Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice

Summary

Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

Abstract

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, and as the disease progresses, the patient develops symptoms of dilated cardiomyopathy, arrhythmia, and congestive heart failure. The DMD^MDX mutant mice do not express dystrophin, and are commonly used as a mouse model of DMD. In our recent study, we observed that intramyocardial injection of wide type (WT)-myogenic progenitor cells-derived exosomes (MPC-Exo) transiently restored the expression of dystrophin in the myocardium of DMD^MDX mutant mice, which was associated with a transient improvement in cardiac function suggesting that WT-MPC-Exo may provide an option to relieve the cardiac symptoms of DMD. This article describes the technique of MPC-Exo purification and transplantation into hearts of DMD^MDX mutant mice.

Introduction

Duchenne muscular dystrophy (DMD) is an X-linked recessive, progressive neuromuscular disease caused by a mutation in DMD gene and the loss of functional dystrophin¹. Dystrophin is expressed primarily in skeletal muscle and myocardium, and is less expressed in smooth muscle, endocrine glands and neurons^2,3. DMD is the most common type of muscular dystrophy with an incidence of one per 3,500 to 5,000 newborn boys worldwide^4,5. Individuals typically develop progressive muscle necrosis, loss of independent walking by early adolesce....

Protocol

Animals were handled according to approved protocols and animal welfare regulations of the Institutional Animal Care and Use Committee of the Medical College of Georgia at Augusta University.

1. Isolation and Purification of MPC-derived Exosomes

1. Seed 5 x 10⁶ C2C12 cells in a 15 cm cell culture dish with 20 mL complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin G and 100 μg/mL streptomycin. Incub.......

Discussion

The method of isolating pure exosomes is essential for studying the function of exosomes. One of the common techniques for exosome isolation is polyethylene glycols (PEGs) mediated precipitation^17,18,25. Exosomes can be precipitated in PEGs, and pelleted by low-speed centrifugation. PEG-mediated purification is very convenient, low-cost, it does not need any advanced equipment, but there is concern about the purity of exosomes s.......

Materials

Name	Company	Catalog Number	Comments
0.22-μm Filter	Fisherbrand	09-720-004
15-cm Cell Culture Dish	Thermo Fisher Scientific	157150
24-gauge catheter	TERUMO	SR-OX2419CA
31-gauge insulin needle	BD	328291
4% paraformaldehyde	Affymetrix	AAJ19943K2
50 mL Centrifuge Tubes	Thermo Fisher Scientific	339652
6-0 suture	Pro Advantage by NDC	P420697
Alexa Fluor 488 goat anti-rabbit IgG	Thermo Fisher Scientific	A-11008
Antibiotic Antimycotic Solution	Corning	30-004-CI
Anti-Dystrophin antibody	Abcam	ab15277
Antigen retriever	Aptum Biologics	R2100-US	Antigen recovery
Autofluorescence Quenching Kit	Vector Laboratories	SP-8400
C2C12 cell line	ATCC	CRL-1772
Centrifuge	Unico	C8606
Change-A-Tip High Temp Cauteries	Bovie Medical Corporation	HIT
Confocal microscopy	Zeiss	Zeiss 780 Upright Confocal
DBA/2J-mdx mice	The Jackson Laboratory	013141
DMEM	Corning	10-013-CM
Fetal Bovine Serum (FBS)	Corning	35-011-CV
Goat serum	MP Biomedicals, LLC	191356
Isoflurane	Patterson Veterinary	07-893-1389
Ketamine	Henry Schein	056344
Mounting Medium with DAPI	Vector Laboratories	H-1500
Mouse Retractor Set	Kent Scientific	SURGI-5001
Polyethylene glycol tert-octylphenyl ether	Fisher Scientific	BP151-100
Rodent ventilator	Harvard Apparatus	55-7066
SW-28 Ti rotor	Beckman	342207
The Vevo 2100 Imaging Platform	FUJIFILM VisualSonics	Vevo 2100	Ultrasound System
Ultracentrifuge	Beckman	365672
Ultra-Clear Tubes	Beckman	344058
Xylazine (XylaMed)	Bimeda-MTC Animal Health Inc.	1XYL003 8XYL006