This work was supported by the Swiss National Science Foundation (grant number 310030-166313), The "Fondation Suisse pour la recherche sur les maladies musculaires," the "Foundation Marcel Levaillant," and the "Fondation pour la recherche ost&#233;o-articulaire."

Human muscle samples (tissues obtained from semitendinous muscles) were collected during orthopedic surgery on healthy patients as surgical waste. All methods relating to the human study were performed in accordance with the guidelines and regulations of the Swiss Regulatory Health Authorities and approved by the Commission Cantonale d&#39;Ethique de la Recherche from the Geneva Cantonal Authorities, Switzerland (protocol CER n&#176; 12-259). Informed and written consents were obtained from all adult subjects involved in the study.
1. Isolation of Myoblasts from Human Skeletal Muscle
<ol>
	<li>During all procedures, maintain sterile conditions by working under a level II tissue culture hood and using sterile medium and sterile buffers. 
	Note: This procedure can be applied to many human skeletal muscles.</li>
</ol>
2. Dissociation Protocol
<ol>
	<li>Transfer muscle samples (2 - 3 g) into a sterile 6 cm plate.</li>
	<li>Wash the muscle samples with 5 - 10 mL of phosphate buffered saline (PBS). Repeat the wash.</li>
	<li>Remove connective and adipose tissues using curved scissors and a clamp.</li>
	<li>Weigh the muscle sample (see step 2.13) and transfer it to a new sterile 6 cm plate.</li>
	<li>Add 5 mL of 0.05% Trypsin-EDTA.</li>
	<li>Using scissors, cut and mince the muscle tissue into small pieces (less than 2 mm).</li>
	<li>Using a 10 mL serological pipette, transfer the minced muscle to a 200 mL dissociation bottle containing a magnetic bar. Repeat this step until 90 mL of 0.05%Trypsin-EDTA has been transferred with all minced muscle to the dissociation box. Close the dissociation bottle and incubate at 37 &#176;C for 60 min under mild agitation.</li>
	<li>Remove the dissociation bottle from the heating bath and stop the enzymatic reaction by adding 10 mL of fetal calf serum (FCS; 10% final volume) to the dissociation bottle. Homogenize the mixture with a pipette.</li>
	<li>Prepare two 50 mL tubes with a 70 &#181;m cell strainer on each. Pass half of the cell suspension through the cell strainer on each tube by pipetting up and down on the filter until the suspension passes through.</li>
	<li>Centrifuge the tubes at 200 x g and 20 &#176;C for 5 min; aspirate and discard the supernatant.</li>
	<li>Resuspend the cells with 10 mL of growth medium (GM; Table 1) and pass the cells through a 40-&#181;m cell strainer placed on one 50 mL tube. Centrifuge the tube at 200 x g and 20 &#176;C for 5 min; aspirate and discard the supernatant.</li>
	<li>Resuspend the cells with 10 mL of GM and centrifuge the tube at 200 x g and 20 &#176;C for 5 min; aspirate and discard the supernatant.</li>
	<li>Resuspend the cells with 1 mL of GM and count the cells using a Neubauer chamber. 
	Note: Usually, a yield of 3 x 105 cells per g of muscle should be harvested. The yield is calculated by dividing the number of cells by the weight of the muscle biopsy (step 2.4).</li>
	<li>Put 2 x 105 cells in a sterile 6-cm plate containing 4 mL of GM and incubate at 37 &#176;C and 7% CO2. Prepare as many plates as necessary.</li>
	<li>Change the GM after 3 days. 
	Note: Freshly dissociated cells need time to adhere to the culture plate. Moreover, the first myogenic cell doubling time is around 50 - 60 h. After the first division, the doubling time is around 20 h.</li>
	<li>After 5 - 7 days, when cells reach 70 - 80% confluence (around 1.5 x 106 cells/per 6 cm plate), start antibody staining and cell sorting (step 3).</li>
</ol>
3. Antibody Staining and Cell Sorting
<ol>
	<li>After rinsing once with PBS, incubate the adherent cells (in the 6-cm plate) with 1 mL of 0.05% Trypsin-EDTA at 37 &#176;C and 7% CO2 for 3 min (or incubate at 5% CO2). Stop the reaction by adding 2 mL of GM and collect the cells in 15 mL tube. After centrifugation (200 x g for 5 min), resuspend the cells in 1 mL of GM.</li>
	<li>Count the cells using a Neubauer chamber.</li>
	<li>Perform the following procedures on ice. 
