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Exosomes Derived From Mesenchymal Stem Cells Accelerate Skeletal Muscle Regeneration
Yoshihiro Nakamura, Shigeru Miyaki, Hiroyuki Ishitobi, Tomoyuki Nakasa, Naosuke Kamei, MD, PhD, Mitsuo Ochi.
Hiroshima University, Hiroshima city, Japan.
Disclosures:
Y. Nakamura: None. S. Miyaki: None. H. Ishitobi: None. T. Nakasa: None. N. Kamei: None. M. Ochi: None.
Introduction: A great deal of reports demonstrated the paracrine actions of Mesenchymal stem cells (MSCs) have shown
therapuetic effects for several diseases and tissue repair. We previously showed that transplantation of bone marrow-derived
MSCs (BMMSCs) promoted the regeneration of injured skeletal muscle (1). However, a number of transplanted BMMSCs didn’t
differentiate into skeletal myofibers. Therefore, our results suggested that secretion from MSC contributed to the regeneration
of skeletal muscle. Although the key topic of paracrine effect used to be protein such as cytokine and chemokine, microvesicles
(MVs) such as exosomes have recently attracted the attention as new players in cell-to-cell communication. Most cells release
MVs, which range from 30 to 1000 nm in diameter, and can be found in body fluids such as blood. Exosomes are one type of
MVs of endocytic origin released to the extracellular environment. These small particles, about 30 -200 nm, are derived from the
fusion of multivesicular bodies to plasma membranes. Interestingly, exosomes can contain mRNA, microRNA (miRNA) and
protein (2, 3, 4). Therefore, exosomes function in cell-to-cell communication factors as carriers of genetic information, and are
associated with the pathogenesis of various diseases and tissue repair. Exosomes derived from cells which have therapeutic
effect may promise for the application of treatment, because the property of exosomes reflects the specialized property of their
original cells. We hypothesized that MSC-derived exosomes function in a novel regulatory factor that contributes to
regeneration of skeletal muscle and elucidate mediator in exosomes.
Methods: Human BMMSCs were cultured with serum-free Dulbecco’s modified Eagle’s medium (DMEM) for 48 h. The
conditioned media were collected. The conditioned media were further ultracentrifuged by 110000 G. Supernatants were
collected as exosome-depleted conditioned media. To obtain exosome, pellets were performed a second ultracentrifugation.
We verified isolated exosomes by western blots, using an antibody against the commonly found exosome biomarker protein,
flotillin-1, CD9 and CD81. The mouse myoblast cell line, C2C12 were seeded and the media were changed to DMEM(control),
MSC-conditioned medium (MSC-CM), MSC-exosome (MSC-exosome) suspension DMEM or MSC-exosome-depleted conditioned
media (MSC-CM (exo-)) containing 2% horse serum to induce myogenic differentiation. To examine myogenesis, quantitative
real time-PCR assays were performed for the MyoD, myogenin and Myh4. We performed immunocytochemistry for myosin
heavy chain using mouse anti F59. Total nuclear numbers were counted to evaluate cell proliferation and the fusion index (ratio
of nuclei in myotubes to all nuclei) was calculated to evaluate the myogenic differentiation. To evaluate the angiogenic functions
of MSC-CM, -exosome and -CM (exo-), we examined whether migration and tube formation in human umbilical vein endothelial
cells (HUVECs). Migration assay was evaluated using HUVECs in upper chamber with membrane. After 4 hours of incubation,
migrated cells in the lower side of a chamber were quantified. An angiogenesis assay kit (KURABO) was used as tube formation
assay. HUVECs co-cultured with human fibroblasts were cultured. After 8 days, the total tube length were measured. 8-week-old
male C57BL/6 mice were injected with cardiotoxin into the belly of tibialis anterior (TA) muscle. PBS or MSC-exosomes were
injected into TA muscle at 1, 3 and 5 days after muscle injury. Mice were killed at 3 or 7 days. Frozen TA muscles were sectioned
in the axial planes. For the evaluation of muscle regeneration, Hematoxylin and eosin staining was performed. The diameter and
the total number of centronucleated myofiber were measured. For the evaluation of the fibrotic area, Masson trichrome
staining was performed. For the evaluation of capillary density on regenerated skeletal muscle, immunofluorescence staining of
CD31 as the marker for endothelial cell was performed on tissue sections. The concentrations of VEGF, FGF-2, IGF-1, MCP-1 and
IL-6 were measured in MSC-CM, -exosome, and -CM (exo-) using the ELISA or BioPlex (BIORAD). MiRNA expression profiles in
MSC, MSC-CM or -exosome were analyzed by miRNA expression assay using nCounter.
