This work was partially supported by a Fund-A-Fellow grant from the Myotonic Dystrophy Foundation (MDF-FF-2014-0013) and the Scientist Development Grant from the American Heart Association (16SDG29320001).

Wildtype and Nestin-GFP transgenic mice were housed in the animal facility at the University of Minnesota. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Minnesota and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
1. Muscle Dissection and Single-cell Isolation
<ol>
	<li>Euthanize adult mice (6-10 weeks, both male and female) with tribromoethanol (250 mg/kg, i.p.) and sterilize their abdomen skin with 70% ethanol. 
	NOTE: Tribromoethanol was used here instead of ketamine for anesthesia/euthanasia, as ketamine is known to interact with the NMDA receptors, which could potentially have an impact on the study.</li>
	<li>Place the mice in the supine position and use a scalpel to make a horizontal incision on the abdominal skin. Peel off the skin by hand, pulling in opposite directions to expose the hindlimb muscles.</li>
	<li>Collect the muscles from both hindlimbs using forceps and scissors. Store them in sterile phosphate-buffered saline (PBS) supplemented with 1% penicillin-streptomycin (P/S) on ice.</li>
	<li>Wash the dissected hindlimb muscles in ice-cold PBS supplemented with 1% P/S two times and transfer them to a sterile 10 cm plate.</li>
	<li>Carefully dissect out nerves, blood vessels, and connective tissue from the muscles using forceps and scissors under a dissection microscope at 2X magnification.</li>
	<li>Finely chop and mince the muscles into small pieces (1-2 mm3) using sterile scissors and blades. If necessary, add a small amount of Dulbecco&#39;s Modified Eagle Medium (DMEM) to ensure that the muscles are not dried out.</li>
	<li>Mechanically break down the tissue by pipetting up and down through a 10 mL serological pipette 10 times.</li>
	<li>Add freshly made digestion solution (DMEM supplemented with 0.2% Type 2 collagenase) to the mixture. Incubate at 37 &#176;C for 2 h with gentle agitation at 35 revolutions/min.</li>
	<li>Triturate using an 18 G needle to homogenize the mixture. Then, centrifuge at 500 x g for 5 min. Discard the supernatant and then resuspend the pellet in 0.25% trypsin/EDTA. Incubate at 37 &#176;C for 10 min. Repeat the centrifugation step two more times.</li>
	<li>Add 10 mL of DMEM supplemented with 20% fetal bovine serum (FBS) to the solution and centrifuge at 500 x g for 5 min. Discard the supernatant and resuspend the pellet in red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA).</li>
	<li>Centrifuge at 500 x g for 5 min. Discard the supernatant and resuspend the pellet in sorting buffer (20 mM HEPES, pH 7.0; 1 mM EDTA; and 1% BSA in Ca/Mg2+-free PBS, pH 7.0). Filter the mixture through a 40 &#181;m cell strainer to obtain a single-cell suspension.</li>
	<li>Centrifuge at 500 x g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of sorting buffer.</li>
	<li>Count the cell number with a hemocytometer and dilute the single-cell suspension to 5 x 106/mL in sorting buffer.</li>
</ol>
2. Cell Staining and Sorting
<ol>
	<li>Prepare controls and the sample as described in Table 1. Stain the single-cell suspension with the respective antibodies on ice for 30 min, as described in Table 1.</li>
	<li>Centrifuge at 500 x g for 5 min and wash the pellets twice with sorting buffer.</li>
	<li>Add DAPI to the single-cell solutions, as indicated in Table 1. Use 5 &#181;g/mL DAPI (final concentration) for the DAPI single-color control and 1 &#181;g/mL DAPI for the PDGFR&#946;-PE-FMO control and the sample. Keep all tubes on ice throughout the experiment.</li>
	<li>Turn on the sorter and the software. Scan and insert a 100 &#181;m sorting chip when prompted.</li>
	<li>Perform the automatic setup (i.e., chip alignment, droplet calibration, side stream calibration, and sort delay calibration) by loading the automatic setup beads when prompted.</li>
	<li>When automatic setup is complete, go to the &#34;Experiment&#34; tab, click on &#34;New,&#34; and select the &#34;Blank Template&#34; from &#34;Public Templates.&#34;</li>
	<li>Under &#8220;Measurement Settings,&#8221; enter &#8220;DAPI&#8221; for &#8220;FL1,&#8221; &#8220;Nestin-GFP&#8221; for &#8220;FL2,&#8221; and &#8220;PDGFR&#946;&#8221; for &#8220;FL3.&#8221; Uncheck the boxes for &#8220;FL4&#8221;-&#8220;FL6&#8221;.</li>
	<li>Check the boxes to activate the 405, 488, and 561 lasers and click on &#34;Create New Experiment.&#34;</li>
	<li>Select the &#34;Start Compensation Wizard&#34; option and follow the &#34;Compensation Wizard&#34; software prompts to set up the compensation.
	<ol>
		<li>Load the unstained control and click &#34;Start.&#34; Click &#34;Detector &#38; Threshold Settings&#34; and adjust the sensor gain of the FSC and BSC detectors to place the population on the scale.</li>
		<li>Adjust the gain levels of the FL1-FL3 fluorescence channels to place the negative populations on the left side of the histograms. Click the &#34;Record&#34; button to record the data.</li>