	Note: The procedure for setting the correct fluorescence compensation on the cytometer is essential and should be similar on any cytometer. Tubes 1 and 2 are used as negative controls. Tubes 3 - 7 are used to set the compensation on the cytometer during the first experiment (Table 2). The final concentration used for each antibody and its corresponding isotype should be the same (around 1 &#181;g/106 cells).</li>
	<li>Dispatch 1 x 105 cells per tube (5-mL tube) for tubes 1 - 7 (Table 2). Place the remaining cell suspension in tube 8 for cell sorting.</li>
	<li>Wash the cells with 1 mL of cold FACS buffer (PBS-5% FCS). Close and centrifuge the tubes at 200 x g and 4 &#176;C for 5 min; aspirate and discard the supernatant.</li>
	<li>Add 200 &#181;L of FACS buffer per tube. Add antibodies to the tube according to Table 2. Incubate on ice for 30 min.</li>
	<li>Wash the cells: add 1 mL of the FACS buffer to each tube. Close and centrifuge the tubes at 200 x g and 4 &#176;C for 5 min. Aspirate and discard the supernatant. Repeat this step one more time.</li>
	<li>Resuspend the cells in 500 &#181;L of FACS buffer. Close the tubes and keep them on ice until cell sorting. Prepare 4 tubes of 1.5 mL, each containing 500 &#181;L of GM, which is necessary to collect the cells during cell sorting. Keep them on ice.</li>
	<li>Sort human myoblasts by flow cytometry (Figure 1). After excluding CD45-positive hematopoietic cells, separate CD45-negative cells based on CD56 expression. Then, gate the CD56-positive population for CD34, CD144, and CD146 (human myoblasts are defined as CD56+/CD146+/CD45-/CD34-/CD144- cells). Reanalyze a small fraction of the sorted cell population to check the purity.</li>
	<li>Pool the tubes containing human myoblasts in one 15-mL tube and wash one time by adding 5 mL of GM. Centrifuge the tube at 200 x g and 20 &#176;C for 5 min; aspirate and discard the supernatant.</li>
	<li>Resuspend the cells in 1 mL of GM and plate the cells on an uncoated 6-cm plate containing 4 mL of GM (2 x 105 cells per plate). Incubate at 37 &#176;C and 7% CO2.</li>
</ol>
4. Cell Maintenance
<ol>
	<li>Change half of the culture medium every 2 days. Remove 2 mL of old GM from the culture dish and add 2 mL of GM. 
	Note: Myoblasts produce growth factors that are beneficial for culture conditions.</li>
	<li>Trypsinize the cells when they reach 70 - 80% confluence to avoid pre-differentiation. After rinsing once with PBS, incubate the adherent cells (in the 6-cm plate) with 1 mL of 0.05% Trypsin-EDTA at 37 &#176;C and 7% CO2 for 3 min. Stop the reaction by adding 2 mL of GM and collect the cells in a 15 mL tube. After centrifugation (200 x g for 5 min), resuspend the cells in 1 mL of GM.</li>
	<li>Count the cells and prepare 6 cm plates with 2 x 105 cells per plate in 4 mL of GM.</li>
	<li>Keep human myoblasts up to 6 passages or freeze them in GM containing 10% DMSO, 1 x 106 cells per mL. Freeze the cells in a progressive freezing box (1 &#176;C/min); for long-term storage, place the cells in liquid nitrogen.</li>
</ol>
5. Transfection of Human Myoblasts with siRNA
Note: Myoblasts are transfected using commercial transfection reagent two days before the induction of the differentiation. The effect of specific siRNA against a gene is always compared to the effect of a control siRNA. siRNAs are double-stranded RNAs that must be kept on ice and manipulated with gloves.
<ol>
	<li>Obtain a 70 - 80% confluent monolayer of human myoblasts in a 6-cm plate (around 1.5 x 106 cells). 
	Note: 5 x 105 cells per transfection are needed to obtain a confluent monolayer of transfected myoblasts after 2 days in GM.</li>
	<li>Prepare glass coverslips (described for the calcium imaging in step 7).</li>
	<li>Warm GM, 0.05% Trypsin-EDTA, and PBS to 37 &#176;C.</li>
	<li>Perform all the following steps in a level II tissue culture hood to maintain sterility.</li>
	<li>In a 1.5-mL tube, dispatch 500 &#181;L of reduced serum medium, 3 &#181;L of transfection reagent, and 1 &#181;L of siRNA at 20 &#181;M. Mix gently and keep at room temperature (RT) for 15 - 20 min.</li>
	<li>During this incubation time, prepare the myoblasts for transfection. After rinsing once with PBS, incubate the adherent cells (in the 6-cm plate) with 1 mL of 0.05% Trypsin-EDTA at 37 &#176;C and 7% CO2 for 3 min. Stop the reaction by adding 2 mL of GM and collect the cells in a 15 mL tube. After centrifugation (200 x g for 5 min), resuspend the cells in 1 mL of GM.</li>
	<li>Count the cells using a Neubauer chamber.</li>
	<li>Resuspend 5 x 105 cells in 200 &#181;L of GM for each transfection (i.e., for 2 transfections, resuspend in 400 &#181;L).</li>
	<li>Add 200 &#181;L of the cell suspension to each 500 &#181;L of siRNA-transfectant mixture. Pipette up and down twice and incubate for 5 min at RT.</li>
	<li>Add the 700 &#181;L (cells + siRNA-transfectant) to a 3.5 cm culture dish containing a glass coverslip. Add GM to get a final volume of 2 mL.</li>
	<li>After 24 h at 37 &#176;C and 7% CO2, completely replace the medium with 2 mL of fresh GM. After another 24 h, a confluent monolayer of transfected cells is usually present; if so, induce myoblast differentiation by replacing the GM with 2 mL of differentiation medium (DM; Table 1).</li>
	<li>Perform calcium imaging experiments 24 - 48 h after the induction of differentiation or on proliferating myoblasts. 