Results: To confirm whether exosomes were isolated from conditioned media by ultracentrifugation, the pellets and
supernatant were analyzed by Western blotting. The exosome marker such as flotillin-1, CD9, and CD81 were present in the
pellets. Conversely, these exosome markers were undetected in the supernatant. We examined whether exosomes induce
myogenesis in C2C12. MSC-CM and -exosome significantly induced the expression of MyoD, myogenin and Myh4 compare with
control. Moreover, total nuclear number and fusion index in C2C12 were significantly increased by MSC-CM and -exosome
compared with control media. These results suggested MSC-CM and -exosome promote both proliferation and differentiation of
myoblast. To examine whether MSC-exosomes induce angiogenenic activity, migration and tube formation was evaluated in
HUVECs. The migration activity was significantly increased by MSC-CM, -exosome and -CM (exo-) in comparison with control.
Tube length was significantly longer in incubation with MSC-CM or -exosome than in incubation with DMEM. These results
suggest MSC-CM and -exosome induce angiogenic activity. We examined the therapeutic effects of MSC-exosome on muscle
regeneration in vivo. At 3 days after muscle injury, TA muscles were completely induced necrosis of myofibers in both control
group and in MSC-exosome group. At 7 days, centronucleated myofibers appeared in both groups. The diameter and the
number of centronucleated myofibers in MSC-exosome group was significantly increased at 7 days compared with the control
group (Fig.1). Masson trichrome staining at 7 days demonstrated the fibrotic area was significantly smaller in MSC-exosome
group than in control group. At vascular staining of CD31 in TA muscles at 7 days, capillary density in MSC-exosome group was
significantly increased than in control group. To identify the mediators in the beneficial exosomes, we measured several muscle
repair related-growth factors in exosomes, and profiled miRNAs in exosomes. MSC-CM and -CM (exo-) included significant
amounts of VEGF, MCP-1, and IL-6, FGF-2, PDGF-BB, and IGF-1 levels were not detected. Whereas, these cytokines and growth
factors in MSC-exosome were not detected. MiRNA profiling showed that 386 miRNAs were abundant in MSC-exosome (> 2.5
fold) compared with MSC-CM (exo-). myogenesis-related and angiogenesis-related miRNA were included in among them.
Discussion: MSC is known to exert its therapeutic effects through secreted growth factors such as VEGF. Although MSC-exosome
included no significant amounts of myogenesis-related and angiogenesis-related growth factors such as IGF-1 and VEGF, we
demonstrate that MSC-exosome promote skeletal muscle repair through activation of myogenesis and angiogenesis. These
results suggest that secretion from MSC besides growth factors and cytokines play an important role in tissue regeneration, and
seems to support our hypothesis that the MSCs derived-exosomes promote muscle regeneration through exo-miRNAs in
exosomes.
Significance: MSC-derived exosomes function in a novel regulatory factor that contributes to regeneration of skeletal muscle.
Present study has the potential to reveal important new regulatory factor/network in tissue repair, and open new biologics for
tissue repair.
Acknowledgments: This project was supported by MEXT/JSPS KAKENHI Grants Number 25253089, 24689057.
References: (1) Valadi et al. Nat Cell Biol, 2007. (2) Kosaka et al. JBC, 2010. (3) Zhang et al. Mol Cell, 2010. (4)Natsu et al. Tissue
Eng, 2004.
ORS 2014 Annual Meeting
Poster No: 0069