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

Summary

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

Abstract

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 PDGFRβ, 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.

Introduction

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 regeneration^2,3,4,5,6,7. 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 amplification^8,9,10,11. In addition to satellite cells, skeletal muscles also contain many other cell populations with myogenic potential^{12,13,14,15,16}, such as platelet-derived growth factor receptor-beta (PDGFRβ)-positive interstitial cells. There is evidence showing that muscle-derived PDGFRβ⁺ cells are able to differentiate into myogenic cells and improve muscle pathology and function^{14,17,18,19,20}. PDGFRβ predominantly labels pericytes²¹, which are perivascular cells with pluripotency^22,23. In addition to PDGFRβ, 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 functions^19,24,25. 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 regeneration^19,24,25. 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 mice^19,24. 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 PDGFRβ in skeletal muscles^19,20,24, we hypothesize that NG2 can be replaced by PDGFRβ for the isolation of pericytes and their subpopulations. This work describes a FACS-based protocol that uses PDGFRβ 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 PDGFRβ 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.

Protocol

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

1. 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.
2. 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.
3. 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.
4. 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.
5. Carefully dissect out nerves, blood vessels, and connective tissue from the muscles using forceps and scissors under a dissection microscope at 2X magnification.
6. 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's Modified Eagle Medium (DMEM) to ensure that the muscles are not dried out.
7. Mechanically break down the tissue by pipetting up and down through a 10 mL serological pipette 10 times.
8. Add freshly made digestion solution (DMEM supplemented with 0.2% Type 2 collagenase) to the mixture. Incubate at 37 °C for 2 h with gentle agitation at 35 revolutions/min.
9. 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 °C for 10 min. Repeat the centrifugation step two more times.
10. 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).
11. 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 µm cell strainer to obtain a single-cell suspension.
12. Centrifuge at 500 x g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of sorting buffer.
13. Count the cell number with a hemocytometer and dilute the single-cell suspension to 5 x 10⁶/mL in sorting buffer.

2. Cell Staining and Sorting

1. 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.
2. Centrifuge at 500 x g for 5 min and wash the pellets twice with sorting buffer.
3. Add DAPI to the single-cell solutions, as indicated in Table 1. Use 5 µg/mL DAPI (final concentration) for the DAPI single-color control and 1 µg/mL DAPI for the PDGFRβ-PE-FMO control and the sample. Keep all tubes on ice throughout the experiment.
4. Turn on the sorter and the software. Scan and insert a 100 µm sorting chip when prompted.
5. 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.
6. When automatic setup is complete, go to the "Experiment" tab, click on "New," and select the "Blank Template" from "Public Templates."
7. Under “Measurement Settings,” enter “DAPI” for “FL1,” “Nestin-GFP” for “FL2,” and “PDGFRβ” for “FL3.” Uncheck the boxes for “FL4”-“FL6”.
8. Check the boxes to activate the 405, 488, and 561 lasers and click on "Create New Experiment."
9. Select the "Start Compensation Wizard" option and follow the "Compensation Wizard" software prompts to set up the compensation.
   1. Load the unstained control and click "Start." Click "Detector & Threshold Settings" and adjust the sensor gain of the FSC and BSC detectors to place the population on the scale.
   2. Adjust the gain levels of the FL1-FL3 fluorescence channels to place the negative populations on the left side of the histograms. Click the "Record" button to record the data.
   3. Load single-color controls one by one when prompted. Click "Start and Record" to record the data. Adjust the gates for the positive populations on the histograms. Click "Next."
   4. Go to "Calculate Matrix" on the "Compensation" tab and click "Calculate" in the "Calculate Compensation Settings" panel to perform the compensation. Click "Finish" to exit the "Compensation Wizard."
10. Load the PDGFRβ-PE-FMO control and click "Start." Draw a polygon gate (Gate A) around the cells of interest under the "All Events" plot.
11. 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 (DAPI^low) 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.
12. Double-click inside Gate C to create a child plot. Change the X-axis to Nestin-GFP and the Y-axis to PDGFRβ-PE. Click the “Record” button to record the data.
13. Load the sample and repeat steps 2.11-2.12. After recording, click on "Pause" to preserve the sample.
14. Define the gating boundaries for PDGFRβ⁺ and Nestin-GFP⁺ cells based on the PDGFRβ-PE-FMO control. Draw gates for the PDGFRβ⁺Nestin-GFP^- and PDGFRβ⁺Nestin-GFP⁺ populations.
15. Under “Sorting Method,” select “2-way Tubes” and assign “PDGFRβ⁺Nestin-GFP^-” and “PDGFRβ⁺Nestin-GFP⁺” 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 “Load Collection” button".
16. Click the "Resume" button to keep the sample running. Click "Start Sort" to collect PDGFRβ⁺Nestin-GFP^- (type I pericytes) and PDGFRβ⁺Nestin-GFP⁺ (type II pericytes) cells.

