Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are studied as a treatment for inflammatory bowel disease. Local injection of MSCs stimulates closure of perianal fistulas in Crohn’s disease.1, 2 Previously, we found that local injections of bone marrow–derived MSCs alleviated experimental colitis in mice.3 MSCs are thought to work via modulating immune responses and stimulating tissue regeneration via secreted proteins and cell–cell contacts. In addition, recent studies have indicated that MSCs also exert effects via exosomes, which are small membrane-enclosed vesicles containing proteins, DNA, and (micro)RNAs.4 The objective of this study was to evaluate if MSC-derived exosomes contribute to the therapeutic effects of local MSC therapy. We investigated whether MSC exosomes stimulate epithelial regeneration and if local application of MSC exosomes, as a cell-free alternative for MSC therapy, can alleviate colitis in epithelial damage–driven models.
MSC exosomes were isolated from murine, bone marrow–derived MSCs (Supplementary Figure 1A and B ), using ultracentrifugation of MSC-conditioned medium (CM), containing 1.2 μg exosomes per milliliter. The presence of MSC exosomes was confirmed by the markers flotillin-1 and alix (Supplementary Figure 1C ), and visualization of 50- to 150-nm vesicles using transmission electron microscopy (Supplementary Figure 1D ). The uptake of fluorescently labeled MSC exosomes by CT26 mouse colonic epithelial cells was confirmed by a red fluorescent signal upon addition of MSC exosomes to CT26 cells (Figure 1A , Supplementary Figure 2A ). To determine the effects of MSC exosomes on epithelial regeneration, CT26 cells were first damaged by exposure to dextran sulfate sodium (DSS) (Supplementary Figure 2B ). A significantly higher cell number was detected when DSS-damaged CT26 cells were cultured with 20 μg/mL MSC exosomes (Figure 1B ). The high-dose MSC exosomes reduced levels of the apoptotic marker cleaved caspase-3 in CT26 cells upon damage with 2% DSS (Figure 1C , Supplementary Figure 2C ), and 3% DSS (Supplementary Figure 2D ), indicating decreased apoptosis. Because epithelial repair is a combination of proliferation and migration, we also assessed the effects of MSC exosomes on cell migration using a scratch assay. CT26 cells treated with CM with exosomes showed the fastest wound closure, but CM without exosomes and 20 μg/mL exosomes also significantly increased wound healing compared with non-CM (Figure 1D , Supplementary Figure 2E ). In addition, also cytokine-stimulated human MSC exosomes showed increased wound closure in human epithelial cells compared with non-CM (Supplementary Figure 2F ). Non-damaged murine epithelial cells stimulated with CM with exosomes showed a slight but significant increase in proliferation in CT26 cultures (Supplementary Figure 2G ). Cell-cycle analysis showed that MSC exosomes increased the percentage of epithelial cells in both the S- and G2-phases (Figure 1E , Supplementary Figure 2H ). Next, we evaluated the effects in 3-dimensional mouse colonic organoids. We confirmed that PKH26-labeled exosomes were taken-up by the epithelial organoids (Figure 1F , Supplementary Figure 3A ) and induced organoid proliferation without changing the number of Ki67-positive cells (Figure 1G , Supplementary Figure 3B and C ). Mucin 2 and cytokeratin 20 (Supplementary Figure 3D ) were down-regulated in colonic organoids cocultured with MSC exosomes, suggesting that the increase in organoid proliferation by MSC exosomes was not leading directly to more differentiation. No differences in expression of the stem cell marker, Leucine-rich repeat-containing G-protein coupled receptor 5, and enteroendocrine marker, chromogranin A, were found (Supplementary Figure 3C ). Finally, we showed that cyclo-oxygenase 2, an enzyme described to be up-regulated in colonic epithelial cells from inflammatory bowel disease patients,5 was down-regulated significantly in colonic organoids 72 hours after exosome treatment (Supplementary Figure 3C).
Next, we used the DSS mouse colitis model to investigate if MSC exosomes are responsible for the beneficial effects of local MSC therapy. DSS-treated mice were injected endoscopically with MSCs (2 × 106), 20 μg MSC exosomes, CM (containing ∼0.24 μg exosomes), or solvent control at day 5. In vitro, 2 × 106 MSCs will produce approximately 9.6 μg of exosomes every 3 days. Local MSC therapy and, to some extent, MSC exosome therapy alleviated DSS-induced colitis, as shown by a higher relative body weight, lower murine endoscopic index of colon severity, lower macroscopic disease score, increased colon length, and decreased epithelial damage, compared with control or CM-treated mice. However, local MSC exosome therapy was less effective compared with MSC therapy (Figure 2A–D , Supplementary Figure 4). This suggests that MSCs also exert their efficacy through other mechanisms or that continuous production of exosomes is needed for profound therapeutic effects. Because locally injected MSCs are thought to be licensed in vivo by the proinflammatory milieu, it might be that cytokine-stimulated MSCs produce more efficient vesicles,6 which also is supported by our human MSC data (Supplementary Figure 2F ). The effects of MSC exosomes might be mediated by microRNAs because it was shown that microRNAs involved in cell death and growth were enriched in exosomes.7 In conclusion, our results show that MSC-derived exosomes may contribute to the amelioration of colitis by stimulation of epithelial repair and decreasing epithelial apoptosis.
The authors thank the staff of the Central Animal Facility of the Leiden University Medical Center for animal care and the group of Professor Clevers, and especially Dr van Es, from the Hubrecht Institute, and Dr Muncan from the Tytgat Institute for providing WNT3a, Noggin, and R-spondin cell lines.