Volume 140, Issue 3, Pages 998-1008.e4 (March 2011) Human Bone Marrow–Derived Clonal Mesenchymal Stem Cells Inhibit Inflammation and Reduce Acute Pancreatitis in Rats  Kyung Hee Jung, Sun U. Song, Tacghee Yi, Myung–Shin Jeon, Sang–Won Hong, Hong–Mei Zheng, Hee–Seung Lee, Myung–Joo Choi, Don–Haeng Lee, Soon–Sun Hong  Gastroenterology  Volume 140, Issue 3, Pages 998-1008.e4 (March 2011) DOI: 10.1053/j.gastro.2010.11.047 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Characterization of hcMSCs isolated from human BM. (A) Fibroblast-like shapes of hcMSCs on plastic culture dishes and crystal violet staining for clear visualization of hcMSCs. (B) Expression of several stem cell markers by using flow cytometry. (C) The multilineage potential of hcMSCs, adipogenic, osteogenic, and chondrogenic differentiation, and molecular markers of gene expression during each differentiation by reverse-transcription PCR. Original magnification, 40× and 100×. Aggrecan, large aggregating proteoglycan; aP2, activating protein 2; Col, collagen α1 chain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPL, lipoprotein lipase; M, marker; OCN, osteocalcin; PPARγ2, peroxisome proliferator-activated receptor γ2. Gastroenterology 2011 140, 998-1008.e4DOI: (10.1053/j.gastro.2010.11.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Effects of hcMSCs in rats with mild and severe AP. (A) Histologic analysis of AP. (B) Decreased apoptotic cells by hcMSCs in mild and severe AP. (C) Pancreas/body-weight ratio and activities of amylase (U/L), lipase (U/L), and MPO (U/mL). Each value represents the mean ± standard deviation of 4 separate experiments. *P < .05 and **P < .01, compared with mild and severe AP groups. Con, control; Con + hcMSCs, hcMSC-alone infusion group; AP, mild and severe AP group; AP + hcMSCs, hcMSCs infusion in mild and severe AP groups. Original magnification, 200×. Gastroenterology 2011 140, 998-1008.e4DOI: (10.1053/j.gastro.2010.11.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Tracking of infused hcMSCs. (A) Pancreas sections of CM-DiI–labeled hcMSCs at 1 × 106, injected into rats with or without mild or severe AP. (B) Human genomic AluI PCR from pancreas tissues. Sample 1, Con; samples 2 and 3, Con + hcMSCs; samples 4 and 5, mild AP + hcMSCs; samples 6 and 7; severe AP + hcMSCs; sample 8, human DNA. (C) Distribution of hcMSCs in various tissues. Distribution was assessed from pancreas, lung, liver, spleen, and kidney sections after CM-DiI–labeled hcMSCs (1 × 106) injection in rats with or without mild or severe AP. The numbers of CM-DiI–labeled hcMSCs represents the mean ± standard deviation of at least 10 fields. Con + hcMSCs, hcMSC-alone infusion group; mild and severe AP + hcMSCs, hcMSCs infusion in mild and severe AP group. Original magnification, 200×. Gastroenterology 2011 140, 998-1008.e4DOI: (10.1053/j.gastro.2010.11.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Combined FISH for human chromosome and immunofluorescent staining. A representative image from combined FISH for a human-specific chromosome after CM-DiI–labeled hcMSCs in rats with mild and severe AP. Mild and severe AP + hcMSCs, hcMSCs infusion in mild and severe AP; hCEN, human chromosome centromere. Original magnification, 200×. Gastroenterology 2011 140, 998-1008.e4DOI: (10.1053/j.gastro.2010.11.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Inflammatory cytokines and mediators after infusion of hcMSCs. (A) mRNA expression levels. (B) TGF-β, TNF-α, and IFN-γ serum levels after hcMSC infusion by enzyme-linked immunosorbent assay. Data are the mean ± standard deviation for at least 3 separate experiments. *P < .05 and **P < .01, compared with mild and severe AP. Con, control; Con + hcMSCs, hcMSC-alone infusion group; AP, mild and severe AP; AP + hcMSCs, hcMSs infusion in mild and severe AP. Gastroenterology 2011 140, 998-1008.e4DOI: (10.1053/j.gastro.2010.11.047) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Suppression of T cells by hcMSCs. (A) PBMCs and lymph node cells were isolated from 2 different rats and human blood, respectively. S, Sprague–Dawley rats; W, Wistar; A, B; PBMC donors; M, hcMSCs; L, lymphocytes. (B) Lymph node cells from rats were stimulated with indicated stimuli and Foxp3+ and (C) Annexin V expression were measured in CD4+ T cells. (D) Inhibition of T-cell infiltration and (E) expression of Foxp3+ by hcMSCs in pancreas tissues. Data represent the mean ± standard deviation for at least 5 fields. *P < .05 and **P < .01, compared with mild and severe AP. Con, control; Con + hcMSCs, hcMSC-alone infusion group; AP, mild and severe AP; AP + hcMSCs, hcMSC infusion in mild and severe AP. Original magnification, 200×. Gastroenterology 2011 140, 998-1008.e4DOI: (10.1053/j.gastro.2010.11.047) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Effect of hcMSCs on different concentrations (×105 and ×106) in rats with mild AP. (A) Pancreas sections of CM-DiI–labeled hcMSCs at 1×105 and 1×106, injected into rats with mild AP. (B) Histologic analysis of AP. (C) Pancreas/body-weight ratio and activities of amylase (U/L), lipase (U/L), and MPO (U/mL). Con, control; Con + hcMSCs, hcMSC-alone infusion group; AP, mild AP group; AP + hcMSCs, hcMSCs infusion in mild AP group. Original magnification, 200×. Gastroenterology 2011 140, 998-1008.e4DOI: (10.1053/j.gastro.2010.11.047) Copyright © 2011 AGA Institute Terms and Conditions