3. Post-sorting Analyses

1. 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.
2. 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 °C with 5% CO₂.
3. 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).
4. 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.
5. 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.
6. Perform immunocytochemistry against perilipin (adipocyte marker) and S-myosin (mature myotube/myofiber marker), as described in previous publications^19,20.
   1. Wash the fixed cells 3 times in PBS for 10 min at room temperature.
   2. 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.
   3. Incubate the cells with anti-perilipin (2 µg/mL) and/or anti-S-myosin (2 µg/mL) antibodies at 4 °C overnight.
   4. Wash the cells 3 times in PBS for 10 min at room temperature.
   5. Incubate the cells with Alexa 555 donkey anti-rabbit (4 µg/mL) and/or Alexa555 donkey anti-mouse (4 µg/mL) antibodies at room temperature for 1 h.
   6. Wash the cells 3 times in PBS for 10 min at room temperature.
   7. 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).

Results

FACS parameters, including laser intensity and channel compensation, are corrected based on the results of unstained control and single-color controls. The PDGFRβ-PE-FMO control is used to set the gating for the PDGFRβ-PE⁺ population (Figure 1A). Among the PDGFRβ-PE^- cells, two populations representing Nestin-GFP⁺ and Nestin-GFP^- cells are clearly separated (Figure 1A). G...

Discussion

Pericytes are multipotent perivascular cells^22,23 located on the abluminal surface of capillaries^21,26. In skeletal muscles, pericytes are able to differentiate along the adipogenic and/or myogenic pathways^19,20,24. Recent studies revealed two subpopulations of pericytes, with different marker expression and dist...

Materials

Name	Company	Catalog Number	Comments
Cell Sorter	Sony	SH800
Automatic Setup Beads	Sony	LE-B3001
DMEM	Gibco	11995
Avertin	Sigma	T48402
Pericyte Growth Medium	ScienCell	1201
MSC Basal Medium (Mouse)	Stemcell Technologies	5501
Adipogenic Stimulatory Supplement (Mouse)	Stemcell Technologies	5503
Fetal Bovine Serum	Gibco	16000
Horse Serum	Sigma	H1270
Collagenase Type 2	Worthington	LS004176
0.25% Trypsin/EDTA	Gibco	25200
Penicillin/Streptomycin	Gibco	15140
PDL	Sigma	P6407
PDGFRβ-PE Antibody	eBioscience	12-1402
Perilipin Antibody	Sigma	P1998
S-Myosin Antibody	DSHB	MF-20
Alexa 555-anti-rabbit antibody	ThermoFisher Scientific	A-31572
Alexa 555-anti-mouse antibody	ThermoFisher Scientific	A-31570
Mounting Medium with DAPI	Vector Laboratories	H-1200
DAPI	ThermoFisher Scientific	D1306
HEPES	Gibco	15630
EDTA	Fisher	BP120
BSA	Sigma	A2058
NH4Cl	Fisher Scientific	A661
KHCO3	Fisher Scientific	P184
PBS	Gibco	14190
18G Needles	BD	305196
10ml Serological Pipette	BD	357551