pyruvate, and 4.5 g/L D-glucose is used.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65433/65433fig01.jpg" /> 
Figure 1: Pre-culture muscle preparation. (A) The skin is removed to reveal the hindlimb muscles, as described in step 3.1. (B,C) All the hindlimb muscles are harvested (B) around and (C) between the bones, as described in step 3.2. (D) The harvested muscles are placed in a 10 cm Petri dish on ice with DMEM drops to keep them moist, as described in step 3.3. (E) The muscles are finely chopped with scissors until a smooth paste is obtained with the consistency depicted in this image. (F) An image of the pellet after the final centrifugation; the blue arrow highlights the pellet, which is against the tube, under the dashed blue line. <a href="https://www.jove.com/files/ftp_upload/65433/65433fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Digestion
NOTE: At the end of the digestion, a centrifuge at 4 &#176;C, a bucket of ice, three cell strainers (100 um, 70 um, 40 um), and three 50 mL tubes (per animal) are needed for section 5.
<ol>
	<li>Prepare and filter the digestion mix as described in step 1.2. Keep the mix on ice.</li>
	<li>Once all muscles are chopped, place the homogenate in a 50 mL tube with 20 mL of digestion mix. Wrap the edges of the lid with flexible film to prevent leaking, and place the tube in a 37 &#176;C shaking water bath at a low to medium speed (50 rpm).</li>
	<li>After 1 h at 37 &#176;C, open the lid, and mix by gently pipetting seven times up and down with a 10 mL pipette to obtain a homogenous mix. Apply new film around the lid, and place it back in the shaking water bath. After 1 h, remove the tube, and turn off the bath. 
	NOTE: For culture, use this incubation time to coat the plates as described in step 7.1 before moving to section 5.</li>
</ol>
5. Filtration
<ol>
	<li>Fill the digestion tube with cold DMEM (supplemented with 1% penicillin-streptomycin) up to 50 mL. Mix by inverting the tube three times. Keep the DMEM in an ice bucket for the next steps.</li>
	<li>Place a 100 &#181;m cell strainer on a new 50 mL tube. Pass the digested mix through the cell strainer into the new tube. Centrifuge at 600 x g for 5 min at 4 &#176;C. Pour out the supernatant into a liquid waste container.</li>
	<li>Resuspend the pellet in 1 mL of cold DMEM (supplemented with 1% penicillin-streptomycin). Fill the tube up to 50 mL with the same DMEM. NOTE: If the centrifuging is skipped, the next pellet will be more difficult to identify and maintain.</li>
	<li>Place a 70 &#181;m cell strainer on a new 50 mL tube. Pass the centrifuged/resuspended mix through the cell strainer into the new tube. Centrifuge at 80 x g for 5 min at 4 &#176;C. 
	NOTE: This step is not mandatory but is recommended to eliminate cell debris.</li>
	<li>Place a 40 &#181;m cell strainer on a new 50 mL tube. Pass the supernatant through the cell strainer into the new tube. Centrifuge at 600 x g for 5 min at 4 &#176;C, pour the supernatant into a liquid waste container, and resuspend the pellet in FBS under the culture hood. The pellet is very small at this step (Figure 1F). 
	NOTE: Filtering through the 40 &#181;m strainer removes debris, which would give a non-specific signal in the later staining of the cultures.</li>
</ol>
6. (Optional) Freezing
NOTE: Section 6 is optional. The protocol can be paused after filtering, but this can reduce the cell survival and culture success.
<ol>
	<li>Add DMSO to obtain a 10% DMSO:90% FBS ratio, and transfer to cryotubes (1 mL of resuspended pellet per 2 mL cryotube).</li>
	<li>Place the cryotube at &#8722;80 &#176;C in a polystyrene box overnight. Move to &#8722;150 &#176;C the next day for long-term storage. 
	NOTE: Short-term storage at &#8722;80 &#176;C is also possible.</li>
	<li>When starting the culture, thaw the cryotube quickly in a 37 &#176;C water bath until the cell suspension has thawed. Mix with 4 mL of DMEM under the culture hood. Spin at 600 x g for 5 min at 4 &#176;C. Pipette out the supernatant, and continue as described in step 7.2.</li>
</ol>
7. Culturing
NOTE: Frozen or fresh cell suspensions can be expected to fill 24-32 wells of three to four 8-well plates.
<ol>
	<li>Coat 8-well plates with the coating solution, which should be thawed at 4 &#176;C or on ice (stock coating solution is normally kept at &#8722;20 &#176;C). Add 0.4 mL of coating solution to one well, and pipette it from well to well. After transferring the coating solution through all the wells, it can be recollected and refrozen for future cultures. Keep the coated plates at 37 &#176;C for 30 min before plating the cells.</li>
	<li>Add DMEM (supplemented with 1% penicillin-streptomycin) supplemented with 4 ng/mL bFGF (see Table of Materials) to the FBS-cell suspension to obtain a 20% FBS:80% DMEM ratio. 
	NOTE: Even though the addition of bFGF can be beneficial in primary myoblast cultures and in satellite-like cell production in bulk cultures, its addition is optional, as its omission in bulk cultures of ~7 days does not severely compromise the cell yields.</li>
	<li>Plate 0.4 mL of the suspension per well (from step 7.2) in the coated 8-well plates. 
