Oral mucosa keratinocytes and their exosomes for epithelial tissue regeneration

Early tumors, including high grade dysplasia and intramucosal invasive cancer, of the esophagus can today be removed using endoscopic resection, often using a technique called endoscopic submucosal dissection (ESD). This treatment is better tolerated and has considerably less mortality and morbidity compared to conventional, more invasive surgery, which usually entails esophagectomy. However, after larger endoscopic dissections, stricture formation is a common complication. Such strictures are usually treated with balloon dilatations, but the procedure often has to be repeated several times and is associated with risks such as perforations.

In recent years, Japanese researchers developed a new method to reduce the risk of strictures. About two weeks before the treatment, an oral mucosa biopsy is taken from the patient from which epithelial cells are isolated and grown on special temperature-responsive polymer-coated surfaces. The polymer changes morphology and wettability properties depending on temperature, which enables non-enzymatic cell harvesting – the cells can be detached as contiguous sheets and with a large amount of extracellular matrix (ECM) maintained. After the ESD, cell sheets can be transplanted to the wound bed without the need for suture or other fixative, it is thought that the remaining ECM acts as a glue. Nine patients were treated in Tokyo and we subsequently transferred the technology to Stockholm where five additional patients were treated. Although one of nine patients in the Japanese cohort and three of five patients in the Swedish cohort still developed strictures, they appeared to be milder and easier to treat than expected. However, the aim of these projects was to evaluate safety and feasibility, further studies are needed to evaluate efficacy of the treatment.

Since some patients still developed strictures despite the cell sheet transplantation, we next aimed to evaluate if exosomes from the cell culture media could be used as a pro-regenerative agent, perhaps in combination with cell sheet therapy. Media was collected from clinical-grade production of cell sheets from eight healthy donors. The media was concentrated by ultra-filtration and exosomes were isolated by size-exclusion chromatography. The exosomes were characterized by western blot (CD9+, Flotillin-1+, GRP94-), electron microscopy and nanoparticle tracking analysis (~125 nm). They reduced the proliferation of skin fibroblasts and stimulated upregulation of gene expression of growth factors relevant for wound healing. We studied the exosomes’ adhesion to esophageal wound bed by topical application to porcine esophageal wounds ex vivo and could detect signal after as little as one minute adhesion time. We also found that the exosomes stimulated wound healing of full-thickness skin wounds in immunocompetent rats, both at the 6th day and 17th day time point.

In conclusion we found that exosomes could be isolated from cell sheet media and that they exhibited pro-regenerative properties even in a xenogeneic setting. Further studies are necessary to evaluate their potential to stimulate mucosal wound healing and reduce stricture formation of the esophagus.

List of scientific papers

I. Jonas E, Sjöqvist S, Elbe P, Kanai N, Enger J, Haas S L, Mohkles-Barakat A, Okano T, Takagi R, Ohki T, Yamamoto M, Kondo M, Markland K, Lim M L, Yamato M, Nilsson M, Permert J, Blomberg P, Löhr J M. Transplantation of tissue-engineered cell sheets for stricture pr28408991 prevention after endoscopic submucosal dissection of the oesophagus. United European Gastroenterology Journal. 2016 Dec;4(6) :741-753.
https://doi.org/10.1177/2050640616631205

II. Sjöqvist S, Ishikawa T, Shimura D, Kasai Y, Imafuku A, Bou-Ghannam S, Iwata T, Kanai N. Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing. Journal of Extracellular Vesicles. 2019 Jan 20;8(1):1565264.
https://doi.org/10.1080/20013078.2019.1565264

III. Sjöqvist S, Kasai Y, Shimura D, Ishikawa T, Iwata T, Kanai N. Gingival keratinocyte-derived exosomes regulate proliferation of fibroblasts and epithelial cells. [Manuscript]