Name: Purification of Progenitors from Skeletal Muscle (Video) | JoVE Journal
Uploaded: 2011-03-16T13:00:00.000+00:00
Duration: 12 min 55 s
Description: Learn to isolate viable myogenic and adipogenic progenitor cells from murine skeletal muscle using enzymatic dissociation and flow cytometry. This method enables direct analysis of cell populations during tissue regeneration.

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5 minutes

1. Tissue Collection:
<ol>
<li>Use mice between 7 and 12 weeks old. Usually, at least two animals are used: one is to set up the FACS single-color controls required for compensation and for fluorescence-minus-one isotype control samples. The other is the actual sample for sorting.</li>
<li>Euthanize the mice by CO2 inhalation or according to an ethics protocol of choice. Place mice on a paper towel, facing up, and spray with 70% ethanol thoroughly to prevent contamination with the mouse fur.</li>
<li>Lift the skin in the abdominal region with forceps, then make a small longitudinal cut with sharp scissors.</li>
<li>Peel the two flaps of skin in opposite directions (up and down) to completely expose the hind limb muscles underneath.</li>
<li>Excise the muscle tissue by inserting and extending the scissors along both sides of the tibia.</li>
<li>Repeat the same process for the femur to extract the muscle tissue.</li>
<li>Place all excised muscle tissue in 60mm petri-dishes, using one dish for the tissue from each mouse.</li>
<li>Repeat the above for all mice.</li>
</ol>
2. Dissociate Tissue Into Single Cells by Enzymatic Digestion:
Throughout this section of the protocol, use sterile reagents and tools and work under sterile conditions.
<ol start="9">
<li>Add 2 mL of Collagenase II ( Sigma, cat#6885, 500u/ mL) and 20 μL of 250mM CaCl2 stock to each Petri-dish.</li>
<li>Tear the muscle tissue into small pieces ( about 1 mm3) with two forceps (one serrated, one with 2 teeth, from Fine Science Tools , cat#11051-10, 11154-10), paying attention to remove and discard as much contaminated non-muscle tissue as possible.</li>
<li>Cover the Petri-dishes and incubate them at 37 degrees Celsius for 30 minutes.</li>
<li>Retrieve the Petri-dishes and use a 3 mL syringe plunger to thoroughly mash the tissue pieces. Use one plunger for each sample to avoid contaminations.</li>
<li>Add some (~ 5 mL) sterile cold PBS to each Petri-dish and transfer the tissue sludge into a 50mL Falcon tube (rinse the Petri-dish if necessary to recover all the tissue)</li>
<li>Shake the Falcon tube vigorously, fill the tube with PBS, centrifuge @800rpm x 5 minutes ( Eppendorf bench top Model :5810R), and aspirate the supernatant (repeat 3 times)</li>
<li>Add 1mL mixture of Collagenase D (Roche, Cat# 11088882001, 1.5u/ mL) /Dispase II (Roche, Cat# :04942078001, 2.4u/ mL) and 10μL of CaCl2 (250mM) to each tube, incubate at 37 degrees Celsius with rotation for one hour. </li>
<li>Add 5-10 mL of cold, sterile PBS, vortex and pipette thoroughly to homogenize the tissue.</li>
<li>Fill up tubes with PBS and mix well.</li>
<li>Transfer the liquid to a 40μm cell strainer placed on top of a 50 mL Falcon tube to filter out the remaining large pieces of tissue. Divide each sample evenly into three 50mL Falcon tubes. Fill up each of the tubes with PBS then centrifuge @1600rpm for 5 minutes at 8 degrees Celsius.</li>
<li>Discard the supernatant and collect cells for staining.</li>
</ol>
3. Antibody Staining and Sorting:
Throughout this section of the protocol, use sterile FACS and collection buffers and work under sterile conditions. Commercial antibodies are generally considered sterile if properly handled.
<ol start="20">
