1. Volume 138, Issue 7, Pages 2519-2530 (June 2010)
Differentiation of Pancreatic Acinar Cells to Hepatocytes Requires an Intermediate Cell Type 
Sung–Yu Wu, Chi–Che Hsieh, Ruei–Ren Wu, Jimmy Susanto, Tsung–Ta Liu, Chia–Rui Shen, Yu Chen, Chien–Chang Su, Fang–Pei Chang, Hsiao–Min Chang, David Tosh, Chia–Ning Shen 
Gastroenterology 
Volume 138, Issue 7, Pages (June 2010)
DOI: /j.gastro
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2. Figure 1
Time course of expression of pancreatic and hepatic markers during transdifferentiation of pancreatic AR42J-B13 cells. (A–D) Immunofluorescent staining of AMY/C/EBPβ, CPA/C/EBPβ, TFN/C/EBPβ, CYP3A1/C/EBPβ, and TFN/Gln Syn in AR42J-B13 cells that were incubated without (A) or with DEX+OSM for 2 (B), 3 (C), and 5 days (D). Cell nuclei were stained with Hoechst dye (blue). (E) Rat hepatocytes were used as positive controls for the liver markers TFN, CYP3A1, and Gln-Syn. (F) Rat exocrine cells were used as positive controls for exocrine markers CPA and AMY.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
The role of the PI3K/Akt pathway in hepatic transdifferentiation of pancreatic cells. (A) AR42J-B13 cells were incubated with DEX+OSM for 15 minutes, 30 minutes, 1 hour, 8 hours, 1 day, 3 days, or 5 days. Total cell lysates were collected at the time points indicated and expression of CPA, pAKT, AKT, C/EBPβ, and Gln-Syn was determined by Western blotting. α-Tubulin was used as a loading control. (B) AR42J-B13 cells were preincubated with or without LY for 30 minutes, before exposure to DEX+OSM for 3 days. Immunofluorescent staining was then performed for the following: C/EBPβ, Gln-Syn, or TFN. Cell nuclei were counterstained with Hoechst dye (blue). (C and D) AR42J-B13 cells were transfected with the pCMS-EGFP vector or pCMS-EGFP-dnAkt (dominant negative Akt) vector and then treated with DEX+OSM for 3 days. (C) Graph showing percentage of GFP-positive cells which are also TFN-positive cells. Bars represent standard deviations. **P < .01 (utilizing the Mann–Whitney test, the difference was statistically significant when compared with controls). (D) Transfected cells (GFP-positive cells, arrows) were immunostained for Gln-Syn, TFN, and C/EBPβ (red). Cell nuclei were stained with Hoechst dye (blue).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

4. Figure 3
Exposure to insulin enhances the conversion from pancreatic cells to hepatocytes. (A) RT-PCR analysis of AR42J and AR42J-B13 cells for amylase, pdx-1, insulin, and ptf1a. β-actin was used as an internal control. (B) Real-time quantitative RT-PCR analysis showing expression of insulin in AR42J and AR42J-B13 cells (day 3). Data represent at least 3 separate experiments normalized to gapdh with AR42J-B13 as the calibrator. (C) Immunostaining for TFN in AR42J-B13 cells that were incubated with DEX+OSM using control rabbit IgG or anti-insulin receptor (anti-IR) antibodies. (D and E) Western blotting analysis of pAKT and Akt in AR42J-B13 cells that were incubated with anti-IR antibodies (D) and in AR42J cells that were incubated with DEX and/or insulin for 1, 2, 4, or 8 hours (E). β-Actin was used as a loading control. (F) AR42J-B13 or AR42J cells were treated with DEX+OSM or DEX+insulin (INS)+OSM for 5 days. TFN, C/EBPβ, HNF-4, and CYP3A1 were detected by immunostaining. Cell nuclei were counterstained with Hoechst dye (blue).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

5. Figure 4
Insulin up-regulates ABCG2 expression during hepatic transdifferentiation. (A–D) AR42J cells were incubated for 5 days with 1 μmol/L DEX with or without either 10 μg/mL insulin (INS) or 10 ng/mL OSM. RT-PCR (A) and quantitative RT-PCR (B) was performed to determine the expression levels of abcg2, c/ebp-β, hnf-4, and cyp3a1. Gapdh was used as a control. Real-time quantitative RT-PCR data shown are representative of at least 3 independent experiments with the DI-treated group used as the calibrator for abcg2, the DEX-treated group (day 3) used as the calibrator for c/ebp-β and hnf-4, and the DIO-treated group used as the callibrator for cyp3a1. Bars represent standard deviations. **P < .01 indicates that the difference between DEX and DEX+INS (DI) or DEX+OSM (DO) and DEX+INS+OSM (DIO) was statistically significant. (C) Cell growth of AR42J or AR42J-B13 cells that were incubated with or without insulin. Cells were counted after 1, 2, or 3 days' culture. Values are expressed as percentage of the initial cell number and represent mean ± standard deviation (SD) of triplicate experiments. (D) AR42J-B13 cells were preincubated with or without LY for 30 minutes at various concentrations before being treated with DEX+OSM for 3 days. Western blotting was used to detect C/EBPβ, Gln-Syn, or TFN. (E) Immunostaining was performed for TFN and ABCG2 in C/EBPβ-transfected AR42J-B13 cells with or without addition of insulin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