	Note: Immunofluorescence is usually performed 48 h after the induction of differentiation.</li>
</ol>
6. Immunofluorescence Staining of Differentiation Markers
Note: Immunofluorescence staining is performed after 48 h in DM, but it can also be performed at other differentiation times. Myogenin and MEF2 are nuclear proteins only expressed in differentiated cells (before fusion into myotubes), and their staining allows for the assessment of the differentiation process. Myosin heavy chain (MyHC) is only expressed in myotubes and is used to estimate the myotube size and fusion index (number of nuclei in myotubes/total number of nuclei).
<ol>
	<li>After 48 h in DM, wash the confluent monolayer of differentiated cells twice with 1 mL of PBS at RT. Do this carefully to avoid detaching the myotubes.</li>
	<li>Fix the cells with 1 mL of 4% paraformaldehyde (PFA) for 15 min at RT. Wash the cells with PBS twice, for 5 min each.</li>
	<li>Permeabilize the cells with 1 mL of PBS containing 0.25% Triton X-100 for 45 min and wash 3 times with 1 mL of PBS.</li>
	<li>Block unspecific binding sites by incubating the cells in PBS supplemented with 0.2% Tween-10 and 2% goat serum (block solution) for 30 min at RT.</li>
	<li>Prepare primary antibodies diluted in block solution (600 &#181;L per 3.5-cm plate). 
	Note: The following primary antibodies are used: mouse anti-myogenin (1:600), rabbit anti-MEF2 (1:300), and mouse anti-MyHC antibody (MF20, 1:1,000). It is possible to mix in the same blocking solution, mouse anti-myosin heavy chain or mouse anti-myogenin, with rabbit anti-MEF2.</li>
	<li>Remove the block solution and add 600 &#181;L of primary antibody solutions per 3.5-cm plate and incubate overnight at 4 &#176;C in a humidified chamber.</li>
	<li>Wash the cells 3 times with 1 mL of PBS. Incubate for 1 h at RT in a dark chamber with 600 &#181;L of the secondary antibodies prepared in the block solution, as follows: goat anti-mouse Alexa 488 (1:1,000), goat anti-rabbit Alexa 546 (1:1,000), and DAPI (1:50, stock solution at 15 &#181;M). 
	CAUTION: DAPI is a toxic compound that should be handle with gloves.</li>
	<li>Aspirate and wash the cells 3 times with 1 mL of PBS.</li>
	<li>Mount glass coverslips using polyvinyl alcohol mounting medium with antifading solution. Keep them at 4 &#176;C and protected from light until imaging the cells.</li>
</ol>
7. Calcium Imaging
<ol>
	<li>Prior culture, prepare glass coverslips 25 mm in diameter. Put the coverslips in 70% ethanol for 48 h to remove any grease and dirt from the surface, which reduces cell adherence to the coverslip.</li>
	<li>After rinsing the coverslipsseveral times with water, put them back in water and sterilize them (autoclaved).</li>
	<li>Put one coverslip per 3.5-cm culture plate and let them dry. Store the Petri dishes for further experiments or use them immediately.</li>
	<li>Add the transfected myoblasts (5 - 6 x 105 cells) to the glass coverslip. 
	Note: Experiments are done 24 - 48 h after the induction of differentiation, or on proliferating myoblasts.</li>
	<li>To perform Ca2+ imaging, wash the cells 2 times with PBS or directly with the medium used for the experiments (calcium-containing medium; Table 3). Load the cells with 2 &#181;M Fura-2/AM plus 1 &#181;M pluronic acid for about 30 min in the dark at RT. 