		<li>Load single-color controls one by one when prompted. Click &#34;Start and Record&#34; to record the data. Adjust the gates for the positive populations on the histograms. Click &#34;Next.&#34;</li>
		<li>Go to &#34;Calculate Matrix&#34; on the &#34;Compensation&#34; tab and click &#34;Calculate&#34; in the &#34;Calculate Compensation Settings&#34; panel to perform the compensation. Click &#34;Finish&#34; to exit the &#34;Compensation Wizard.&#34;</li>
	</ol>
	</li>
	<li>Load the PDGFR&#946;-PE-FMO control and click &#34;Start.&#34; Draw a polygon gate (Gate A) around the cells of interest under the &#34;All Events&#34; plot.</li>
	<li>Double-click inside Gate A to create a child plot. Change the Y-axis to DAPI and draw a polygon gate (Gate B) around live (DAPIlow) cells. Double-click inside Gate B to create a child plot. Change the X-axis to FSC-H and the Y-axis to FSC-W and draw a polygon gate (Gate C) around the singlets to eliminate doublets.</li>
	<li>Double-click inside Gate C to create a child plot. Change the X-axis to Nestin-GFP and the Y-axis to PDGFR&#946;-PE. Click the &#8220;Record&#8221; button to record the data.</li>
	<li>Load the sample and repeat steps 2.11-2.12. After recording, click on &#34;Pause&#34; to preserve the sample.</li>
	<li>Define the gating boundaries for PDGFR&#946;+ and Nestin-GFP+ cells based on the PDGFR&#946;-PE-FMO control. Draw gates for the PDGFR&#946;+Nestin-GFP- and PDGFR&#946;+Nestin-GFP+ populations.</li>
	<li>Under &#8220;Sorting Method,&#8221; select &#8220;2-way Tubes&#8221; and assign &#8220;PDGFR&#946;+Nestin-GFP-&#8221; and &#8220;PDGFR&#946;+Nestin-GFP+&#8221; cells to the left and right collecting tubes, respectively. Mount the sorting buffer-filled 15 mL collecting tubes on the collection stage and click the &#8220;Load Collection&#8221; button&#34;.</li>
	<li>Click the &#34;Resume&#34; button to keep the sample running. Click &#34;Start Sort&#34; to collect PDGFR&#946;+Nestin-GFP- (type I pericytes) and PDGFR&#946;+Nestin-GFP+ (type II pericytes) cells.</li>
</ol>
3. Post-sorting Analyses
<ol>
	<li>Centrifuge the sorted cells at 500 x g for 5 min, resuspend the pellet in 1 mL of pericyte medium (see Materials Table), and count the cell density using a hemocytometer.</li>
	<li>Seed type I and type II pericytes on poly-D-lysine (PDL)-coated coverslips at ~1 x 104 cells/cm2. Grow in pericyte medium for 3 days at 37 &#176;C with 5% CO2.</li>
	<li>On day 3, examine the pericyte morphology (under phase contrast) and endogenous Nestin-GFP expression using a fluorescent microscope (excitation laser: 488 nm, excitation filter: 470/40 nm, and emission filter: 515/30 nm). Take images under a 20X objective (0.45 NA).</li>
	<li>Replace the pericyte medium with adipogenic (mouse MSC basal medium + adipogenic stimulatory supplement) and myogenic (DMEM + 2% horse serum) medium to initiate adipogenic and myogenic differentiation, respectively, as described previously20. Change the medium every 2-3 days.</li>
	<li>Fix the cells on day 17 (14 days after adipogenic/myogenic differentiation) in 4% paraformaldehyde (PFA) for 20 min at room temperature. 
	NOTE: Caution, PFA is a carcinogen.</li>
	<li>Perform immunocytochemistry against perilipin (adipocyte marker) and S-myosin (mature myotube/myofiber marker), as described in previous publications19,20.
	<ol>
		<li>Wash the fixed cells 3 times in PBS for 10 min at room temperature.</li>
		<li>Add blocking buffer (PBS supplemented with 5% donkey serum, 3% BSA, and 0.3% Triton X-100) and incubate at room temperature for 1 h.</li>
		<li>Incubate the cells with anti-perilipin (2 &#181;g/mL) and/or anti-S-myosin (2 &#181;g/mL) antibodies at 4 &#176;C overnight.</li>
		<li>Wash the cells 3 times in PBS for 10 min at room temperature.</li>
		<li>Incubate the cells with Alexa 555 donkey anti-rabbit (4 &#181;g/mL) and/or Alexa555 donkey anti-mouse (4 &#181;g/mL) antibodies at room temperature for 1 h.</li>
		<li>Wash the cells 3 times in PBS for 10 min at room temperature.</li>
		<li>Mount the immunostained cells with mounting medium containing DAPI (see the table of materials). Examine perilipin and S-myosin expression using a fluorescent microscope (excitation laser: 543 nm; 540/45 nm excitation and 600/50 nm emission) and take images under a 40X objective (0.60 NA).</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+are+essential+for+skeletal+muscle+regeneration:+the+cell+on+the+edge+returns+centre+stage.">Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage.</a> Development. 139 (16), 2845-2856 (2012).</li><li>von Maltzahn, J., Jones, A. E., Parks, R. J., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7+is+critical+for+the+normal+function+of+satellite+cells+in+adult+skeletal+muscle.">Pax7 is critical for the normal function of satellite cells in adult skeletal muscle.</a> Proc Natl Acad Sci U S A. 110 (41), 16474-16479 (2013).</li><li>Lepper, C., Partridge, T. A., Fan, C. 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All authors have no conflict of interest to disclose.

