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 migra....

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 wit....

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). Gating boundarie.......

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

List of materials used in this article

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
18 G Needles	BD	305196
10 mL Serological Pipette	BD	357551
OneComp eBeads	eBioscience	01-1111