HucMSC-Exo Promote Mucosal Healing in Experimental Colitis by Accelerating Intestinal Stem Cells and Epithelium Regeneration via Wnt Signaling Pathway

Abstract

Background: Mucosal healing has emerged as a crucial therapeutic goal for inflammatory bowel diseases (IBD). Exosomes (Exo) as a potential acellular candidate for stem cell therapy might be competent to promote mucosal healing, while its mechanism remains unexplored.

Methods: Exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) were subjected to experimental colitis mice intraperitoneally to estimate the role in mucosal healing and the regeneration of intestinal stem cells (ISCs) and epithelium. The intestinal organoid model of IBD was constructed utilizing tumor necrosis factor (TNF)-α for subsequent function analysis in vitro. Transcriptome sequencing was performed to decipher the underlying mechanism and Wnt-C59, an oral Wnt inhibitor, was used to confirm that further. Finally, the potential specific components of hucMSC‑exo were investigated based on several existing miRNA expression datasets.

Results: HucMSC-exo showed striking potential for mucosal healing in colitis mice, characterized by decreased histopathological injuries and neutrophil infiltration as well as improved epithelial integrity. HucMSC-exo up-regulated the expression of leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), a specific marker for ISCs and accelerated the proliferation of intestinal epithelium. HucMSC-exo endowed intestinal organoids with more excellent capacity to grow and bud under TNF-α stimulation. More than that, the fact that hucMSC-exo activated the canonical Wnt signaling pathway to promote mucosal healing was uncovered by not only RNA-sequencing but also relevant experimental data. Finally, bioinformatics analysis of the existing miRNA expression datasets indicated that several miRNAs abundant in hucMSC-exo involved widely in regeneration or repair related biological processes and Wnt signaling pathway might be one of the most important signal transduction pathways.

Conclusion: Our results suggested that hucMSC-exo could facilitate mucosal healing in experimental colitis by accelerating ISCs and intestinal epithelium regeneration via transferring key miRNAs, which was dependent on the activation of Wnt/β-catenin signaling pathway.

Keywords: Wnt/β-catenin signaling pathway; exosome; inflammatory bowel disease; mesenchymal stem cells; mucosal healing.

Figure 1

HucMSC-exo accelerated mucosal healing in DSS-induced colitis mice. (A) Schematic diagram for the construction of DSS-induced colitis model and hucMSC-exo treatment. (B) The survival curves of mice in DSS-induced colitis model (n refers to the number of animals in each group, n=14). (C) Changes in mice weight (n=10). (D) DAI scores of mice (n=10). (E) Macroscopic images of colon tissues. (F) Colon length in different groups (n=10). (G) H&E staining and histological scores of mice colon sections in DSS-induced colitis model (n=10, scale bar=500 µm, 40×; scale bar=200 µm, 100×). (H) The neutrophil infiltration in colon tissues measured by IHC staining of MPO (n=5, scale bar=100 µm, 400×). (I) MPO activity in colon tissues evaluated by colorimetry (n=6). (J) Fold change in serum FITC-dextran between each group showing the intestinal permeability (n=6). (K) mRNA levels of Il10, Il17, Ifng and Tnfa in colon tissues estimated by real-time PCR (n=5). Data are presented as mean ± SD. Mouse survival curves were plotted by the Kaplan–Meier method and analyzed by Log rank test. The Mann–Whitney U-test (non-normal distribution) or Unpaired Student’s t-test (normal distribution) was used to compare the variables between two groups. P<0.05 was considered as statistically significant. *P<0.05, **P<0.01 and ***P<0.001.

Figure 2

HucMSC-exo accelerated mucosal healing in TNBS-induced colitis mice. (A) Schematic diagram for the construction of TNBS-induced colitis model and hucMSC-exo treatment. (B) The survival curves of mice in TNBS-induced colitis (n=13). (C) Changes in mice weight (n=10). (D) DAI scores of mice (n=10). (E) Macroscopic images of colon tissues. (F) Colon length in different groups (n=10). (G) H&E staining and histological scores of mice colon sections in TNBS-induced colitis model (n=10, scale bar=500 µm, 40×; scale bar=200 µm, 100×). (H) The neutrophil infiltration in colon tissues measured by IHC staining of MPO (n=5, scale bar=100 µm, 400×). (I) MPO activity in colon tissues evaluated by colorimetry (n=6). (J) Fold change in serum FITC-dextran between each group showing the intestinal permeability (n=6). (K) mRNA levels of Il10, Il17, Ifng and Tnfa in colon tissues estimated by real-time PCR (n=5). Data are presented as mean ± SD. Mouse survival curves were plotted by the Kaplan–Meier method and analyzed by Log rank test. The Mann–Whitney U-test (non-normal distribution) or Unpaired Student’s t-test (normal distribution) was used to compare the variables between two groups. P<0.05 was considered as statistically significant. *P<0.05 ***P<0.001 and ns indicates P>0.05.

Figure 3

HucMSC-exo promoted the regeneration of intestinal stem cells and epithelium in experimental colitis mice. (A and B) The mRNA and protein expression levels of ISCs marker Lgr5 in colon tissues from indicated groups estimated by real-time PCR or Western blot respectively (n=5, corresponding full-length blots are presented in Supplementary Figure 4). (C) The proliferation potential of the remaining colon epithelium was measured by IHC staining of Ki-67 (n=5, scale bar=100 µm, 400×). (D) Detected cell proliferation at the level of DNA synthesis by EdU incorporation assay (n=5, scale bar=50 µm, 400×). Data were shown as mean ± SD. The Mann–Whitney U-test (non-normal distribution) or Unpaired Student’s t-test (normal distribution) was used to compare the variables between two groups. P<0.05 was considered as statistically significant.*P<0.05, **P<0.01, ***P<0.001.