	NOTE: Calculate 30 cm2 of culture per animal for frozen and fresh preparations.</li>
	<li>Incubate the cultures at 37 &#176;C with 5% CO2 for up to 10 days, changing the medium every day after the culture begins to change to a yellowish color (usually 5-7 days). 
	NOTE: To quantify cells in the S phase of the cell cycle13, add 10&#160;&#181;M EdU 2 h before fixation. To capture the first S phase, add 10 &#181;M EdU from the plating, and fix at 40 h of culture.</li>
</ol>
8. Fixation
NOTE: Sections 8-10 should be conducted at room temperature unless otherwise stated.
<ol>
	<li>Pipette out the culture medium, and fix the cells with 4% PFA (0.15 mL/well). 
	CAUTION: Add PFA under a chemical hood. 
	NOTE: If all the wells are fixed at the same time, incubate with PFA at room temperature for 10 min. If the wells are fixed at different time points, add PFA to the wells to be fixed, and keep the plate in the incubator at 37 &#176;C for 5 min.</li>
	<li>Pipette out the PFA, and add PBS for 10 s (0.15 mL/well). Pipette out the PBS, and add fresh PBS for 5 min (0.15 mL/well). 
	NOTE: If all the wells are fixed at the same time, incubate with PBS at room temperature. If the wells are fixed at different time points, add PBS to the fixed wells, and keep the plate in the incubator at 37 &#176;C for 5 min. Then, add 0.4 mL of PBS, and keep the plate in the incubator for up to 1 week.</li>
</ol>
9. Permeabilization and blocking
<ol>
	<li>When ready to stain, pipette out the PBS, and permeabilize with 0.5% TritonX 100 in PBS (0.15 mL/well) for 8 min. Pipette out the TritonX 100, rinse with PBS for 10 s (0.15 mL/well), pipette out the PBS, and wash with PBS for 5 min (0.15 mL/well).</li>
	<li>Block with 5% IgG-free BSA in PBS for 30-60 min (0.15 mL/well).</li>
</ol>
10. Staining
<ol>
	<li>Pipette out the BSA, and add the primary antibody mix diluted in PBS (0.15 mL/well) (see the Table of Materials; dilutions: anti-CD31 1:100, anti-FOSB 1:200, anti-GFP 1:1,000, anti-KI67 1:1,000, anti-MyHC 1:400, anti-MYOD 1:200, anti-MYOG 1:150, anti-PAX7 1:100, anti-PDGFRa 1:50) for overnight incubation at 4 &#176;C. 
	NOTE: After the antibody incubation, collect the antibody mix, add sodium azide, and keep at 4 &#176;C or &#8722;20 &#176;C (as per the antibody manufacturer&#39;s instructions) for future reuse.</li>
	<li>Pipette out the antibody mix, rinse with PBS for 10 s (0.15 mL/well), pipette out the PBS, and wash with PBS for 5 min (0.15 mL/well).</li>
	<li>Pipette out the washing PBS, add the secondary antibody mix (goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555, goat anti-rat Alexa Fluor 647, goat anti-mouse Alexa Fluor 555, goat anti-chicken Alexa Fluor 488, all used at dilutions of 1:500-1,000) and nucleus marker (e.g., DAPI) diluted in PBS (0.15 mL/well) (see Table of Materials), and incubate for 1 h at room temperature, protected from light.</li>
	<li>Pipette out the secondary antibody mix, rinse with PBS for 10 s (0.15 mL/well), pipette out the PBS, wash with PBS for 5 min (0.15 mL/well), pipette out the PBS, and mount. 
	NOTE: If 8-well plates with removable separators are used, peel off the separators before mounting.</li>
</ol>

Harvesting Mouse Skeletal Muscle to Culture Stem Cells and Their Niche

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The authors declare no conflicts of interest.

Adult skeletal muscle function is underpinned by a finely orchestrated set of cellular interactions and molecular signals. Here, a method is presented that allows for the study of these parameters in an ex vivo setting that closely resembles the physiological microenvironment.
Several groups have reported in vitro methods to culture myogenic cells. These methods aimed to isolate satellite cells to study their myogenic progenitor properties. Two main approaches are used to iso...

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

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

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

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

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

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

Author Spotlight: Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components 

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

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

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

unfractionated-bulk-culture-mouse-skeletal-muscle-to-recapitulate

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Biology

1.2K Views.  Univ Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France. Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

Video: Author Spotlight: Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components 

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

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 ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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 ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

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 ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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 ...