<li>For single color and isotype control samples, resuspend the sample in 1mL FACS buffer and divide the suspended cells in nine 1.5mL pre-labelled Eppendorf tubes (100 μL each tube).
<ol>
<li>Set up single color staining mixes according to the following table: 
ATTENTION: The final concentration of isotype control antibodies in the stain should match that of the primary antibody they are the control for. For example, if anti-Sca-1 is used for 1 μg/106 cells, its corresponding isotype must be used at the same concentration. 
<table border="1">
 <tbody>
 <tr>
 <td>Tube #</td>
 <td>Name of tube</td>
 <td>Company</td>
 <td>Cat#</td>
 <td>clone</td> 
 <td>isotype</td>
 <td>dilution</td>
 </tr>
 <tr>
 <td>1</td>
 <td>Non-stain</td>
 <td> </td>
 <td> </td>
 <td> </td>
 <td> </td>
 <td> </td>
 </tr>
 <tr>
 <td>2</td>
 <td>Hoechst33342</td>
 <td>sigma</td>
 <td>B2261</td>
 <td> </td>
 <td> </td>
 <td>2-5ug/ml</td>
 </tr>
 <tr>
 <td>3</td>
 <td>PI</td>
 <td>sigma</td>
 <td>P4864</td>
 <td> </td>
 <td> </td>
 <td>1ug/ml</td>
 </tr>
 <tr>
 <td rowspan="2">4</td> 
 <td rowspan="2">CD31-FITC CD45-FITC</td> 
 <td>ebioscience</td>
 <td>11-0311-85</td>
 <td>390</td>
 <td>Rat IgG2a</td>
 <td>500</td>
 </tr>
 <tr>
 <td>House made</td>
 <td> </td>
 <td>I3/2</td>
 <td>Rat IgG2b</td>
 <td>500</td>
 </tr>
 <tr>
 <td>5</td>
 <td>Sca1-PECY7</td>
 <td>ebioscience</td>
 <td>25-5981-82</td>
 <td>D7</td>
 <td>Rat IgG2a</td>
 <td>5000</td>
 </tr>
 <tr>
 <td>6</td>
 <td>α-7 APC</td>
 <td>House made</td>
 <td> </td>
 <td>R2F2</td>
 <td>Rat IgG2b</td>
 <td>1000</td>
 </tr>
 </tbody>
</table>
</li>
<li>Set up isotype control staining mixes using the following table: 
 <table border="1">
<tbody>
 <tr>
 <td>Tube #</td>
 <td>Name of tube</td>
 <td>Hoechst</td>
 <td>PI</td>
 <td>CD31- FITC</td>
 <td>CD45- FITC</td>
 <td>Scal- PE-CY7</td>
 <td>a-7- APC</td>
 <td>Rat- IgG2b- APC</td>
 <td>Rat- IgG2a- pecy7</td>
 <td>Rat- IgG2a- FITC</td>
 <td>Rat- IgG2b- FITC</td>
 </tr>
 <tr>
 <td>7</td>
 <td>Iso- CD31 /CD45</td>
 <td>+</td>
 <td>+</td>
 <td>-</td>
 <td>-</td>
 <td>+</td>
 <td>+</td>
 <td>-</td>
 <td>-</td>
 <td>+</td>
 <td>+</td>
 </tr>
 <tr>
 <td>8</td>
 <td>Iso- Scal- pecy7</td>
 <td>+</td>
 <td>+</td>
 <td>+</td>
 <td>+</td>
 <td>-</td>
 <td>+</td>
 <td>-</td>
 <td>+</td>
 <td>-</td>
 <td>-</td>
 </tr>
 <tr>
 <td>9</td>
 <td>Iso- α-7-APC</td>
 <td>+</td>
 <td>+</td>
 <td>+</td>
 <td>+</td>
 <td>+</td>
 <td>-</td>
 <td>+</td>
 <td>-</td>
 <td>-</td>
 <td>-</td>
 </tr>
 </tbody>
</table>
</li>
<li>Pipet the single color and isotope control staining mixes (we usually adjust the concentration of antibodies so that between 5 and 10 μL of staining mix are added to each sample) into the appropriate Eppendorf tube from Step 20. Mix well and incubate on ice for 1 hour.</li></ol></li>
<li>Stain cells from sample mouse with 800μL of the antibody cocktail (below) for FACS cell sorting. Mix well and incubate on ice for 1 hour. 
Antibody cocktail: Hoechst, PI, CD31-FITC, CD45-FITC, Sca1-PECY7, α-7 APC 
ATTENTION: The optimal ratio of antibodies to cells must be determined by individually titrating each antibody, as significant differences may exist between batches. The optimal amount of antibody to be used should be expressed in μgs of antibody per 106 target cells and kept constant when using the same batch of antibody. Clearly, if the number of target cells does not change significantly between experiments, or if the staining volume is scaled with the number of cells, a fixed dilution of each antibody can also be used.</li>
<li>Wash: Add approximately 1mL of FACS buffer to each of the 9 control tubes; add approximately 20mL of FACS buffer to the sample tube.</li>