Pericytes are multipotent perivascular cells22,23 located on the abluminal surface of capillaries21,26. In skeletal muscles, pericytes are able to differentiate along the adipogenic and/or myogenic pathways19,20,24. Recent studies revealed two subpopulations of pericytes, with different marker expression and distinct differentiation potentials19,24,25. Type I (NG2+Nestin-) pericytes are adipogenic, while type II (NG2+Nestin+) pericytes are myogenic. The isolation of these subpopulations for in vitro studies requires the NG2-DsRed and Nestin-GFP double-transgenic mouse line. The dependence of this purification protocol on double-transgenic mice significantly limits its application and thus research on the biology of pericyte subpopulations.
This video article illustrates an alternative method for the isolation of type I and type II pericytes from mouse skeletal muscles. This new method still relies on a FACS-based technique for cell separation. However, instead of utilizing transgenic NG2-DsRed fluorescence, it targets the endogenous PDGFR&#946; signal. This is based on the observation that NG2-DsRed expression co-localizes with PDGFR&#946; in skeletal muscle19. Compared to the original method, this protocol has several advantages. First, it does not require the NG2-DsRed genetic background, although the Nestin-GFP background is still needed. Second, instead of targeting NG2, this protocol uses PDGFR&#946;, a well-characterized marker with validated and commercially available antibodies. Third, it allows for the simultaneous isolation of both type I and type II pericytes. For example, PDGFR&#946;-PE+Nestin-GFP- and PDGFR&#946;-PE+Nestin-GFP+ cells isolated using this protocol demonstrate distinct differentiation capabilities. Specifically, PDGFR&#946;-PE+Nestin-GFP-, but not PDGFR&#946;-PE+Nestin-GFP+, cells differentiate into adipocytes in adipogenic condition, whereas PDGFR&#946;-PE+Nestin-GFP+, but not PDGFR&#946;-PE+Nestin-GFP-, cells undergo myogenic differentiation under myogenic condition. These data are consistent with previous reports showing that NG2-DsRed+Nestin-GFP- cells (type I pericytes) are adipogenic and NG2-DsRed+Nestin-GFP+ cells (type II pericytes) are myogenic19,24, suggesting that isolated PDGFR&#946;-PE+Nestin-GFP- and PDGFR&#946;-PE+Nestin-GFP+ cells are indeed type I and type II pericytes, respectively. Together, these results suggest that this new protocol allows for the relatively easy isolation/purification of pericyte subpopulations from mouse skeletal muscles, and possibly other tissues. This will promote studies on pericyte biology and their therapeutic translation for various disorders.
It should be noted, however, that there is a limitation of this method due to the use PDGFR&#946;. It has been shown in a previous study that PW1+ interstitial cells (PICs) also express PDGFR&#946;20. Therefore, isolated PDGFR&#946;-PE+Nestin-GFP- and PDGFR&#946;-PE+Nestin-GFP+ populations may contain PICs. However, the contribution of these PICs to pericyte differentiation is limited, given that pericytes outnumber PICs in skeletal muscles20 and that type I pericytes do not undergo myogenesis19,25.
Using this method, type I and type II pericytes were obtained at yields of 9.5% and 2.1%, respectively. These numbers were comparable to the reported yields of 2.8% and 3.4%, respectively, in the double-transgenic mice19. The slight difference may be due to different gating strategies or digestion protocols. These results again suggest that the proposed protocol can be used to isolate subtypes of pericytes from skeletal muscles.
The critical steps of this protocol include the following points: (1) Make sure that the skeletal muscles are fully minced and able to easily pass through a 10 mL serological pipette. Large muscle blocks interfere with enzymatic digestion and significantly reduce yield. (2) Use freshly made collagenase solution for muscle digestion and allow gentle agitation (35 revolutions/min) during incubation. (3) Keep the controls and sample on ice throughout the staining and sorting steps. (4) Use FMO controls to set up gating boundaries.
In addition to differentiation capability, type I and type II pericytes also show different morphology. Specifically, type I pericytes have round cell bodies, with short processes and relatively large nuclei. Type II pericytes, on the other hand, usually have small cell bodies, with long and thin processes and small nuclei. The morphological differences suggest that type I and type II pericytes are intrinsically different, which is consistent with the heterogeneous nature of pericytes21. What causes/maintains the difference between type I and type II pericytes, as well as the underlying molecular mechanisms, remain unclear and require further investigation. This isolation protocol will help to answer these important questions.

