Cellular and Molecular Gastroenterology and Hepatology
Elsevier
image
Mesenchymal Stromal Cell–Derived Exosomes Contribute to Epithelial Regeneration in Experimental Inflammatory Bowel Disease
Volume: 9, Issue: 4
DOI 10.1016/j.jcmgh.2020.01.007
  • PDF   
  • XML   
  •       

Table of Contents

Highlights

Notes

Barnhoorn, Plug, Jonge, Molenkamp, Bos, Schoonderwoerd, Corver, van der Meulen-de Jong, Verspaget, and Hawinkels: Mesenchymal Stromal Cell–Derived Exosomes Contribute to Epithelial Regeneration in Experimental Inflammatory Bowel Disease

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).

MSC exosomes stimulate epithelial regeneration in vitro. (A) Images of CT26 cells treated for 12 hours with PKH26-labeled exosomes. (B) Percentage of Hoechst-positive DSS-damaged CT26 cells after treatment with the indicated conditions. Data represent the average of 3 independent experiments in triplicate. One-way analysis of variance, Dunnett multiple comparison with non-CM. (C) Western blot analysis for cleaved caspase-3 in 2% DSS-treated CT26 cells under the indicated conditions. Representative blot from 3 independent experiments. (D) Relative wound closure (after 27 h) of CT26 cells stimulated with the indicated conditions. Data represent the mean of 3 independent experiments in triplicate. One-way analysis of variance, the Dunnett multiple comparison with non-CM. (E) Percentage of CT26 cells in G1- and G2-phases after co-culture with exosomes. Representative data are shown from 2 independent experiments in triplicate. Student t test was used. (F) Images of green fluorescent protein-positive colon organoids cultured with PKH26-labeled exosomes for 1 week. Images are representative of 2 independent experiments. (G) MTS assay of colon organoids cultured with exosomes. Data represent 2 independent experiments performed in triplicate. Student t test. ∗P ≤ .05, ∗∗P < .01, and ∗∗∗P < .001. MW, molecular weight; PBS, phosphate-buffered saline; w, with; wo, without.
Figure 1
MSC exosomes stimulate epithelial regeneration in vitro. (A) Images of CT26 cells treated for 12 hours with PKH26-labeled exosomes. (B) Percentage of Hoechst-positive DSS-damaged CT26 cells after treatment with the indicated conditions. Data represent the average of 3 independent experiments in triplicate. One-way analysis of variance, Dunnett multiple comparison with non-CM. (C) Western blot analysis for cleaved caspase-3 in 2% DSS-treated CT26 cells under the indicated conditions. Representative blot from 3 independent experiments. (D) Relative wound closure (after 27 h) of CT26 cells stimulated with the indicated conditions. Data represent the mean of 3 independent experiments in triplicate. One-way analysis of variance, the Dunnett multiple comparison with non-CM. (E) Percentage of CT26 cells in G1- and G2-phases after co-culture with exosomes. Representative data are shown from 2 independent experiments in triplicate. Student t test was used. (F) Images of green fluorescent protein-positive colon organoids cultured with PKH26-labeled exosomes for 1 week. Images are representative of 2 independent experiments. (G) MTS assay of colon organoids cultured with exosomes. Data represent 2 independent experiments performed in triplicate. Student t test. ∗P ≤ .05, ∗∗P < .01, and ∗∗∗P < .001. MW, molecular weight; PBS, phosphate-buffered saline; w, with; wo, without.

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.

