Name: isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement
Uploaded: 2017-07-26T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

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...

<ol>
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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...

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.

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			<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>
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			<td height="25" style="height:25px;width:321px;">Gentamycin</td>
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			<td height="25" style="height:25px;width:321px;">Trypsin-EDTA 0.05%</td>
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			<td height="25" style="height:25px;width:321px;">70 &#956;m cell strainer</td>
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			<td style="width:235px;">Falcon</td>
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			<td height="25" style="height:25px;width:321px;">Falcon 5&#160;ml tube (FACS)</td>
			<td style="width:235px;">Falcon</td>
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			<td height="25" style="height:25px;width:321px;">Culture medium: OPTI-MEM</td>
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			<td height="25" style="height:25px;width:321px;">Mouse anti-MyHC *</td>
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			<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 m...

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 ...

The overall goal of this procedure is to obtain a pure population of human myoblasts from adult skeletal muscle. These cells are used for functional studies such as assessment of myogenic differentiation and measurement of store-operated calcium entry. The main advantage of this technique is that it allows a rapid, simple and reproducible isolation of human primary myoblasts.These myoblasts are the new ex-vivo to study the calcium signaling during muscle regeneration. This method can help answer key questions in the muscle field such as, identifying the ionic channels and the transcription factors involved during muscle differentiation. For this method can provide insight into gene functions during human myogenic differentiation.It can also be applied to other studies such as, potential for using myogenic stem cells to treat muscular dystrophies. Transfer the muscle sample into a sterile six centimeter plate, wash the sample with PBS twice. Remove any remaining adipose tissue and weigh the sample.Next, add trypsin and mince the muscle into pieces smaller than two millimeters in size. Once minced, use a 10 milliliter pipette to transfer the tissue into a 200 milliliter dissociation bottle containing a stir bar, then continue collecting minced tissue in trypsin solution until the dissociation bottle is filled to 90 milliliters. Now, incubate the tissue in a water bath at 37 degrees celsius for 60 minutes with gentle stirring.After an hour, add 10 milliliters of FCS to stop the reaction and homogenize the mixture using trituration. Now, load 70 micron strainers onto two 50 milliliter tubes and pass half of the cell suspension through each strainer by pipetting the suspension into the filters. Next, make a suspension of the cells in growth medium and plate 200, 000 cells onto six centimeter plates with four milliliters of medium.Incubate the plates, changing the medium every third day. After five to seven days, the cells should be 70%to 80%confluent, then, stain them. To begin, rinse the plate of confluent cells with PBS.Then, add one milliliter of trypsin solution and let the enzyme work for three minutes. Stop the reaction by adding two milliliters of medium and collect the cells in a 15 milliliter tube. Then, spin the cells down and re-suspend them in one milliliter of medium.Next, take a cell count while storing the cells on ice. For the facts analysis, load seven tubes with 100, 000 cells each. Load the remaining cells into an eighth tube for sorting.Wash the cells with one milliliter of cold fax buffer, then, centrifuge the tubes and aspirate and discard the supernatant. Add back 200 microliters of fax buffer with the appropriate antibodies and let the cells incubate on ice for 30 minutes. After 30 minutes, wash the cells again and re-suspend them in 500 microliters of fax buffer, then sort the cells using a flow cytometer.After excluding the CD45 positive hematopoietic cells, separate the CD45 negative cells based on CD56 expression. Then, gate the CD56 positive population for CD34, CD144 and CD146. Re-analyse a fraction of the sorted population, to check the purity.Now, pull the human myoblasts and wash them with five milliliters of medium. Then re-suspend them in one milliliter of medium. Transfer the suspension to a six centimeter plate with four milliliters of medium and incubate the plate at 37 degrees celsius with 7%carbon dioxide.For this procedure, have cultured myoblasts and make fresh transvection medium. Incubate the medium at room temperature for 15 to 20 minutes. During the medium incubation, prepare the myoblasts.First, rinse them once with PBS. Then, trypsinise the adherent cells. Stop the reaction by adding two milliliters of medium.Then, collection the cells into a 50 milliliter tube. Spin them down and re-suspend them in one milliliter of medium. For each transvection, prepare 500, 000 cells in 200 microliters of medium.To each 500 microliters of transvection medium, add a 200 microliter aliquot of cells and then mix using only two strokes of the pipette gun. Then, let the reactions proceed for five minutes at room temperature. After five minutes, transfer the reactions to 3.5 centimeter culture dishes, each containing one glass cover slip.Next, add 1.3 milliliters of medium for a final volume of two milliliters and incubate the dishes. After 48 hours of differentiation, the cells can be immunostained or analyzed using calcium imaging. For either option, first carefully wash the cells twice using one milliliter of room temperature PBS to wash.Try to avoid detaching the myotubes. For a calcium imaging, cover the washed cells with two micromolar fura-2 AM plus one micromolar threonic acid in calcium containing medium. Then, let them incubate for about 30 minutes before mixing.Next, wash the cells twice with unmodified calcium containing medium and then, incubate them for 10 to 15 minutes for the esterification. Now, remove the cover slip from a culture plate and install it into the experimental chamber. Load the chamber with calcium containing medium, place it under a microscope, then, start the data recording.Fura-2 is excited alternately at 340 and 380 nanometers. The emission light is collected at 510 nanometers. Diving the 340 nanometer nanometer images by the 380 images, provides the image ratios as the fluctuations and calcium concentration are relatively low, acquire one image ratio every two seconds.After two or three minutes, replace the medium with calcium free medium. Then, for one or two minutes, monitor the activity of the store operated calcium in the cells. Next, add one micromolar thapsigargin to the cells to deplete their internal calcium stores.Wait eight to 10 minutes, then, rapidly replace the medium with the calcium containing medium and record for five minutes while calcium enters the cells via the suc e channels. After terminating the experiment, analyze the images. Select an area where there are no cells for region one, which defines the background, then, select an area in the cytoplasm for region two.The two regions can be drawn on any of the images in the series. Next, go to the run experiments tab and select reference images for the 340 and 380 channels, select region one, then click on subtract background. Now, run the entire experiment to remove the background from all the images.Once completed, click log data option to open a spreadsheet in which the time and the ratio data are logged. Muscle cells were grown up, expanded and sorted using fax. Myoblasts represented more than 60%of the analyzed population.The primary human myoblasts were then cultured in differentiation medium and immunostained for the transcription factor of MEF2 and myosin heavy chain proteins. A majority of cells expressed myosin heavy chain and formed myotubes with fusion index values of about 60%the other 40%of the cells remained undifferentiated mononucleated cells, also known as reserve cells. The described store operated calcium entry experiment were successfully performed on the differentiated cells.Performing the same experiment to on cells transvected with small interfering RNA, directed against calcium permeable channels of the TRPC family, shows that the knockdown had a measurable impact on store operated calcium entry. After watching this video, you should have a good understanding of how to isolate human myoblasts from adult skeletal muscle. We use these cells to study the successive steps of muscle differentiation and in particular, deactivation of the transcription factors and calcium channels.Once mastered, this technique can be done in less than three hours if it is performed properly. It is a rapid, simple, reproducible method that obtains the yield of human myoblasts. While attempting this procedure, it is important to prevent the myoblast cultures from reaching full confidence, as there will be unwanted spontaneous differentiation.

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