Muscular dystrophy is a muscle-degenerative disorder that has no effective treatments so far. The development of therapies that promote tissue regeneration has always been of great interest. Tissue regeneration and repair after damage depend on resident stem cells/progenitor cells1. Satellite cells are committed myogenic precursor cells that contribute to muscle regeneration2,3,4,5,6,7. Their clinical use, however, is hampered by their limited migration and low survival rate after injection, as well as by their decreased differentiation capability after in vitro amplification8,9,10,11. In addition to satellite cells, skeletal muscles also contain many other cell populations with myogenic potential12,13,14,15,16, such as platelet-derived growth factor receptor-beta (PDGFR&#946;)-positive interstitial cells. There is evidence showing that muscle-derived PDGFR&#946;+ cells are able to differentiate into myogenic cells and improve muscle pathology and function14,17,18,19,20. PDGFR&#946; predominantly labels pericytes21, which are perivascular cells with pluripotency22,23. In addition to PDGFR&#946;, many other markers, including Neuron-Glial 2 (NG2) and CD146, are also used to identify pericytes21. It should be noted, however, that none of these markers is pericyte-specific21. Recent studies revealed two subtypes of muscle pericytes, called type I and type II, which express different molecular markers and carry out distinct functions19,24,25. Biochemically, type I pericytes are NG2+Nestin-, while type II pericytes are NG2+Nestin+19,24. Functionally, type I pericytes can undergo adipogenic differentiation, contributing to fat accumulation and/or fibrosis, whereas type II pericytes can differentiate along the myogenic pathway, contributing to muscle regeneration19,24,25. These results demonstrate that: (1) type I pericytes may be targeted in the treatment of fatty degenerative disorders/fibrosis, and (2) type II pericytes have great therapeutic potential for muscular dystrophy. Further investigation and characterization of these populations require an isolation protocol that enables the separation of type I and type II pericytes at a high level of purity.
Currently, the isolation of pericyte subpopulations relies on NG2-DsRed and Nestin-GFP double-transgenic mice19,24. The availability of NG2-DsRed mice and the quality of most NG2 antibodies limit the widespread use of this method. Given that all NG2+ pericytes also express PDGFR&#946; in skeletal muscles19,20,24, we hypothesize that NG2 can be replaced by PDGFR&#946; for the isolation of pericytes and their subpopulations. This work describes a FACS-based protocol that uses PDGFR&#946; staining and the Nestin-GFP signal. This method is less demanding for investigators because: (1) it does not require the NG2-DsRed background and (2) it uses commercially available PDGFR&#946; antibodies, which are well-characterized. In addition, it enables the simultaneous isolation of type I and type II pericytes at high purity, allowing for further investigation into the biology and therapeutic potential of these pericyte subpopulations. Following purification, these cells can be grown in culture, and their morphologies can be visualized. This work also shows that type I and type II pericytes isolated using this method, like those purified from double-transgenic mice, are adipogenic and myogenic, respectively.