	Note: Pluronic acid helps to solubilize Fura-2. Fura-2/AM and pluronic acid are dissolved in the calcium-containing medium.</li>
	<li>Wash the cells twice in the same medium but without Fura-2/AM and pluronic acid and leave them in the dark for an additional 10 - 15 min to allow for the de-esterification of Fura-2/AM.</li>
	<li>If the loading efficiency of Fura-2 is too low, add more Fura-2 (e.g., 4 &#181;M) and/or incubate the cells for a longer time (e.g., 40 min).</li>
	<li>Remove the coverslip from the culture plate and install it in the experimental chamber. Add ~1 mL of calcium-containing medium (depending upon the volume of the experimental chamber). Install it on the microscope stage and start the recording. 
	Note: The fluorescent Ca2+-sensitive probe Fura-2 is excited alternately at 340 nm and 380 nm, and the emission light is collected at 510 nm. As the fluctuations of the Ca2+ concentration are relatively slow, acquire 1 ratio every 2 s.</li>
	<li>To activate SOCE, the following classical thapsigargin/Ca2+ re-addition protocol is used: leave the cells for 2-3 min in calcium-containing medium. Replace the medium with calcium-free medium for 1-2 min.
	<ol>
		<li>Add 1 &#181;M thapsigargin to deplete the internal calcium stores. 
		Note: Thapsigargin is an irreversible blocker of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pump activity.After 8 - 10 min, rapidly replace the medium with the calcium-containing medium. Ca2+ will enter the cells through the SOCE channels. Wait around 5 min before terminating the experiments. 
		CAUTION: Thapsigargin is a toxic compound that should be handled with gloves.</li>
	</ol>
	</li>
</ol>
8. Ca2+ Measurement Analysis
Note: Perform the analysis with the acquisition software.
<ol>
	<li>Open the experiment. Draw region 1 in an area where there are no cells (background region), and draw the other regions in the cytoplasm of the cells to be analyzed (the regions can be drawn on any image: the 340-nm, the 380-nm, or the ratio image). Go to &#34;Run experiments&#34; &#62; &#34;reference images.&#34; Under the 340 and 380 channels, select &#34;region 1&#34; and then click on &#34;Subtract background.&#34;</li>
	<li>Run the entire experiment (F4: forward) to ensure that nothing interferes with the background region, such as a fluorescent dust spot that travels through the background region, and that the cells are not moving during the time of the experiments. 
	Note: The regions of interest that are selected should not include dark areas; otherwise, the ratio value will appear noisy, as part of the background will be included in the measurement.</li>
	<li>Click on &#34;Log data&#34; and play the entire experiment again. 
	Note: This will open a spreadsheet in which the time and the ratio are logged. It is also possible to save the individual 340 nm and 380 nm wavelengths.</li>
	<li>To get information about SOCE, extract the two important parameters from the experiment: the amplitude of the Ca2+ re-addition and the slope of the Ca2+ entry phase (Figure 3).</li>
	<li>Obtain the amplitude by subtracting the ratio value just before Ca2+ re-addition from the maximal amplitude of the Ca2+ elevation. Obtain the maximal slope by fitting a linear regression of the first seconds after Ca2+ re-addition. 
	Note: The area under the curve of the thapsigargin response is also an informative parameter to extract, as it reflects the amount of free calcium stored in the SR.</li>
</ol>

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The authors have no competing financial interests.

The isolation and culture of human myoblasts from adult skeletal muscle offers an in vitro model to study muscle differentiation and muscle regeneration. In this paper, we provide a protocol that allows for the purification of high yields of human myoblasts in a simple and cost-limited way. In addition, this technique provides reliable and reproducible results in terms of percentage of myoblasts isolated and their myogenic differentiation efficiency. Indeed, we obtained around 60% of human myoblasts after cell sorting. These cells can be maintained in culture for up to 6 passages (around 30 doublings) and keep their capacities to differentiate. Furthermore, myogenic differentiation occurs rapidly after changing the medium from GM to DM, as after 2 to 3 days in DM, myoblast fusion is maximal. In this model, around 60% of the myoblasts fuse into myotubes after 2 days in DM. Another advantage is that these primary human cell cultures are easily transfected with siRNA. The GM and DM media used in our laboratory are homemade. Alternatively, commercial media may be used, but we never tried their efficiency in human primary cultures.
