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OP22 Mesenchymal stromal cell-derived exosomes stimulate epithelial regeneration in vitro and reduce experimental colitis

M. Barnhoorn*1, L. Plug1, E. Muller - de Jonge1, E. Bos2, A. van der Meulen - de Jong1, H. Verspaget1, L. Hawinkels1

1Leiden University Medical Center, Gastroenterology and Hepatology, Leiden, The Netherlands, 2Leiden University Medical Center, Cell and Chemical Biology, Leiden, The Netherlands

Background

Local injection of mesenchymal stromal cells (MSCs) stimulates the closure of perianal fistulas in inflammatory bowel disease (IBD) and was therefore recently approved for clinical use in Europe. MSCs are generally believed to work by modulation of immune responses and stimulation of tissue regeneration. MSCs are thought to communicate with neighbour cells through secreted proteins and via direct cell-to-cell contact. However, recent literature shows that they can also communicate via MSC-derived exosomes. In this project, we investigated the effect of MSC–exosomes on epithelial regeneration and whether local MSC therapy in experimental colitis could be mediated by MSC-derived exosomes. Simultaneously, we explored MSC–exosome therapy as a cell-free alternative for MSC therapy.

Methods

Exosomes were isolated from bone marrow-derived murine MSCs using ultracentrifugation. The presence of exosomes was verified using electron microscopy and western blotting for the exosome markers flotillin-1 and alix. To evaluate the epithelial regenerative capacity of exosomes in vitro, a dextran sodium sulphate (DSS)-induced cell death assay in CT26 epithelial cells was used, as well as a scratch cell migration assay. The damaged epithelial cells were treated with low (2 ng/ml) and high (20 ng/ml) concentrations of exosomes, MSC-conditioned medium (CM) with/without exosomes and non-CM. An MTS assay was used to evaluate the effects of exosomes on proliferation of non-damaged epithelial cells. To examine the therapeutic effects of MSC-derived exosomes in vivo, exosomes, MSCs, or PBS were locally applied to the distal colon in DSS-treated mice.

Results

Exosomes were successfully isolated from the CM of MSCs, as shown by high flottilin-1 and alix expression, and could be visualised using electron microscopy. PKH-labelled exosomes showed fusion with epithelial cells in vitro after 24 h. MSC–CM and a high-exosome concentration were found to increase epithelial cell survival/proliferation in the in vitro DSS assay and cell migration in the scratch assay, and also enhanced the proliferation of non-damaged epithelial cells compared with non-CM and a low concentration of exosomes. Furthermore, in vivo experiments showed that endoscopic injections with a high dose of exosomes partially reduced DSS-induced colitis, demonstrated by a higher relative body weight and lower endoscopic disease score compared with PBS-treated mice. Yet, the MSC–exosomes were not as effective as the MSC therapy in vivo.

Conclusion

We showed that a high dose of MSC-derived exosomes is able to counteract epithelial damage in vitro and partially reduce colitis in vivo. These results pave the way for further exploring cell-free MSC-related therapy by using MSC–exosomes in the treatment of IBD.