<table><tbody><tr><td>Cell Sorter</td><td>Sony</td><td>SH800</td><td></td></tr><tr><td>Automatic Setup Beads</td><td>Sony</td><td>LE-B3001</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995</td><td></td></tr><tr><td>Avertin&amp;nbsp;</td><td>Sigma</td><td>T48402</td><td></td></tr><tr><td>Pericyte Growth Medium</td><td>ScienCell</td><td>1201</td><td></td></tr><tr><td>MSC Basal Medium (Mouse)</td><td>Stemcell Technologies</td><td>5501</td><td></td></tr><tr><td>Adipogenic Stimulatory Supplement (Mouse)</td><td>Stemcell Technologies</td><td>5503</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000</td><td></td></tr><tr><td>Horse Serum</td><td>Sigma</td><td>H1270</td><td></td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>0.25% Trypsin/EDTA&amp;nbsp;</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>PDL</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>PDGFR&amp;beta;-PE Antibody</td><td>eBioscience</td><td>12-1402</td><td></td></tr><tr><td>Perilipin Antibody</td><td>Sigma</td><td>P1998</td><td></td></tr><tr><td>S-Myosin Antibody</td><td>DSHB</td><td>MF-20</td><td></td></tr><tr><td>Alexa 555-anti-rabbit antibody&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>A-31572</td><td></td></tr><tr><td>Alexa 555-anti-mouse antibody</td><td>ThermoFisher Scientific</td><td>A-31570</td><td></td></tr><tr><td>Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2058</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Fisher Scientific</td><td>A661</td><td></td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P184</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190</td><td></td></tr><tr><td>18 G Needles</td><td>BD</td><td>305196</td><td></td></tr><tr><td>10 mL Serological Pipette</td><td>BD</td><td>357551</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111</td><td></td></tr></tbody></table>

isolation of type i and type ii pericytes from mouse skeletal muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

FACS parameters, including laser intensity and channel compensation, are corrected based on the results of unstained control and single-color controls. The PDGFR&#946;-PE-FMO control is used to set the gating for the PDGFR&#946;-PE+ population (Figure 1A). Among the PDGFR&#946;-PE- cells, two populations representing Nestin-GFP+ and Nestin-GFP- cells are clearly separated (Figure 1A). Gating boundaries for the PDGFR&#946;-PE+Nestin-GFP+ and PDGFR&#946;-PE+Nestin-GFP- populations are set based on the gating for PDGFR&#946;-PE+ and Nestin-GFP+ (Figure 1A). These boundaries are used to define and sort PDGFR&#946;-PE+Nestin-GFP+ (type II pericytes) and PDGFR&#946;-PE+Nestin-GFP- (type I pericytes) cells from the sample (Figure 1B). Isolated PDGFR&#946;-PE+Nestin-GFP- and PDGFR&#946;-PE+Nestin-GFP+ cells account for 9.5% and 2.1% of the total cells in the single-cell solution, respectively.
FACS-isolated type I and type II pericytes demonstrate morphological differences after three days in culture. Type I pericytes display amoeboid morphology, with round cell bodies and short processes (Figure 2). Type II pericytes, however, show ramified morphology, characterized by small cell bodies and long, thin processes (Figure 2). At this time, most type II pericytes remain as Nestin-GFP+, while type I pericytes are Nestin-GFP- (Figure 2).
In addition, type I, but not type II, pericytes differentiate into perilipin-expressing adipocytes after 14 days in adipogenic medium (Figure 3). Type II, but not type I, pericytes differentiate into S-myosin-expressing myotubes after 14 days in myogenic medium (Figure 4). These results strongly indicate that type I pericytes are adipogenic, while type II pericytes are myogenic.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55904/55904fig1.jpg" /> 
Figure 1: Gating boundaries and representative sorting. (A) Fluorescent plot of the PDGFR&#946;-PE-FMO control, demonstrating the PDGFR&#946;-PE+Nestin-GFP- and PDGFR&#946;-PE+Nestin-GFP+ gating boundaries. (B) Representative fluorescent plot of the sample showing the distribution of the PDGFR&#946;-PE+Nestin-GFP- (type I pericytes) and PDGFR&#946;-PE+Nestin-GFP+ (type II pericytes) cells. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55904/55904fig1large.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/55904/55904fig2.jpg" /> 
Figure 2: Morphology and Nestin-GFP expression in sorted type I and type II pericytes. Sorted type I and type II pericytes were seeded on coverslips and grown in pericyte medium for 3 days. Type I pericytes showed round cell bodies, with short processes, and no endogenous GFP signal under a fluorescent microscope (excitation Laser: 488 nm; excitation and emission filters: 470/40 nm and 515/30 nm, respectively). Type II pericytes demonstrated small cell bodies, with long and thin processes, and a strong endogenous GFP signal under a fluorescent microscope under the same settings. Scale bar = 100 &#181;m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55904/55904fig2large.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/55904/55904fig3.jpg" /> 
Figure 3: Adipogenic differentiation of sorted type I and type II pericytes. Sorted type I and type II pericytes were grown in pericyte medium for 3 days. They were then differentiated in adipogenic medium for 14 days. The cells were fixed and immunostained with anti-perilipin antibody followed by Alexa555 donkey anti-rabbit antibody. Immunocytochemistry showed that type I, but not type II, pericytes expressed the adipocyte marker perilipin (red) under a fluorescent microscope (excitation laser: 543 nm; excitation and emission filters: 540/45 nm and 600/50 nm, respectively). Scale bar = 50 &#181;m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55904/55904fig3large.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/55904/55904fig4.jpg" /> 
Figure 4: Myogenic differentiation of sorted type I and type II pericytes. Sorted type I and type II pericytes were grown in pericyte medium for 3 days and were then differentiated in myogenic medium for 14 days. The cells were fixed and immunostained with anti-S-myosin antibody and Alexa555 donkey anti-mouse antibody. Immunocytochemistry showed that type II, but not type I, pericytes expressed the mature myotube/myofiber marker S-myosin (red) under a fluorescent microscope (excitation laser: 543 nm; excitation and emission filters: 540/45 nm and 600/50 nm, respectively). Scale bar = 50 &#181;m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55904/55904fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Tubes</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Unstained control</td>
			<td>Single cell suspension from wildtype mice</td>
		</tr>
		<tr>
			<td>DAPI single-color control (dead cell exclusion)</td>
			<td>Single cell suspension from wildtype mice + DAPI (5 &#956;g/mL)</td>
		</tr>
		<tr>
			<td>GFP single-color control</td>
			<td>Single cell suspension from Nestin-GFP mice</td>
		</tr>
		<tr>
			<td>PE single-color control</td>
			<td>OneComp eBeads + PDGFR&#946;-PE antibody (4 &#956;g/mL)</td>
		</tr>
		<tr>
			<td>PE-FMO control</td>
			<td>Single cell suspension from Nestin-GFP mice + DAPI (1 &#956;g/mL)</td>
		</tr>
		<tr>
			<td>Sample</td>
			<td>Single cell suspension from Nestin-GFP mice + PDGFR&#946;-PE antibody (4 &#956;g/mL) + DAPI (1 &#956;g/mL)</td>
		</tr>
	</tbody>
</table>
Table 1: Staining protocol for the single-color controls, PDGFR&#946;-PE-FMO control, and muscle sample.

Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

isolation-of-type-i-and-type-ii-pericytes-from-mouse-skeletal-muscles

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.1K Views.  University of Minnesota. This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Video: Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

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

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Neuroscience

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Biology

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Medicine

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation assay.

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2&#180;-deoxyuridine incorporation assay.

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...