6. Figure 5
ABCG2 is expressed during hepatic transdifferentiation. (A) Expression of CPA, C/EBPβ, ABCG2, or Gln-Syn in AR42J-B13 cells that were incubated without or with DEX+OSM for 1, 2, 3, or 5 days was determined by Western blotting. α-Tubulin was used as a loading control. (B–D) AR42J-B13 cells were exposed to DEX+OSM for 3 days (B and D–F) or for 1, 2, or 3 days (C). (B) Double immunostaining for AMY/ABCG2, C/EBPβ/ABCG2, or TFN/ABCG2. The cell nucleus was counterstained with Hoechst dye (blue). (C) FACS analysis for expression of ABCG2 and TFN. (D) Hoechst dye efflux by transdifferentiated cells resulted in the SP-phenotype (gated green region). (E) Immunofluorescent staining for ABCG2, AMY, and TFN on SP or non-SP cells. (F) RT-PCR for abcg2, ptf1a, transferrin, and nestin in SP-isolated or non-SP isolated cells. Gapdh was used as a control.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Differentiation potential of SP cells. (A–D) SP and non-SP cells were isolated from AR42J-B13 (A) or AR42J (B–D) cells that were incubated with DEX+insulin (INS) for 3 or 5 days. (A) SP cells were cultured for 4 and 7 days with or without EGF and NIC. RT-PCR was performed to determine expression of abcg2, nestin, and insulin. Gapdh was used as a control. (B) SP and non-SP cells were cultured with or without GLP-1 for 5 days, and RT-PCR was performed to determine expression of abcg2, insulin, pc2, glut2, and c/ebp-β. Gapdh was used as a control. (C) SP cells were cultured with 10 nmol/L GLP-1 for 5 or 7 days before immunofluorescent staining for INS and C-peptide, respectively. (D) INS secretion assay following a 10 nmol/L/2-hour glucose challenge.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Hepatic transdifferentiation of adult mouse pancreatic acinar cells. (A) EGFP-labeled pancreatic acinar cells isolated from adult Elas-CreER;Z/EG mouse pancreata were incubated with 1 μmol/L DEX for 7 days. Immunostaining was performed for AMY, C/EBPβ, TFN, and ABCG2 (red). The cell nucleus was counterstained with Hoechst dye (blue). (B) FACS analysis of ABCG2 expression in mouse acinar cells treated with DEX+insulin (INS), DEX+OSM, or DEX+INS+OSM for 3 days. (C) Pancreatic acinar cells isolated from adult mouse pancreas were incubated with DEX, DEX+INS (DI), or DEX+OSM (DO) for 7 days or with DEX for 12 days. Real-time quantitative RT-PCR was performed to determine the expression level of abcg2, c/ebpβ, and transferrin. Gapdh was used as a control. Data are representative of 2 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Direct transdifferentiation of adult mouse pancreatic acinar cells to hepatocytes and ABCG2-positive intermediates. (A) Mouse primary exocrine cells isolated from Elas-CreER;Z/EG mice injected with tamoxifen and cultured for 2 to 4 days. FACS analysis was performed to determine the expression of EGFP and ABCG2. (B) EGFP-labeled pancreatic acinar cells isolated from Elas-CreER;Z/EG mice were incubated for 2 days with DEX+INS+OSM. The SP phenotype (gated region) was examined by using flow cytometry to determine Hoechst dye efflux. Preincubation with verapamil suppressed the SP-phenotype and the confirmed gated region was generated from Hoechst dye efflux. (C) Mouse primary exocrine cells isolated from Elas-CreER;Z/EG mice injected with tamoxifen were cultured for 4 days. EGFP-labeled pancreatic acinar cells were sorted using flow cytometry and cultured for 9 days with DEX. Immunostaining was performed for TFN and ABCG2 (red). Cell nuclei were counterstained with Hoechst dye (blue). (D) Mouse primary exocrine cells isolated from Z/EG mice were infected with Ad-ABCG2-CreERT2 (multiplicity of infection = 12.5) for 1 day followed by incubation with DEX+4-hydroxytamoxifen for 3 days and then DEX alone for a further 9 days. Immunostaining was performed for detecting TFN and C/EBPβ (red). Cell nuclei were counterstained with Hoechst dye (blue).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions