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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 DMDMDX 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 DMDMDX 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 DMDMDX 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 neurons2,3. DMD is the most common type of muscular dystrophy with an incidence of one per 3,500 to 5,000 newborn boys worldwide4,5. 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.......

Representative Results

A flow chart for isolating and purifying exosomes from C2C12 cells is shown in Figure 1A. To confirm the presence of exosomes, we performed transmission electron microscopy analysis. Transmission electron microscopy image (Figure 1B) shows the morphology of the bright and round shape vesicles of C2C12 derived exosomes. Western blot analysis confirmed the presence of exosome markers, including CD63 and TSG101 (

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 precipitation17,18,25. 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.......

Acknowledgements

Tang were partially supported by the American Heart Association: GRNT31430008, NIH-AR070029, NIH-HL086555, NIH-HL134354.

....

Materials

NameCompanyCatalog NumberComments
0.22-μm FilterFisherbrand09-720-004
15-cm Cell Culture DishThermo Fisher Scientific157150
24-gauge catheterTERUMOSR-OX2419CA
31-gauge insulin needleBD328291
4% paraformaldehyde AffymetrixAAJ19943K2
50 mL Centrifuge TubesThermo Fisher Scientific339652
6-0 suturePro Advantage by NDCP420697
Alexa Fluor 488 goat anti-rabbit IgGThermo Fisher ScientificA-11008
Antibiotic Antimycotic SolutionCorning 30-004-CI
Anti-Dystrophin antibodyAbcamab15277
Antigen retriever Aptum BiologicsR2100-USAntigen recovery
Autofluorescence Quenching Kit Vector LaboratoriesSP-8400
C2C12 cell lineATCCCRL-1772
CentrifugeUnicoC8606
Change-A-Tip High Temp CauteriesBovie Medical CorporationHIT
Confocal microscopyZeissZeiss 780 Upright Confocal
DBA/2J-mdx miceThe Jackson Laboratory013141
DMEMCorning 10-013-CM
Fetal Bovine Serum (FBS)Corning 35-011-CV
Goat serum MP Biomedicals, LLC191356
IsofluranePatterson Veterinary07-893-1389
KetamineHenry Schein056344
Mounting Medium with DAPI Vector LaboratoriesH-1500
Mouse Retractor SetKent ScientificSURGI-5001
Polyethylene glycol tert-octylphenyl etherFisher ScientificBP151-100
Rodent ventilatorHarvard Apparatus55-7066
SW-28 Ti rotorBeckman342207
The Vevo 2100 Imaging PlatformFUJIFILM VisualSonicsVevo 2100Ultrasound System 
UltracentrifugeBeckman365672
Ultra-Clear TubesBeckman344058
Xylazine (XylaMed)Bimeda-MTC Animal Health Inc.1XYL003 8XYL006

References

  1. Yiu, E. M., Kornberg, A. J. Duchenne muscular dystrophy. Journal of Paediatrics and Child Health. 51 (8), 759-764 (2015).
  2. Nudel, U., et al. Duchenne muscular dystrophy gene product is not identical in muscle and brain.

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Duchenne Muscular DystrophyExosomesMyogenic Progenitor CellsCardiac FunctionEchocardiographyUltracentrifugationIntramyocardial Transplantation

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