Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing

ABSTRACT

The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer.

KEYWORDS:

• Extracellular vesicles
• exosomes
• wound healing
• oral keratinocytes
• clinical samples
• therapy
• regenerative medicine

Geolocation information

This work was performed in Tokyo and Iwate, Japan and in Stockholm, Sweden.

Acknowledgments

This work was supported by The Swedish Society of Medicine, Erik och Edith Fernströms stiftelse för medicinsk forskning, Misao-Yanagihara-Grant for regenerative medicine research and JSPS KAKENHI Grant Number JP18H02985. We thank Dr Takahashi and Mr Okada at The Cancer Institute, Japanese Foundation for Cancer Research for help with Nano Tracking Analysis. We also thank Mr Kinji Ishida and Tomohito Hanasaka at Technical Support Center for Life Science Research (LSR) of Iwate Medical University, Iwate, Japan for the help with TEM observations. Finally, we are grateful to CellSeed Inc for providing the clinical-grade media.

Disclosure statement

Tokyo Women’s Medical University has a pending patent regarding the use of oral mucosa-derived exosomes with SS, NK and T Iwata as inventors

Supplementary material

Supplemental data for this article can be accessed here

Additional information

Funding

This work was supported by the Swedish Society of Medicine, Erik och Edith Fernströms stiftelse för medicinsk forskning, Misao-Yanagihara-Grant for regenerative medicine research and JSPS KAKENHI [Grant Number JP18H02985].