Journal: Hepatology (Baltimore, Md.)

Article Title: Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA

doi: 10.1002/hep.30542

Figure Lengend Snippet: Regulation of proliferative, apoptotic, and fibrosis effects by LSCEVs in human cholangiocytes. (A) A bromodeoxyuridine (BrdU) cell proliferation assay kit (Colorimetric; Novus Biologicals, LLC, Centennial, CO) was used, and 10 μM BrdU was added to 4,000 cells/well (H69 cells) into 96-well plates and incubated for 48 hours in Dulbecco’s modified Eagle’s medium (DMEM) deprived of fetal bovine serum in the presence of normal HHEVs or human MSCEVs or human LSCEVs, with various treatments as indicated. Endothelial growth factor-induced (10 ng/mL) proliferation was also evaluated in H69 cells incubated with or without ribonuclease (RNase)-pretreated LSCEVs (30 μg/mL). The absorbance was measured at 450 nm using the iD5 Multi-Mode Microplate Reader from Molecular Devices (San Jose, CA). (B) LSCEV treatment also blocked LPS-induced cholangiocyte proliferation. In the presence of LPS, H69 cells exhibited a doubling time of 46 hours, whereas LSCEV-treated cells (30 μg/mL) exhibited a doubling time of approximately 67 hours (P < 0.05 at 48 hours and 72 hours control versus LSCEV-treated cells). Data are presented as the mean number of cells ± SEM from three independent experiments. (C) Release of stem cell factor and granulocyte-macrophage colony-stimulating factor by 1 × 105 H69 cells incubated for 24 hours or 48 hours with 30 μg/mL LSCEVs compared with H69 cells incubated with HHEVs (control). (D) The percentage of apoptotic cells after 48-hour TNF-α stimulation (100 ng/mL) was evaluated by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. TNF-α-treated H69 cells were incubated with different kinds of EVs or RNase-treated LSCEVs, or LSCEVs pretreated with trypsin or 100 μg/mL of sHA. (E) Effect of LSCEVs on TGF-β-induced fibrosis marker α-SMA in H69 cholangiocytes. H69 cholangiocytes were treated with TGF-β (25 mM) in the presence of EVs as indicated for 48 hours. Viable cells were collected, and the total RNA was isolated for real-time quantitative PCR analysis. (F) Effect of LSCEVs on TGF-β-inhibited senescence of human HSCs. HSCs were treated with EVs plus TGF-β (25 mM) for 48 hours. The cellular senescence index was measured by fluorescence intensity of β-gal cellular senescence assay. *P < 0.05 relative to HHEV or respective controls. #P < 0.05 relative to LSCEV or TGF-β controls. Abbreviations: NS, not significant; sHA, soluble hyaluronic acid.

Article Snippet: Thus, both the protective effects of LSCEV against LPS-induced ductular reaction and TGF-β-induced fibrosis in cholangiocytes, plus the activation in HSCs, might contribute to its recovery activity. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Regulation of proliferative, apoptotic, and fibrosis effects by LSCEVs in human cholangiocytes. (A) A bromodeoxyuridine (BrdU) cell proliferation assay kit (Colorimetric; Novus Biologicals, LLC, Centennial, CO) was used, and 10 μM BrdU was added to 4,000 cells/well (H69 cells) into 96-well plates and incubated for 48 hours in Dulbecco’s modified Eagle’s medium (DMEM) deprived of fetal bovine serum in the presence of normal HHEVs or human MSCEVs or human LSCEVs, with various treatments as indicated.

Techniques: BrdU Cell Proliferation Assay, Incubation, Modification, TUNEL Assay, Marker, Isolation, Real-time Polymerase Chain Reaction, Fluorescence