Figure 4

HucMSC-exosomes promoted the regeneration of ISCs and intestinal epithelium in organoids in ex-vivo colitis model. 100 µg/mL hucMSC-exo were administrated continuously for 48 h to evaluate its effects on intestinal organoids. (A–D) The regeneration of ISCs was measured by organoid growth rate (24 h and 48 h, n refers to the number of organoids we consecutively observed in each group, n=8), budding rate (48 h) and average buddings number (48 h, n refers to the number of independent experiments, n=4). Yellow triangles marked buddings in organoid, scale Bar=500 µm, 50×. (E and F) The proliferation potential of epithelium in organoids was measured by IF staining of Ki-67 and EdU incorporation assay (48 h, n=4, scale Bar=500 µm, 40×), results were expressed as the ratio of the integrated density of Ki-67 (red) or EdU (green) to DAPI (blue). Data were shown as mean ± SD. The Mann–Whitney U-test (non-normal distribution) or Unpaired Student’s t-test (normal distribution) was used to compare the variables between two groups. P<0.05 was considered as statistically significant. *P<0.05 and ns indicates P>0.05.

Figure 5

HucMSC-exosomes activated the Wnt/β-catenin signaling pathway in experimental colitis mice. The mRNA of colon tissues from 5 mice in DSS+PBS group and 5 mice in DSS+EXO group were enrolled for RNA sequencing. (A) Volcano map of differentially expressed genes (DEGs) with p ≤ 0.05 and fold change ≥ 2. (B) Enriched biological processes for genes in DSS+EXO group versus DSS+PBS group. (C) Enriched pathways associated with ISCs regeneration and mucosal healing. (D) Gene set enrichment analysis. NES, normalized enrichment score. It is generally considered that the gene set under the pathway of |NES| > 1 is meaningful. (E and F) The protein expressions level of WNT2, active β-catenin and total β-catenin were analyzed by Western blot (n=5, corresponding full-length blots are presented in Supplementary Figure 4). (G) The localization and expression of active β-catenin (red) was analyzed by IF staining of active β-catenin (n=5, scale bar=50 µm, 400×). The purple region produced by the mixture of red and blue (DAPI) was thought to be the place where β-catenin entered the nucleus. Data were shown as mean ± SD. The Mann–Whitney U-test (non-normal distribution) or Unpaired Student’s t-test (normal distribution) was used to compare the variables between two groups. P<0.05 was considered as statistically significant. *P<0.05, **P<0.01, ***P<0.001, ns indicates P>0.05.

Figure 6

Wnt-C59 blocked the therapeutic efficacy of hucMSC-exosomes partially. (A) Experimental scheme for the pathway inhibited model. (B–F) exhibited the survival curves (n=10), mice weight, DAI scores, macroscopic images of colonic tissues and colon length of mice in different groups respectively (n=6). (G and H) H&E staining and corresponding histological scores of mice colon sections (n=6, scale bar=500 µm, 40×; scale bar=200 µm, 100×). (I and J) The neutrophil infiltration in the colon tissues measured by IHC staining of MPO (n=5, scale bar=100 µm, 400×). (K) Fold change in serum FITC-dextran between each group (n=6). Data were shown as mean ± SD. Mouse survival curves were plotted by the Kaplan–Meier method and analyzed by Log rank test. The Unpaired Student’s t-test was used to compare the variables between two groups. P<0.05 was considered as statistically significant. *P<0.05, **P<0.01 and ns indicates P>0.05.

Figure 7

Wnt-C59 blocked the promoting effects of hucMSC-exosomes on regeneration of ISCs and intestinal epithelium partially. (A) Western blot analysis of the protein expression level of WNT2, active β-catenin, total β-catenin and LGR5 in the pathway inhibited model (n=5, corresponding full-length blots are presented in Supplementary Figure 4). (B) The localization and expression of active β-catenin (red) was analyzed by IF staining of active β-catenin. The purple region produced by the mixture of red and blue (DAPI) was thought to be the place where β-catenin entered the nucleus. The proliferation potential of colon epithelium was measured by IHC staining of Ki-67 ((C) n=5, scale bar=100 µm, 400×) and EdU incorporation assay ((D) n=5, scale bar=50 µm, 400×). Data were shown as mean ± SD. The Mann–Whitney U-test (non-normal distribution) or Unpaired Student’s t-test (normal distribution) was used to compare the variables between two groups. P<0.05 was considered as statistically significant. *P<0.05, **P<0.01, ***P<0.001, ns indicates P>0.05.

Figure 8

Identification of hucMSC-exo-specific microRNAs based on public data acquisition and bioinformatic analysis. (A) HucMSCs and hucMSC-exo miRNAs abundance analysis using GSE46989, GSE69909 and GSE159814 from the GEO DataSet as well as sequencing data from Yi Zhang. (B) Venn diagram for the intersection of the 20 most abundant miRNAs from three different datasets and the overlap miRNAs. (C) Gene Ontology analysis of the TargetScan-, miRDB- and TarBase-predicted mRNA targets for the four overlap miRNAs in three datasets. (D) KEGG analysis of the predicted mRNA targets.

Figure 9

The schema of hucMSC-exosomes to promote mucosal healing.