Locally applied MSC exosomes partially alleviate experimental colitis. (A) Relative body weights of mice with DSS-induced colitis, endoscopically treated with the indicated conditions. Means ± SEM. One-way analysis of variance, Dunnett multiple comparison with PBS. (B) Difference in murine endoscopic index of colitis severity (MEICS) between day 10 and day 5 for the treatment groups. One-way analysis of variance, Dunnett multiple comparison with PBS. (C) Macroscopic colonic disease score at day 10. One-way analysis of variance, the Dunnett multiple comparison with PBS. (D) Percentage of distal colon covered by cytokeratin-positive epithelial cells. Data represent 2 independent mouse experiments, n = 7–19 mice/group. ∗P < .05. PBS, phosphate-buffered saline; w, with.
Figure 2
Locally applied MSC exosomes partially alleviate experimental colitis. (A) Relative body weights of mice with DSS-induced colitis, endoscopically treated with the indicated conditions. Means ± SEM. One-way analysis of variance, Dunnett multiple comparison with PBS. (B) Difference in murine endoscopic index of colitis severity (MEICS) between day 10 and day 5 for the treatment groups. One-way analysis of variance, Dunnett multiple comparison with PBS. (C) Macroscopic colonic disease score at day 10. One-way analysis of variance, the Dunnett multiple comparison with PBS. (D) Percentage of distal colon covered by cytokeratin-positive epithelial cells. Data represent 2 independent mouse experiments, n = 7–19 mice/group. ∗P < .05. PBS, phosphate-buffered saline; w, with.

References

1 

    Panes J.Lancet 388: 2016. , pp.1281-1290

2 

    Molendijk I.Gastroenterology 149: 2015. , pp.918-927 e6

3 

    Barnhoorn M.Inflamm Bowel Dis 24: 2018. , pp.1755-1767

4 

    Phinney D.G.Gastroenterology 35: 1998. , pp.297-306

5 

    Singer I.I.Gastroenterology 115: 1998. , pp.297-306

6 

    Harting M.T.Stem Cell 36: 2018. , pp.79-90

7 

    Ferguson S.W.Sci Rep 8: 2018. , pp.1419

Supplementary References

1 

    Molendijk I.J Crohns Colitis 10: 2016. , pp.953-964

2 

    Molendijk I.Gastroenterology 149: 2015. , pp.918-927 e6

3 

    van Haaften C.J Exp Clin Cancer Res 34: 2015. , pp.38

4 

    Sato T.Gastroenterology 141: 2011. , pp.1762-1772

5 

    Barnhoorn M.Inflamm Bowel Dis 24: 2018. , pp.1755-1767

6 

    Becker C.Gut 54: 2005. , pp.950-954

7 

    Cooper H.S.Lab Invest 69: 1993. , pp.238-249

8 

    Hawinkels L.J.Oncogene 33: 2014. , pp.97-107

Acknowledgment

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.

Notes

Author contributions: M. C. Barnoorn designed the study, performed data acquisition, analysis, and interpretation, and drafted the manuscript; L. Plug performed data acquisition, analysis, and interpretation; E. S. M. Muller-de Jonge, D. Molenkamp, E. Bos, and W. E. Corver acquired and analyzed the data; M. J. A. Schoonderwoerd interpreted the data and critically revised the manuscript for intellectual content; A. E. van der Meulen-de Jong and H. W. Verspaget designed and advised in the execution of the study and critically revised the manuscript for intellectual content; and L. J. A. C. Hawinkels interpreted the data, designed and supervised the study, and critically revised the manuscript for intellectual content.
Conflicts of interest: The authors disclose no conflicts.
https://www.researchpad.co/tools/openurl?pubtype=article&doi=10.1016/j.jcmgh.2020.01.007&title=Mesenchymal Stromal Cell–Derived Exosomes Contribute to Epithelial Regeneration in Experimental Inflammatory Bowel Disease&author=M.C. Barnhoorn,L. Plug,E. S. M. Muller-de Jonge,D. Molenkamp,E. Bos,M.J.A. Schoonderwoerd,W.E. Corver,A.E. van der Meulen-de Jong,H.W. Verspaget,L.J.A.C. Hawinkels,&keyword=CM, conditioned medium,DSS, dextran sulfate sodium,MSC, mesenchymal stromal cell,&subject=Research Letter,