One drawback of the method mentioned is the use of trypsin during tissue enzymatic digestion. This procedure removes a large fraction of the cell surface markers and implies that we leave the cells in culture for some days before performing FACS . Collagenase could be used instead of trypsin to better preserve the membrane proteins and allow for the sorting of human myoblasts immediately after tissue dissociation. However, the cost of collagenase is significantly higher than trypsin, and an amplification step would still be required after cell sorting to obtain sufficient cell number for the subsequent experiments. Another limitation of the technique is the &#34;quality&#34; of the muscle samples obtained from orthopedic surgery. Indeed, the pieces of tissue obtained could be more or less damaged due to surgery, and the time between the surgery and the cell dissociation in the laboratory can vary from day to day. However, for myoblast isolation, purification, and differentiation into myotubes, we routinely obtain satisfactory results. Finally, the age of the patient can be an issue, as the amount and regenerative capacity of satellite cells decline with age20. Thus, we restrict our experiments to young patients (less than 40 years old).
As mentioned, myoblasts differentiate rapidly and efficiently, which is a great advantage of studying the initial events of skeletal muscle differentiation. However, as myotubes are formed rapidly, they also have a tendency to spontaneously contract and detach from the culture plates. This precludes the analysis of the later fusion events. To study these later events, one would need to coat the culture plate with extracellular matrix to improve the cell adherence and to decrease myotube detachment21.
Calcium imaging using the fluorescent probe Fura-2 is a widely used, reproducible technique that is easy to perform. The loading of Fura-2 is efficient, and the use of a ratiometric probe such as Fura-2 has several advantages over a single-wavelength dye. It normalizes changes of focus or differences in the loading between cells and within a cell (e.g., nucleus versus cytosol). The protocol we described to elicit and measure SOCE is regularly used in many different cellular systems. The silencing of different channels or calcium regulators permits to elucidation of the molecular composition of SOCE and the relative importance of the different players, both in myoblasts and in myotubes. An alternative approach using Fura-2 and Ca2+ imaging is the application of the Mn2+ quench technique. Mn2+ enters the cells through Ca2+-permeable channels and quenches the fura-2 fluorescence. The measure of the quench rate gives a more direct measure of the SOCE22,23. This, however, requires the utilization of slightly different equipment. Ca2+ signaling could also be performed using genetically encoded Ca2+ indicators, which have the great advantage of being precisely localized in different cell compartments. The drawbacks are the requirement for cell transfection and the dynamics of the signal, which is usually less than with chemical dyes24. To measure Ca2+ in organelles like mitochondria or the SR, the genetic probes are the gold standard. However, for cytosolic Ca2+ measurements, the benefit of using Fura-2 over genetic probes remains.

Human skeletal muscles are composed of groups of contractile, multinucleated muscle fibers resulting from the fusion of myogenic precursor cells. Skeletal muscles have the capacity to regenerate after injury thanks to the presence of SCs, the skeletal muscle stem cells located between the plasma membrane of myofibers (sarcolemma) and the basal lamina. In uninjured muscle, SCs are mostly present in a quiescent state. In response to mechanical stress or injury, SCs become activated (myoblasts), proliferate, and undergo either differentiation to form new myofibers or self-renewal to replenish the SC pool1,2. Over the years, several techniques were developed to isolate SCs and their progeny, the myoblasts, from skeletal muscle biopsies. With the greater understanding of the cell surface markers expressed on these cells and the methodology of fluorescence-activated cell sorting (FACS)3,4,5, it is now possible to isolate pure populations of human myoblasts from muscle samples.
Calcium signaling regulates skeletal muscle development, homeostasis, and regeneration. In particular, the Ca2+ entry that is activated following intracellular store depletion, called SOCE, is of great importance6 for these processes. Upon cell stimulation, the Ca2+ level in the endo/sarcoplasmic reticulum (ER/SR) decreases, which in turn activates plasma membrane Ca2+ channels that permit Ca2+ entry to refill the ER/SR7. The two main proteins involved in SOCE are the ER/SR Ca2+-sensing stromal interaction molecule 1 (STIM1) protein and the plasma membrane Ca2+ channel Orai1. Skeletal muscle abundantly expresses these two proteins, as well as other proteins of the same families (STIM2 and Orai2-Orai3)8,9 and several Ca2+-permeable channels of the transient receptor potential canonical (TRPC) family10,11,12,13. Ca2+ entry through the SOCE pathway is of great importance for muscle formation/regeneration14. Mutations of STIM1 or Orai1 proteins have deleterious effects on muscle function, leading mainly to muscle hypotonia15. Animal models with SOCE impairment also display reduced muscle mass and force, together with enhanced fatigability6,16,17. As mentioned, other STIM and Orai proteins, as well as many TRPC channels, are expressed in skeletal muscle, and their respective roles have not been determined so far. By knocking down their expression level, it is thus possible to investigate their implications in SOCE and their roles during human skeletal muscle differentiation.
