1. Volume 152, Issue 5, Pages 1174-1186 (April 2017)
Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice 
Su Yeon An, Yu Jin Jang, Hee-Joung Lim, Jiyou Han, Jaehun Lee, Gyunggyu Lee, Ji Young Park, Seo-Young Park, Ji Hyang Kim, Byung-Rok Do, Choongseong Han, Hee-Kyung Park, Ok-Hee Kim, Myeong Jun Song, Say-June Kim, Jong-Hoon Kim 
Gastroenterology 
Volume 152, Issue 5, Pages (April 2017)
DOI: /j.gastro
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2. Figure 1
The secretomes of UCMSCs and hpUCMSCs attenuate liver fibrosis. (A) A schematic representation of the experimental design. (B) Photomicrographs of liver sections stained with Sirius red (upper) and immunohistochemical stains using anti-α-SMA (red) and F4/80 (green) antibodies and DAPI (blue) (lower) at day 3 after injection of the secretomes (scrtm) of either UCMSCs or hpUCMSCs. Normal livers were obtained from animals that received neither TAA nor scrtm. Sham livers received TAA followed by vehicle medium injections. Scale bars, 100 μm. (C and D) Fold changes of fibrotic areas and α-SMA-immunoreactive areas at day 3 (C) and day 7 (D) after scrtm injections. The percentage of positive areas of total image area (% of pixel) were measured using NIH imageJ and shown as relative values to those in normal liver tissues that were arbitrarily set as 1. Values are means ± SEM from randomly selected fields of liver sections obtained from 3 to 6 different animals per group *P < .05 and **P < .01 compared with sham controls. *** P < .05 compared with UCMSC-scrtm. (E) Sirius red stains of liver tissues at day 7 after scrtm injections. Scale bars, 100 μm.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Regulation of fibrosis-related genes in fibrotic livers after secretome injections (A). Heat maps of four major categories of fibrosis-related genes in TAA-induced fibrotic liver tissues. Mouse fibrosis PCR array was conducted in fibrotic liver tissues 3 days after scrtm injections. The results are shown as average levels of the relative expression (n = 3 for each group) (B and C). Details of the expression of TGFβ and Smads. The heat map and dataset for all 84 genes are provided in Supplementary Figure 4 and Supplementary Table 1.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
UCMSC- and hpUCMSC-secretomes inhibit HSC activation in vitro. (A) Phase-contrast and immunofluorescence images of hTert-HSCs. hTert-HSCs were treated with either UCMSC-scrtm or hpUCMSC-scrtm in the presence of TGFβ1 under serum-free (SF) conditions. Analyses were conducted 48 hours after the indicated treatments. Scale bars, 100 μm. (B) Quantification of α-SMA-positive areas. Percentages of immunoreactive areas were expressed as relative values to those in control groups grown under SF condition in the absence of TGFβ1. (C and D) Representative Western blots for α-SMA and pSMAD2 (C) and for the expression α-SMA after treatment with different dosages of UCMSC-scrtm and hpUCMSCs-scrtm in the presence of TGFβ1 (D). (E) Quantitation of pSMAD2 and α-SMA levels from three independent Western blot analyses. (F) qPCR analysis of the relative expression of α-SMA, collagen 1a1 (COL1a1), and collagen 1a2 (COL1a2). The data were normalized to GAPDH expression and expressed as relative values compared with control. *P < .05 and **P < .01 versus the TGFβ1 treatment. ***P < .05 compared with UCMSC-scrtm.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Predictions of anti-fibrotic candidate molecules in the hpUCMSC-secretome. (A) Thirty-two proteins with > 5-fold abundance in the hpUCMSC-scrtm compared with the UCMSC-scrtm. Relative protein abundance was determined by comparing the normalized ratio of the number of peptides identified in Nano-Chip-LC/QTOF-MS analyses. The complete list of common proteins identified in both the UCMSC-scrtm and the hpUCMSC-scrtm are shown in Supplementary Table 2. (B) Western blot analysis of the cell-free secretomes showing the secretion levels of decorin, PEDF, and MFGE8. Ponceau S was used to evaluate protein loading for the scrtm. (C) Potential network interactions of decorin, PEDF, and MFGE8 in fibrogenic processes. The fibrosis network was constructed by integrating the three candidate molecules with other major TGFβ-induced fibrosis-associated molecules, such as TGF receptor, Smads, α-SMA, collagens, and TIMPs, into the MetaCore GeneGo mapping tool. The resulting network was computationally generated based on the databases curated from publications. The colors of the lines denote activation (green), inhibition (red), and unspecified interactions (gray).
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
MFGE8 antagonizes TGFβ-induced HSC activation in vitro. (A) Western blot analysis of α-SMA in human primary HSCs after treatment with indicated human recombinant proteins. (B) Dose titration analysis of MFGE8 on inhibition of TGFβ1-mediated activation in human primary HSCs. Cells were treated with TGFβ1 in the presence of different doses of MFGE8 as indicated for 48 hours. (C and D) Western blot (C) and qPCR (D) analyses showing the expression of TGFβ receptor type I and II receptors and Smad7 after indicated treatments. **P < .01 versus the TGFβ1 treatment. (E) A representative Western blot showing the secretion of MFGE8 from MSCs obtained from different human tissues (UCMSCs, umbilical cord; SHEDs, exfoliated deciduous teeth; BMMSCs, bone marrow). ‘hp’ indicates MSCs that were primed by hepatic induction. (F) Validation of the levels of MFGE8 secretion from MSCs of different tissues. The levels of MFGE8 in cell-free secretomes were determined by ELISA before and after hepatic priming (hp). Values represent means ± SEM of 3 different secretomes from three different batches per each cell line. **P < .01 compared with naïve MSCs of different tissues.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Anti-fibrotic effects of MFGE8 in the liver of C57BL/6 mice intoxicated with TAA. (A) Histological analyses of TAA-induced fibrotic liver after administration of secretome and MFGE8. Liver sections were obtained 3 days after treatment with sham (medium vehicle; n = 12), hpUCMSC-scrtm (n = 12), hpUCMSC-scrtm plus MFGE8 neutralizing antibodies (20 μg/mL) (n = 4), or MFGE8 recombinant protein (160 μg/kg) (n = 20). The level of fibrosis and HSC activation was compared after staining with hematoxylin and eosin (H&E), Sirius red, Masson’s trichrome (MT), and α-SMA antibody. Scale bars, 100 μm. (B and C) Quantification of histological analyses. Percentages of fibrotic (Sirius red) and immunoreactive (α-SMA) areas were measured using NIH image J and expressed as relative values to those in normal livers. (D) Degree of hepatic fibrosis measured by hydroxyproline content in TAA-induced fibrotic tissues at day 3 and 7 after administration of rhMFGE8. *P < .05 and **P < .01 compared with sham controls. ***P < .01 compared with secretome injection.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Levels of MFGE8 in liver tissues and serum of patients with cirrhosis. (A) Immunohistochemical analysis of normal and cirrhotic liver tissues for the expression of MFGE8. Scale bars, 100 μm. (B and C) Western blot analysis showing the expression of MFGE8 in normal and cirrhotic liver tissues. Protein samples in different lanes were obtained from different individuals (n = 6, each group). Densitometric Western blot data were relatively normalized to actin levels and the expression level of MFGE8 was shown as relative values to normal tissues in (C). (D) ELISA analyses showing serum levels of MFGE8 in normal and patients with cirrhosis (n = 16, each group).
Gastroenterology  , DOI: ( /j.gastro )
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