<li>Centrifuge the Eppendorf tubes @ 3000rpm for 5 minutes ( Eppendorf bench top centrifuge, Model:5417C ). Centrifuge the sample tube @1600rpm for 5 minutes (Eppendorf bench top centrifuge, Model:5810R).</li>
<li>Aspirate and discard the supernatant from all the tubes.</li>
<li>Resuspend cells in the FACS buffer (1mL for each of the controls, 4mL for the sorting sample), Pipette cells into "FACS tubes" through the cell strainer cap (BD Falcon cat# 352235, 40μm pore size) to remove remaining clumps that may affect the cytometer.</li>
<li>Set up the FACS Sorter with single color controls and isotype controls.</li>
<li>Collect sorted cells according to the following table in a 3.5 mL collection buffer (95% DMEM, 5%FBS) separately. Normally, when harvesting both hind limbs, a single wild type mouse could yield about 150,000 myogenic progenitor cells or 100,000 adipogenic progenitor cells, with the viability above 95%, as judged by reanalyzing sorted cells by FACS at the end of the procedure (Figure 1) 
Definition of myogenic and adipogenic progenitors based on surface staining 
 <table border="1">
<tbody>
 <tr>
 <td>Antibody/stain </td>
 <td>Myogenic progenitor (MP)</td>
 <td>Adipogenic progenitor (AP)</td>
 </tr>
 <tr>
 <td>Hoechst</td>
 <td>+</td>
 <td>+</td>
 </tr>
 <tr>
 <td>PI</td>
 <td>-</td>
 <td>-</td>
 </tr>
 <tr>
 <td>CD31-FITC</td>
 <td>-</td>
 <td>-</td>
 </tr>
 <tr>
 <td>CD45-FITC</td>
 <td>-</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Sca1-PECY7</td>
 <td>-</td>
 <td>+</td>
 </tr>
 <tr>
 <td>α-7 APC</td>
 <td>+</td>
 <td>-</td>
 </tr>
 </tbody>
</table>
</li>
</ol>
4. Transplantation:
<ol start="28">
<li>Centrifuge sorted cells @1500rpm for 5 minutes, remove the supernatant and transfer cells to autoclaved Eppendorf tubes (1.5 mL),
rinse cells with 500 μL of sterile PBS (serum free!), then centrifuge @ 3000rpm for 5 minutes. Maintaining all reagents in sterile conditions and working in a tissue culture hood, resuspend myogenic progenitors in PBS, or adipogenic progenitor in Matrigel ( BD cat# 354234) at approximately 106 cells /mL.</li>
<li>Transplant myogenic progenitors or adipogenic progenitor to recipient mice.
<ol>
<li>Anaesthetize the mouse with isoflouran according to your institution policy.</li>
<li>Shave the hair around the injection region, then sterilize the skin by rubbing with 70% ethanol</li>
<li>Inject 20μL of myogenic progenitors or adipogenic progenitors (= 20k cells) using a 3/10 CC insulin syringe ( BD cat# 309300).</li></ol></li>
<li>After an appropriate time (three weeks works well, but myofibers expressing donor-derived transgenic markers are usually evident within one week after transplant), anaesthetize the recipient mice from step 29 by intraperitoneal injection of 400 μL Avertin (Sigma Cat# T04840-2, 25 mg/ mL), then perfuse transcardially with 50mL PBS (contains 10 μM of EDTA) first, followed by 15mL of 4% PFA. Collect target tissue, post-fix with 4% PFA overnight if required, then transfer to 20% sucrose overnight. Embed in OCT, freeze and cryosection.</li>
</ol>
5. Representative Results:
When MP are transplanted into mouse skeletal muscle, they readily fuse with pre-existing myofibers. Therefore any genetic label present in the donor cells will be easily detectable in the fibers that received them. Figure 2 shows an example where the transplanted cells expressed human alkaline phostphatase, revealed histochemically.
 When AP are transplanted the result is highly dependent on the environment of the transplantation site: when transplanted subcutaneously these cells give origins to adipocytes and myofibroblasts (see figure 2). In many other sites, including muscle, they do not survive unless fatty degeneration has been induced3.
 
 <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="Flow cytometry analysis diagram; FL8-A: APC vs FL6-A: PECγ; cell population gating." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
 Figure 1. Sorting strategy for the isolation of progenitor populations from skeletal muscle. (A) Sorting strategy: Viable cells were identified based on forward scatter and side scatter. Hoechst staining was used to exclude anuclear debris and propidium iodide (PI) staining to exclude dead cells. Hematopoietic (CD45) and endothelial (CD31) cells were excluded from the sorting gates. The α7+ Hoechst+ PI- CD45- CD31- Scal- subset contains all myogenic progenitors (MP). The Scal+ Hoechst+ PI- CD45- CD31- α7- population contains adipogenic progenitors (AP). (B) Fluorescence-minus-one (FMO) isotype controls confirm the specificity of the stain. (C) Purity checks of MP and AP subsets after sorting.
 <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="Histology tissue sections stained for cellular structure identification; microscopy experiment results." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
 Figure 2. Representative results following MP and AP transplantation. AP were transplanted subcutaneously (top panel) and MP intramuscularly (bottom panel). In both cases, donor cells originated from a mouse expressing transgenic human alkaline phosphatase, identified by the brown staining.

<ol><li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+stem+cells%5BTitle%5D)+AND+%22Curr+Opin+Genet+Dev%22%5BJournal%5D)">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li>
<li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+stem+cells%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li>
<li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+injury+activates+resident+fibro%2Fadipogenic+progenitors+that+facilitate+myogenesis%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li>
<li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibro%2Fadipogenic+progenitors%3A+A+double-edged+sword+in+skeletal+muscle+regeneration%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li>
<li>Sherwood, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+adult+mouse+myogenic+progenitors%3A+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li>
<li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li>
<li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li>
</ol>

No conflicts of interest declared.

The goal of this protocol is to strike a reasonable balance between high yields and high viability of the purified cells. The most critical step in ensuring that healthy cells are recovered is, predictably, the enzymatic dissociation of the starting tissue. The handling of the tissue should be particularly gentle, which is hard to demonstrate or appreciate even in an audiovisual format. Another key factor is the length of the procedure. The longer it takes to go from the donor to the recipient animal, the lower the viability and therefore, the engraftment efficiency. Should there be any question about viability that is not immediately answered by looking at the frequency of PI positive event in the purified cell samples after sorting, we suggest that a limiting dilution assay to measure clonogenicity is performed3. Typical sorts from undamaged muscle routinely yield about 1 in 15-20 cells capable of initiating myogenic or fibro/adipogenic colonies in vitro. While essentially 100% of the colonies initiated from MPs contain differentiated myotubes, only about one third of colonies obtained from FAPs contain adipocytes in addition to smooth muscle actin positive myofibroblasts.

