Additional file 1 of MFGE8 in exosomes derived from mesenchymal stem cells prevents esophageal stricture after endoscopic submucosal dissection in pigs
Creators: name: Lai, Huasheng
name: Yip, Hon-Chi
name: Gong, Yu
name: Chan, Kai-Fung
name: Leung, Kevin Kai-Chung
name: Chan, Melissa Shannon
name: Xia, Xianfeng
name: Chiu, Philip Wai-Yan
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Virology

description: Additional file 1: Figure S1. The characterization of ADMSCs and the detection of ADMSCs differentiation. A Morphology of ADMSCs at passage 0. B Flow cytometry for CD markers including CD29, CD44, CD90, CD105 and negative marker CD45. C Differentiation in vitro for adipogenesis, osteogenesis, and chondrogenesis. Scale bars, 50 μm. Figure S2. Scheme of fabricating the ChGel sponge scaffold. Figure S3. Biocompatibility of ChGel sponge scaffold. A, B Comparison of cell viability between PBS group and ChGel group through live/dead staining assay in RAW264.7 and 3T3-L1 cells respectively (scale bars, 50 μm). Figure S4 Treatment with ChGelMSC-Exo alleviates fibrosis, inflammatory and promotes angiogenesis at the submucosal layer of esophagus. A–F The quantification of the western blot on α-SMA, collagen-I, p-Smad2, IL-10, IL-1β and VEGF on day 7, 14 and 21. All data are shown as the means ± SEM. Statistical significance was analyzed by one-way ANOVA followed by a Tukey post hoc analysis. *P &lt; 0.05, **P &lt; 0.01. Figure S5. Effect of MSC-Exo on macrophage phenotype polarization. A Morphology of BMDMs and RAW 264.7; Flow cytometry was conducted to detect the macrophage markers (F4/80 and CD68) in BMDMs and RAW264.7 respectively. B Red fluorescence signals in Raw264.7 showed the process of cellular uptake of PKH26-labelled MSC-Exo. C–E The ability of MSC-Exo on promoting M2-like macrophage polarization was through a dosage-dependent manner, as tested by immunofluoresence, Western blot and PCR analysis respectively. F, G Immunofluorescence and flow cytometry were conducted to analyze the expression of CD206 on the previously polarized M1 macrophages subsequent to exposure to MSC/MSC-Exo. All data are shown as the means ± SEM. Statistical significance was analyzed by one-way ANOVA followed by a Tukey post hoc analysis. *P &lt; 0.05, **P &lt; 0.01. Scale bars, 50 μm. Figure S6. Concentration titration and time effect of TGFβ1 on fibroblast-myofibroblast transition. A, B Analysis of the fibroblasts subject to the treatment with the varying concentration of TGFβ1 by immunofluorescence and western blot. C Western blot analysis of the TGFβ1 stimulation on fibroblasts across different time points. All data are shown as the means ± SEM. Statistical significance was analyzed by one-way ANOVA followed by a Tukey post hoc analysis among multiple groups. *P &lt; 0.05, **P &lt; 0.01. Scale bar, 25 μm. Figure S7. Exosomal MFGE8 activates the STAT3 pathway and induces the M2 macrophage polarization. A Go (Gene Ontology) analysis of LC-MS/MS results was conducted to elucidate the protein functions of ADMSC-Exo. B The top 10 most enriched pathways were identified by KEGG pathway analysis. C Representative intensity value of the exosomal proteins with relatively high expression, which exceeded a 2-fold increase in comparison to Asporin. D Protein levels of p-STAT3 and STAT3 in the control (BMDMs without any treatments), MSC-Exo treatment (BMDMs cultivated with MSC-Exo), MSC-Exo+Anti-MFGE8 (BMDMs cultivated with MSC-Exo and Anti-MFGE8) and MSC-Exo+S3I-201 (BMDMs cultivated with MSC-Exo and S3I-201) groups; Comparison of p-STAT3/STAT3 ratio was conducted among these four groups. All data are shown as the means ± SEM. Statistical significance was analyzed by one-way ANOVA followed by a Tukey post hoc analysis among multiple groups. *P &lt; 0.05, **P &lt; 0.01. Figure S8. miR-148a-3p promotes proliferation and angiogenesis in HMVECs. A Expression of miR-148a-3p was detected by qPCR in HMVECs transfected with miR-148a-3p mimic or inhibitor. B MTS assay to test proliferation ability of HMVECs transfected with miR-148a-3p mimic or inhibitor. C The angiogenesis capacity of HMVECs transfected with miR-148a-3p mimic or inhibitor was evaluated by tube formation assay. 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. *P &lt; 0.05, **P &lt; 0.01. Scale bars, 100 μm. Table S1. Full gene names for pig cytokines and chemokines. Table S2. Full name of genes related to fibrosis.