Research ArticleRegenerative Medicine

Mesenchymal stem cells promote healing of nonsteroidal anti-inflammatory drug-related peptic ulcer through paracrine actions in pigs

See allHide authors and affiliations

Science Translational Medicine  30 Oct 2019:
Vol. 11, Issue 516, eaat7455
DOI: 10.1126/scitranslmed.aat7455
  • Fig. 1 A porcine model of NSAID-related gastric ulcer is induced by administration of indomethacin before and after creation of artificial ulcers by ESD.

    (A) Scheme of adipose-derived mesenchymal stem cells (ADMSCs) or MSC-derived condition medium (MSC-CM) treatment for promoting healing of NSAID-related gastric ulcer in pigs. (B) Endoscopic view of gastric ulceration after dosing with indomethacin for 10 days before ESD. (C) Comparison of the ulcer healing process between pigs with indomethacin ingestion and normal controls. The ulcer index was calculated as the product of the longest and shortest diameters of the ulcers. Pigs: n = 3 in the normal control group, and n = 11 in the indomethacin ingestion group. All data are shown as the means ± SEM. Change in ulcer index was analyzed by repeated-measures ANOVA with a post hoc multivariate test for each time point. PO, per os.

  • Fig. 2 Endoscopic submucosal injection of ADMSCs facilitates healing of NSAID-related gastric ulcer by day 7.

    (A) Details of the endoscopic submucosal dissection (ESD) specimen and necropsy at day 21. (B and C) Endoscopy to measure the ulcer size in pigs. (D) Hematoxylin & eosin (H&E) staining and scoring in terms of ulcer margin height, glandular architecture, granulation tissue depth, and leukocyte infiltration. Scale bars, 100 μm. Pigs, n = 5. All data are shown as means ± SEM. Change in ulcer index was analyzed by repeated-measures ANOVA with a post hoc multivariate test for each time point. Comparison between paired ulcers was made with dependent samples t test. *P < 0.05 and **P < 0.01.

  • Fig. 3 Treatment with ADMSCs promotes cellular proliferation and angiogenesis and reduces inflammation.

    (A and B) Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and CD31. Scale bars, 100 μm. (C to F) The expression of vascular endothelial growth factor (VEGF), IL-10, and IL-1β in ulcer samples was measured by Western blot. Myeloperoxidase (MPO) activity was determined by ELISA. (G and H) Representative images (pig 1) of engraftment of stem cells (red) in vivo and fluorescence counterstaining for α-smooth muscle antigen (α-SMA) (green) and pan-cytokeratin (green). Scale bars, 200 μm. Pigs, n = 5. All data are shown as means ± SEM. Comparison between paired ulcers was made with dependent samples t test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. DAPI, 4′,6-diamidino-2-phenylindole. *P < 0.05 and **P < 0.01.

  • Fig. 4 Stem cell–secreted factors alleviate indomethacin-induced cytotoxicity and enhance angiogenesis and cell migration in vitro.

    (A) RT2 Profiler PCR array for MSC-derived cytokines and chemokines. (B and C) MTS assay testing the cell viability upon exposure to indomethacin (IND), with or without MSC-CM treatment. (D) Representative photographs of tube formation of human microvascular endothelial cells (HMVECs) on the Matrigel-coated 24-well plate after 8 hours of exposure to phosphate-buffered saline (PBS), MSC-CM, or VEGF. The number of meshes, junctions, and branches formed per high-power field (HPF), and the total length of branches per HPF were calculated by ImageJ software. (E) Representative images of vertical scratch made on the monolayer of human gastric epithelial cells GES-1 at 0, 12, and 24 hours posttreated with PBS or MSC-CM. The wound area was quantified using ImageJ software, and the percentage change was calculated as 100× (open wound area at 12 or 24 hours)/(open wound area at 0 hour). All data are shown as the means ± SEM. Statistical significance was analyzed by independent samples t test or one-way ANOVA followed by a Tukey post hoc analysis between two or multiple groups, respectively. Please refer to table S3 for the full gene names. *P < 0.05 and **P < 0.01.

  • Fig. 5 Endoscopic submucosal injection of MSC-CM enhances healing of NSAID-related gastric ulcer in pigs.

    (A) Endoscopic surveillance for ulcer healing. (B) Details of the ESD specimens. (C) H&E staining and scoring in terms of the ulcer margin height, glandular architecture, granulation tissue depth, and leukocyte infiltration. Scale bars, 100 μm. (D) Immunohistochemistry (IHC) for CD31. Scale bars, 100 μm. (E and F) The expression of VEGF in ulcer tissue was determined by Western blot and real-time quantitative PCR, respectively. (G and H) Expression of IL-1β and IL-10 in ulcer samples was examined by Western blot. (I) MPO activity was measured by ELISA. (J to N) Quantitative real-time PCR for antioxidative enzymes including catalase, superoxidase dismutase 1 (SOD1), and SOD2. The activities of catalase and SOD in ulcer tissues were measured using ELISA kits. Pigs, n = 6; pigs, n = 3 for CD31 IHC at day 7. All data are shown as means ± SEM. Change in ulcer index was analyzed by repeated-measures ANOVA with a post hoc multivariate test for each time point. Comparison between paired ulcers was made with dependent samples t test. *P < 0.05 and **P < 0.01.