<table><tbody><tr><td>Collagenase II</td><td>Sigma-Aldrich</td><td>C-6885,</td><td>500 u/ml</td></tr><tr><td>Collagenase D</td><td>Roche Group</td><td>11088882001</td><td>1.5 u/ml</td></tr><tr><td>Displace II</td><td>Roche Group</td><td>04942078001</td><td>2.4 u/ml</td></tr><tr><td>cell strainer</td><td>BD Biosciences</td><td>352340</td><td>40 &amp;#x03BC;m</td></tr><tr><td>Tube with cell strainer</td><td>BD Biosciences</td><td>352235</td><td>40 &amp;#x03BC;m</td></tr><tr><td>H&amp;#8211;chst33342</td><td>Sigma-Aldrich</td><td>B2261</td><td>2-5 ug/ml</td></tr><tr><td>CD31-FITC</td><td>eBioscience</td><td>11-0311-85</td><td>Clone 390</td></tr><tr><td>CD45-FITC</td><td>Home made</td><td></td><td>Clone I3/2</td></tr><tr><td>Sca1-PECY7</td><td>eBioscience</td><td>25-5981-82</td><td>Clone D7</td></tr><tr><td>&amp;#945;-7 integrin APC</td><td>Home made</td><td>Inquire with Rossi lab</td><td>R2F2</td></tr></tbody></table>

purification of progenitors from skeletal muscle

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 ...

