Additional file 1: of TSG-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing M2 macrophage switch in mice

Supplementary materials. Canine adipose tissue-derived mesenchymal stem cells: isolation, culture, and characterization. (PDF 16 kb

Additional file 4: of TSG-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing M2 macrophage switch in mice

Figure S3. mRNA expression level of TSG-6 of naive cAT-MSCs, cAT-MSCs treated with transfection reagents only (cAT-MSC + transfection reagent), cAT-MSCs transfected with a scrambled siRNA (siCTL-cAT-MSC), or cAT-MSCs transfected with TSG-6 siRNA (siTSG6-cAT-MSC) was determined by real-time RT-PCR. Results are presented as the mean Âą standard deviation of three independent experiments. ***P < 0.001. (TIFF 1818 kb

Additional file 5: of TSG-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing M2 macrophage switch in mice

Table S1. Primers used for this study. (PDF 33 kb

Additional file 2: of TSG-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing M2 macrophage switch in mice

Figure S1. cAT-MSCs have high expression of CD29, CD73, CD44, and CD90, and do not express CD31, CD34, or CD45. (TIFF 3502 kb

Additional file 3: of TSG-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing M2 macrophage switch in mice

Figure S2. cAT-MSCs have the ability to differentiate into adipocytes (Oil Red O staining), osteocytes (Alizarin Red S staining), and chondrocytes (Alcian Blue staining). Scale bars = 200 Îźm. (TIFF 9973 kb

Production of urea in hepatocyte-like cells derived from RFP- and Oct4/Sox2-ATMSCs after 28 days hepatogenic differentiation.

<p>After hepatogenic induction, urea production was significantly increased in cultures of differentiated RFP- and Oct4/Sox2-ATMSCs. Thus, the amount of urea produced by hepatocyte-like cells derived from Oct4/Sox2-ATMSCs was significantly higher than that of RFP-ATMSCs. Undifferentiated ATMSC and HepG2 Cells were used as negative and positive control, respectively. The experiments were repeated at least three times and similar findings were observed. Data represent the mean ± SD of three independent experiments. Statistical analysis was performed by ANOVA followed by Tukey’s post hoc test of the means (significant, **<i>p</i> < 0.01).</p

Immunophenotyping of RFP- and Oct4/Sox2-transfected ATMSCs.

<p>RFP-transfected ATMSCs and Oct4/Sox2-transfected ATMSCs at passage 5 were immunophenotyped for CD29, CD31, CD34, CD44, CD45, CD73, CD90, and CD105 by flow cytometry. The expression of ATMSC surface markers characteristic of MSCs was maintained.</p

Period acid Schiff (PAS) staining of RFP- and Oct4/Sox2-ATMSCs after 28 days hepatogenic differentiation.

<p>(A) Detection of glycogen in the cytoplasm of MSCs subjected to the liver differentiation protocol was demonstrated by PAS staining. PAS-positive substances stain pink in the cytoplasm of cells. (B) The number of PAS-positive cells is expressed as percentage of the total number of counted cells and was significantly higher in Oct4/Sox2-ATMSCs than that of RFP-ATMSCs. The experiments were repeated at least three times and similar findings were observed. Data represent the mean ± SD of three independent experiments. Statistical analysis was performed by student <i>t</i>-test (significant, *<i>p</i> < 0.05).</p

PCR analysis and immunofluorescence of liver markers after 28 days hepatogenic differentiation.

<p>(A) The mRNA expression level of albumin (ALB) was strongly expressed in hepatogenically differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein (AFP) was lower than that of RFP-ATMSCs. The expression levels of transferrin were not significantly different in both cells. Undifferentiated ATMSCs and HepG2 were used as negative and positive controls, respectively. (B) Hepatocyte-like cells from RFP- and Oct4/Sox2-ATMSCs are confirmed by immunofluorescence staining for AFP and ALB. Nuclei were counterstained with Hoecst33342.</p

Analysis of the Oct4 and Sox2 expression in Oct4/Sox2-ATMSCs.

<p>(A) RT-PCR analysis revealed that the mRNA expression of Oct4 and Sox2 in Oct4/Sox2-ATMSCs were significantly higher than that in RFP-ATMSCs at 24 h post-transfection. (B) Western blot analysis showed high levels of Oct4 and Sox2 expression in Oct4/Sox2-ATMSCs. Data are representative of three independent experiments, with similar results. Statistical analysis was performed by student <i>t</i>-test (significant, **<i>p</i> < 0.01).</p