  • Fig. 6 Treatment with MSC-CM induces the proliferation of wound-resident cells.

    (A) IHC for PCNA. Scale bars, 50 μm. (B to E) Immunofluorescence double staining for pan-cytokeratin, CD31, α-SMA (white arrows indicate the proliferative myofibroblasts; yellow arrows indicate the proliferative vascular smooth muscle cells), and leucine-rich repeat–containing G protein–coupled receptor 5 (Lgr5) (yellow arrows indicate the proliferative Lgr5+ cells) with PCNA. Scale bars, 75 μm. Pigs, n = 6. All data are shown as means ± SEM. Comparison between paired ulcers was made with dependent samples t test. *P < 0.05 and **P < 0.01.

  • Fig. 7 IHC for PCNA shows the proliferative organization of the UACL at the base of ulcer margin.

    Periodic acid–Schiff (PAS) staining was used to identify the UACL. Scale bars, 200 μm (low-power field) and 50 μm (high-power field).

  • Fig. 8 Treatment with MSC-CM up-regulates the transcriptions of genes related to wound healing in ulcer tissue obtained at day 7.

    (A) Heatmap of the expression of 84 genes in the MSC-CM group and the control group (n = 2 ulcers per group). (B) Volcano plot of the differentially regulated genes between the MSC-CM group and the control group. (C to E) Heatmaps of differentially regulated genes involved in the processes crucial to gastric ulcer healing, including inflammation, reepithelization, and extracellular matrix remodeling. Please refer to table S4 for the full gene names. Pigs, n = 6. All data are shown as means ± SEM. Statistical significance was analyzed by independent samples t test. *P < 0.05 and **P < 0.01.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/516/eaat7455/DC1

    Materials and Methods

    Fig. S1. ADMSCs are characterized by flow cytometry and differentiation in vitro.

    Fig. S2. Fluorescence images show stem cell engraftment after transplantation in the stomach in pigs.

    Fig. S3. Summary of the functional interactions between the identified genes and the MAPK and Akt signaling.

    Fig. S4. ADMSC treatment does not reverse the indomethacin-induced inhibition of the COX2-PGE2 axis but activates the ErK/MAPK and the PI3K/Akt pathways.

    Fig. S5. Treatment with MSC-CM induces the phosphorylation of ErK and Akt on human gastric epithelial cells GES-1 in vitro.

    Fig. S6. Blocking the ErK and Akt signaling alleviates the cell viability, migration, and angiogenic activity enhanced by treatment with MSC-CM.

    Fig. S7. Coadministration of MSC-CM does not enhance the efficacy of PPI treatment for NSAID-related gastric ulcer in pigs.

    Fig. S8. Proposed actions of ADMSCs in enhancing healing of NSAID-related gastric ulcer.

    Table S1. Raw data for ulcer index and the percentage change in ulcer size compared with day 1.

    Table S2. Quantification of cell identity of PCNA+ cells.

    Table S3. Full gene names for pig cytokines and chemokines.

    Table S4. Full name of genes related to wound healing.

    Data file S1. Raw data (provided as separate Excel file).

    References (53, 54)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. ADMSCs are characterized by flow cytometry and differentiation in vitro.
    • Fig. S2. Fluorescence images show stem cell engraftment after transplantation in the stomach in pigs.
    • Fig. S3. Summary of the functional interactions between the identified genes and the MAPK and Akt signaling.
    • Fig. S4. ADMSC treatment does not reverse the indomethacin-induced inhibition of the COX2-PGE2 axis but activates the ErK/MAPK and the PI3K/Akt pathways.
    • Fig. S5. Treatment with MSC-CM induces the phosphorylation of ErK and Akt on human gastric epithelial cells GES-1 in vitro.
    • Fig. S6. Blocking the ErK and Akt signaling alleviates the cell viability, migration, and angiogenic activity enhanced by treatment with MSC-CM.
    • Fig. S7. Coadministration of MSC-CM does not enhance the efficacy of PPI treatment for NSAID-related gastric ulcer in pigs.
    • Fig. S8. Proposed actions of ADMSCs in enhancing healing of NSAID-related gastric ulcer.
    • Table S1. Raw data for ulcer index and the percentage change in ulcer size compared with day 1.
    • Table S2. Quantification of cell identity of PCNA+ cells.
    • Table S3. Full gene names for pig cytokines and chemokines.
    • Table S4. Full name of genes related to wound healing.
    • Legend for data file S1
    • References (53, 54)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Stay Connected to Science Translational Medicine

Navigate This Article