The overall goal of the following experiment is to isolate viable myogenic and adipogenic progenitor cells from mirroring skeletal muscle. This is achieved by first harvesting muscle tissue from euthanized mice. As a second step, the sample is enzymatically digested, which dissociates the tissue into single cells.Next fluorescence activated cell sorting or facts is used in order to isolate the progenitor cells. Results are obtained that show viability and differentiation of the isolated cells when transplanted into recipient mice based on histochemical staining for the genetic label, which in this example is human alkaline phosphatase. The main advantage of this technique over preexisting techniques like prepl is that the cells can be isolated directly from the animal at different time points.For example, after inducing tissue damage, which means there is no selection on the cell population and the cell population can be analyzed at the molecular level to look at the different stages that the cells cross during the process of in vivo regeneration. So this technique can help answer a critical question in the tissue regeneration field, and in particular, what role each cell type plays during in vivo regeneration and how do these cell types interact with each other to rebuild a healthy tissue? The implication of this technique extends toward the therapy of diseases such as muscular dystrophy.As the study of this progenitors can eventually enable regenerative strategies for this type of diseases. In addition, the implications of this technique extend to a number of other tissues ranging from heart to adipose because at least the fibro adipogenic progenitors have been found in every single tissue we tested so far. Generally, newcomers to this technique will struggle because harsh dissociation techniques will reduce cell viability and will create too much debris, which will then interfere with the clean separation of the cells later on.We first had the idea for this method when we decided to apply flow cytometry, which is usually used for APOE cells to the study of stem cells in solid tissues. Visual demonstration of this technique is important as the tissue dissociation steps are hard to learn because it's very difficult to describe them properly. Demonstrating this technique today will be linky, a research assistant in my laboratory who first developed and optimize the technique For this procedure, at least two mice are required.One mouse is used for cells to provide the necessary single color and antibody isotype controls for facts, and the other will provide the sample to be sorted. To begin, obtain mice between seven and 12 weeks old that have been previously euthanized in a sterile laboratory hood. Place the euthanized mouse on a sterile paper towel, face up and spray the fur thoroughly with 70%ethanol to prevent contamination with forceps in one hand and sharp scissors in the other.Lift the skin in the abdominal region and make a small longitudinal incision. Grasp the skin on each side of the incision and peel back the skin to completely expose the hind limb muscles. Next, excise the muscle tissue by inserting the closed scissors beneath the muscle and above the tibia and then opening the scissors.The back edge of the blades will gently force the separation of the muscle from the tibia. Repeat for both sides of the tibia and place the tissue in a 60 millimeter Petri dish. Now excise the muscle tissue from the femur in the same manner and add the tissue to the petri dish.Repeat for each mouse using one petri dish per mouse. Next, dissociate the tissue into single cells. Add two milliliters or 1000 units of collagenase, two and 20 microliters of 250 millimolar calcium chloride stock solution to each Petri dish of muscle tissue.Then working quickly and carefully. Use two gray forceps, one serrated, and one with two teeth to tear the tissue into one cubic millimeter pieces. Remove and discard as much non-muscle tissue as possible.Cover the Petri dishes and incubate at 37 degrees Celsius for 30 minutes. Following the incubation, retrieve the dishes and return them to the sterile laboratory hood. Remove the plunger from a three milliliter syringe and use it to mash the tissue.Use one plunger per plate to avoid cross-contamination. Add approximately five milliliters of cold sterile PBS to each Petri dish and transfer the contents of the dish to a 50 milliliter conical tube. Rinse with an additional five milliliters of PBS to recover all tissue and add it to the conical tube.Wash and centrifuge the samples three times according to the protocol. Next, add one milliliter of a solution comprised of collagenase D dys space two and calcium chloride to each tube and incubate at 37 degrees Celsius with rotation For one hour. After the incubation, add five milliliters of cold sterile PBS to the tubes, vortex the tubes briefly and tritrate several times to dissociate clumps.Fill the tubes with PBS mix well and place on ice To remove any remaining large pieces of tissue, place a 40 micron cell strainer over a 50 milliliter conical tube on ice and filter the cell suspension. Divide the flow through evenly into three 50 milliliter conical tubes filled with cold, sterile PBS and centrifuge at 1600 RPM for five minutes at eight degrees Celsius. Following centrifugation, carefully remove the S and place the cells on ice for staining and sorting of myogenic and adipogenic progenitor cells.First, set up the six single color staining mixed controls according to the accompanying written protocol. These controls include no stain hulk stain, propidium iodide, CD 31 and CD 45 FSE labeled antibodies, SC one P size seven labeled antibody and alpha seven A PC labeled antibody. Next, set up the three antibody isotype control staining mixes according to the written protocol.Once the staining mixes have been prepared, labeled nine 1.5 milliliter einor tubes for the single color and isotype control samples. Resus suspend and combine the cells from the three conical tubes in a total volume of one milliliter of fax buffer. Next, dispense 100 microliters of the cells to each labeled einor control tube and pipette between five and 10 microliters of the staining mixes depending on the concentration of the antibodies to the corresponding control tubes.Re suspend the cells from the sample mouse in 200 microliters of fax buffer and combine in one of the 50 milliliter conical tubes. Then add 800 microliters of the antibody