SOCE measurement can be performed using two different approaches: electrophysiological current recordings and Ca2+ measurements. Electrophysiology is certainly a more direct method, as it measures the current of interest and its associated electrophysiological signature. However, this technique is very difficult to apply to muscle cells, mainly because of the large size of the muscle cells and the small size of the endogenous SOCE current. Cytosolic Ca2+ imaging is technically very accessible, but the measure is more indirect, as the Ca2+ level measured in the cytosol reflects both the entry of Ca2+ and the re-pumping out of the cell or in the internal stores.
A methodology that includes isolating myoblasts from small pieces of human skeletal muscle after enzymatic digestion, amplification, and FACS is explained in this paper. The process of muscle differentiation and the immunofluorescence protocol to follow the expression of differentiation markers over time are described18. Finally, the measurement of SOCE and the role of different ion channels in Ca2+ signaling and skeletal muscle differentiation are explained.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Ham&#8217;s F10</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">31550-023</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">FCS</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">10270-106</td>
			<td style="width:201px;">Lot 41G5532K</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">BSA</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">O5482</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">fetuin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">F3385</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">EGF</td>
			<td style="width:235px;">Corning</td>
			<td style="width:187px;">354001</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Dexamethansone</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">D1756</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Insulin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">I9278</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Creatine</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">27900</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Pyruvate</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">P4562</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Uridine</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">U3003</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Gentamycin</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">15710-049</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Trypsin-EDTA 0.05%</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">25300-062</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">70 &#956;m cell strainer</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352350</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">40 &#956;m cell strainer</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352340</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 50&#160;ml tube</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352070</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 15&#160;ml tube</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352096</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Falcon 5&#160;ml tube (FACS)</td>
			<td style="width:235px;">Falcon</td>
			<td style="width:187px;">352058</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Culture medium: OPTI-MEM</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">31985-047</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Transfectant reagnt: RNAiMax</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">1756064</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PBS</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">14190-094</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mounting medium</td>
			<td style="width:235px;">Fluka</td>
			<td style="width:187px;">10981</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti-MyHC *</td>
			<td style="width:235px;">DSHB</td>
			<td style="width:187px;">MF20&#160;</td>
			<td style="width:201px;">concentrate</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti-myogenin</td>
			<td style="width:235px;">BD Pharmigen</td>
			<td style="width:187px;">556358</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Rabbit anti-MEF2</td>
			<td style="width:235px;">Santa Cruz Biotech</td>
			<td style="width:187px;">C-21</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Goat anti-mouse Alexa488</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">A11029</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Goat anti-rabbit Alexa 546</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">A11035</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD56-Alexa488</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557699</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD146-PECy7</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562135</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD45-PE-CF594</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562279</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD34-APC</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">345804</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Mouse anti human CD144-PE</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">560410</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Alexa Fluor 488 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557702</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PECy7 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">557872</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PE-CF594 mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">562292</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">APC mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">550854</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">PE mouse IgG1k</td>
			<td style="width:235px;">BD Pharmingen</td>
			<td style="width:187px;">556650</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">DAPI</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">D9542</td>
			<td style="width:201px;">CAUTION:&#160; toxic compound</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Paraformaldehyde</td>
			<td style="width:235px;">Fluka</td>
			<td style="width:187px;">762400</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Fura-2 AM</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">F1201</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Pluronic F-127</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">P3000MP</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">Thapsigargin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">T9033</td>
			<td style="width:201px;">CAUTION&#160;: toxic compound&#160;</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">Ratio imaging acquisition software: Metafluor 6.3</td>
			<td style="width:235px;">Molecular Device</td>
			<td style="width:187px;"></td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="24">
			<td colspan="2" height="24" style="height:24px;">*&#160;: MF 20 was deposited to the DSHB by Fischman, D.A.&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

After the enzymatic dissociation of a human muscle sample, cells were amplified in GM. Human myoblasts, defined as CD56+/CD146+/CD45-/CD34-/CD144- cells, were obtained after FACS. Myoblasts represented more than 60% of the analyzed population (Figure 1). Primary human myoblasts were replated, grown to confluence, and cultured in DM for 48 h. After immunostaining for the expression of the transcription factor MEF2 and the muscle-specific protein myosin heavy chain (MyHC), we observed that a majority of cells formed myotubes in vitro (MyHC-positive cells), with fusion index values of 60.9 &#177; 1.3% (n = 5; Figure 2). We also observed that around 40% of human myoblasts, called reserve cells, escaped this terminal differentiation and remained undifferentiated mononucleated cells. For functional studies, we analyzed the Ca2+ response of myoblasts or myotubes 48 h after the initiation of differentiation. Figure 3 shows a representative protocol used to induced SOCE and the key parameters to extract from such a recording. To discern information related to the molecules involved in SOCE, cells were transfected with siRNA against the molecule of interest. Figure 4 shows the impact of silencing Ca2+-permeable channels of the TRPC family on SOCE in myoblasts.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55918/55918fig1.jpg" /> 
Figure 1: FACS of Human Myoblasts. Dissociated human cells were immunostained with antibodies for the following cell surface markers: CD45, CD56, CD34, CD144, and CD146. After excluding CD45-positive hematopoietic cells, CD45-negative cells were separated based on CD56 expression. The CD56-positive population was then gated for CD34, CD144, and CD146. Human myoblasts were defined as CD56+/CD146+/CD45-/CD34-/CD144- cells, as previously described19,4. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55918/55918fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55918/55918fig2.jpg" /> 
Figure 2: Human Myoblast Differentiation. (A) A confluent monolayer of human myoblasts was exposed to DM for 48 h; fixed; and stained with antibodies against MEF2 (red), MyHC (green), and DAPI (blue) to identify nuclei. Scale bar = 50 &#181;m. (B) The fusion index represents the number of nuclei incorporated in myotubes divided by the total number of nuclei on a certain field. Data are mean &#177; SEM of 5 independent experiments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55918/55918fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55918/55918fig3.jpg" /> 
Figure 3: Representative Calcium Measurement of Thapsigargin-induced SOCE. Myotubes were loaded with Fura-2 and the cytosolic Ca2+ concentration was measured. (A) The cells sit for a few minutes in a calcium-containing medium, followed by 2 min in a calcium-free medium. 1 &#181;M thapsigargin is then used for 8 min, followed by the calcium re-addition. The 3 important parameters to extract from such recording are: the area under the curve of the thapsigargin response, the slope of the SOCE, and the amplitude of the SOCE. (B) Quantification of the 3 parameters . The area under the curve provides information about the amount of Ca2+ stored in the ER/SR, and the two other parameters allow for the quantification of the SOCE. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55918/55918fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55918/55918fig4.jpg" /> 
Figure 4: Involvement of TRPC1 and TRPC4 in SOCE. (A) Cytosolic Ca2+ concentration was assessed using Fura-2 in proliferating myoblasts transfected with siTRPC1 or siTRPC1 and siTRPC4. SOCE was triggered by internal store depletion with 1 &#181;M thapsigargin in the absence of external Ca2+ (Ca2+ free) and measured during the re-addition of 2 mM external Ca2+. Each trace represents the mean of a representative coverslip. (B) Quantification of the effects of siRNA on SOCE amplitude. This figure has been modified from reference12, with permission. Bars are mean &#177; SEM, *p &#60;0.05. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55918/55918fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Growth medium (GM):</td>
			<td colspan="2">Concentration</td>
			<td></td>
			<td colspan="2">Differentiation medium (DM):</td>
			<td colspan="2">Concentration</td>
		</tr>
		<tr>
			<td colspan="2">Ham&#39;s F10</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">DMEM</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td colspan="2">FCS</td>
			<td colspan="2">15%</td>
			<td></td>
			<td colspan="2">bovine serum albumin</td>
			<td colspan="2">0.5 mg/ml</td>
		</tr>
		<tr>
			<td colspan="2">bovine serum albumin</td>
			<td colspan="2">0.5 mg/ml</td>
			<td></td>
			<td colspan="2">epidermal growth factor</td>
			<td colspan="2">10 ng/ml</td>
		</tr>
		<tr>
			<td colspan="2">fetuin</td>
			<td colspan="2">0.5 mg/ml</td>
			<td></td>
			<td colspan="2">insulin</td>
			<td colspan="2">0.01 mg/ml</td>
		</tr>
		<tr>
			<td colspan="2">epidermal growth factor</td>
			<td colspan="2">10 ng/ml</td>
			<td></td>
			<td colspan="2">creatine</td>
			<td colspan="2">1 mM</td>
		</tr>
		<tr>
			<td colspan="2">dexamethasone&#160;</td>
			<td colspan="2">&#160;0.39 &#181;g/ml</td>
			<td></td>
			<td colspan="2">pyruvate</td>
			<td colspan="2">100 &#181;g/ml</td>
		</tr>
		<tr>
			<td colspan="2">insulin</td>
			<td colspan="2">0.04 mg/ml</td>
			<td></td>
			<td colspan="2">uridine</td>
			<td colspan="2">50 &#181;g/ml</td>
		</tr>
		<tr>
			<td colspan="2">creatine</td>
			<td colspan="2">1 mM</td>
			<td></td>
			<td colspan="2">gentamycin&#160;</td>
			<td colspan="2">10 &#181;g/ml</td>
		</tr>
		<tr>
			<td colspan="2">pyruvate</td>
			<td colspan="2">100 &#181;g/ml</td>
			<td></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td colspan="2">uridine</td>
			<td colspan="2">50 &#181;g/ml</td>
			<td></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td colspan="2">gentamycin&#160;</td>
			<td colspan="2">5 &#181;g/ml</td>
			<td></td>
			<td colspan="2"></td>
			<td colspan="2"></td>
		</tr>
	</tbody>
</table>
Table 1: Medium Composition for Myoblast Culture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tubes</td>
			<td colspan="6">Antibodies</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="6">None</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="6">Isotype AlexaFluor&#160;488-, Isotype PE-CF594, Isotype PE, Isotype PECy7Isotype&#160;APC</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="6">Mouse anti-human&#160;CD56-AlexaFluor&#160;488</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="6">Mouse anti-human&#160;CD45 PE-CF594</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="6">Mouse anti-human&#160;CD144-PE</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="6">Mouse anti-human CD146-PECy7</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="6">Mouse anti-human&#160;CD34-APC</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="6">Mouse anti-human&#160;CD56-AlexaFluor&#160;488-, Mouse anti-human CD45 PE-CF594, Mouse anti-human CD144-PE, Mouse anti-human&#160;CD34-APC, Mouse anti-human&#160;CD146-PECy7</td>
		</tr>
	</tbody>
</table>
Table 2: Antibodies used for the Cell Sorting.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Calcium-containing medium</td>
			<td colspan="2">concentration</td>
			<td></td>
			<td colspan="2">Calcium-free medium</td>
			<td colspan="2">concentration</td>
		</tr>
		<tr>
			<td colspan="2">NaCl</td>
			<td colspan="2">135 mM</td>
			<td></td>
			<td colspan="2">NaCl</td>
			<td colspan="2">135 mM</td>
		</tr>
		<tr>
			<td colspan="2">KCl</td>
			<td colspan="2">5 mM</td>
			<td></td>
			<td colspan="2">KCl</td>
			<td colspan="2">5 mM</td>
		</tr>
		<tr>
			<td colspan="2">MgCl2</td>
			<td colspan="2">1 mM</td>
			<td></td>
			<td colspan="2">MgCl2</td>
			<td colspan="2">1 mM</td>
		</tr>
		<tr>
			<td colspan="2">CaCl2</td>
			<td colspan="2">2 mM</td>
			<td></td>
			<td colspan="2">EGTA</td>
			<td colspan="2">1 mM</td>
		</tr>
		<tr>
			<td colspan="2">Hepes</td>
			<td colspan="2">10 mM</td>
			<td></td>
			<td colspan="2">Hepes</td>
			<td colspan="2">10 mM</td>
		</tr>
		<tr>
			<td colspan="2">Glucose</td>
			<td colspan="2">10 mM</td>
			<td></td>
			<td colspan="2">Glucose</td>
			<td colspan="2">10 mM</td>
		</tr>
		<tr>
			<td colspan="2">pH 7.45 adjusted with NaOH</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">pH 7.45 adjusted with NaOH</td>
			<td colspan="2"></td>
		</tr>
	</tbody>
</table>
Table 3: Medium Composition for Calcium Imaging Experiments.

Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

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

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 ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

10.0K Views.  Geneva University Hospitals & Faculty of Medicine. 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.

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

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 ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very important to obtain adult stem cells from different tissues such as adipose, umbilical cord, bone marrow and blood. Among the possible sources, dental pulp is particularly interesting because it is easy to obtain in respect of bioethical considerations. Indeed, human Dental Pulp Stem Cells (hDPSCs) are a type of adult stem cells able to differentiate in neuronal-like cells and can be obtained from the third molar of healthy patients (13-19 ages). In particular, the dental pulp was removed with an excavator, cut into small slices, treated with collagenase IV and cultured in a flask. To induce the neuronal differentiation, hDPSCs were stimulated with EGF/bFGF for 2 weeks. Previously, we have demonstrated that during the differentiation process the content of cellular prion Protein (PrPC) in hDPSCs increased. The cytofluorimetric analysis showed an early expression of PrPC that increased after neuronal differentiation process. Ablation of PrPC by siRNA PrP prevented neuronal differentiation induced by EGF/bFGF. In this paper, we illustrate that as we enhanced the isolation, separation and in vitro cultivation methods of hDPSCs with several easy procedures, more efficient cell clones were obtained and large-scale expansion of the mesenchymal stem cells (MSCs) was observed. We also show how the hDPSCs, obtained with methods detailed in the protocol, are an excellent experimental model to study the neuronal differentiation process of MSCs and subsequent cellular and molecular processes.

Here we present a protocol for human Dental Pulp Stem Cells isolation and propagation in order to evaluate the prion protein expression during neuronal differentiation process.

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...