cocktail. Mix each of the control tubes and sample tubes.Well incubate them on ice for one hour. Following incubation, wash the cells by adding one milliliter of fax buffer to each of the nine control tubes and approximately 20 milliliters of fax buffer to the sample tube. Centrifuge the control einor tubes at 3000 RPM for five minutes.Centrifuge the sample conical tube at 1600 RPM for five minutes. Then aspirate and discard the snat following centrifugation. Resuspend the control cells in each eend orph tube with one milliliter of fax buffer and resuspend the sample cells in four milliliters of fax buffer.Add propidium iodide to the sample cells and isotype controls as well as to the pi. Single color control tube. Pipet the cells into fax tubes through the cell strainer cap to remove remaining clumps that may affect the cytometer.Set up the fax instrument with a single color and isotype controls. Sort the sample cells according to the accompanying written protocol and collect each population separately. And a 3.5 milliliter collection tube Viable myogenic progenitors are the population that is hulked and alpha seven a PC positive and PI CD 31 FE CD 45 FE and ska.One PE size seven negative viable adipogenic progenitors are hulked and ska one PE size seven positive and pi CD 31 FE CD 45 FE and alpha seven a PC negative. A single wild type mouse may yield about one times 10 to the fifth adipogenic progenitors, and 1.5 to two times 10 to the fifth. Myogenic progenitors with greater than 95%viability.To prepare cells for transplantation into host mice, the cells are washed. First centrifuge distorted cells at 1500 RPM for five minutes. Remove the supernatant, resuspend the cells in 500 microliters of PBS and transfer them to einor tubes.Centrifuge the tubes at 3000 RPM for five minutes and remove the supernatant. Then resus suspend myogenic progenitors in PBS or adipogenic progenitors in matri matrigel at approximately 10 to the six cells per milliliter. After preparing the anesthetized host mice for each donor cell type, according to the written protocol, use a three tenths cubic centimeter insulin syringe to inject 20 microliters approximately 20, 000 cells of myogenic or adipogenic progenitors.Allow the appropriate amount of time for the transplanted cells to differentiate usually about three weeks. Then isolate the target tissue and prepare it for cryosectioning as described in the protocol in this figure, the sorting strategy is demonstrated to isolate the adipogenic and myogenic progenitors viable cells were identified based on forward scatter and side scatter hawk staining was used to exclude a nuclear debris and propidium iodide or PI staining was used to exclude dead cells, hematopoietic and endothelial cells, or CD 45 and CD 31 cells respectively were then excluded from the sorting gates. Finally, the subset that is alpha seven positive.SKA one negative contains all myogenic progenitors and the population that is alpha seven negative. SKA one positive contains all adipogenic progenitors fluorescence minus one isotype controls confirm the specificity of the stain. In addition, purity checks of the adipogenic and myogenic subsets were performed.After sorting in this representative example, adipogenic progenitor donor cells expressing transgenic, human alkaline phosphatase injected subcutaneously can be identified in host tissue by histochemistry as indicated by brown staining. Myogenic progenitor donor cells expressing transgenic. Human alkaline phosphatase injected intramuscularly were identified in this tissue section as shown in brown.Using histochemistry Once master, this technique can be done in five hours if it performed properly. If more samples are processing, the time will increase. While attempting this procedure, it's important to remember to work quickly and gently.Therefore, avoid processing too many samples at a time. Prolonging the processing time will greatly reduce the cell variability following this procedure. Other methods like colono SC OG expression analysis by quantitative R-T-P-C-R can also perform in order to answer additional questions like, how do these pro cells response to damage in vivo after its development?This technique paved the way to other researchers in the field of tissue regeneration to explore the crosstalk between the pro cells in damaged tissue. After watching this video, you should have good understanding of how to have this tissues dissociated without damage. The progenitor cells identify the progenitor cells based on their service markers and analyze their developmental potential in vivo.Thank you for watching. Good luck with your experiment.

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Research

JoVE Journal

Biology

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...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

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.

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of Myospheres From hESCs by Epigenetic Reprogramming

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

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

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.

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

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.

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

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.

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

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.

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

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2´-deoxyuridine incorporation assay.

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

This protocol provides a standardized method for isolating and enriching primary myoblasts from adult and neonatal mouse skeletal muscle through tissue dissociation, sequential filtration, preplating, and defined culture conditions, enabling the establishment and maintenance of satellite cell-derived myoblast cultures for downstream experimental applications.

Isolation of Skeletal Muscle Satellite Cells for In Vitro Myogenesis Studies

Learn to isolate viable myogenic and adipogenic progenitor cells from murine skeletal muscle using enzymatic dissociation and flow cytometry. This method enables direct analysis of cell populations during tissue regeneration.

Purification of Progenitors from Skeletal Muscle

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