Fig 1.
Cre-mediated lineage tracing approach.
(A) Elastase-Cre Tg mice, in which the elastase promoter drives the expression of Cre in pancreatic acinar cells, were crossed with RTFN-Pdx1-EGFP mice. The double heterozygous ERTF-Pdx1-EGFP mice lost the loxP-flanked neor gene in the acinar cells. Pdx1 and EGFP were reversibly induced in these cells by withdrawing Dox, which had been added to the drinking water to suppress their expression. (B) Schematic illustration of our hypothesis that if acinar-to-β-cell transdifferentiation/reprogramming could be induced by the sustained high expression of Pdx1, we might find EGFP-positive insulin-producing cells in the exocrine pancreas and/or in islets in the ERTF-Pdx1-EGFP mice. (C) Time course and histology. Dox was withdrawn from the drinking water of 4-5-week-old ERTF-Pdx1-EGFP mice, and histological changes of the pancreas were examined 3–9 weeks later (hematoxylin and eosin staining). Lower right panel: The areas surrounded by dotted lines represent tubular complexes. Arrow shows an increase of mesenchymal cells and connective tissue. Asterisks indicate islets. Bars = 50 μm.
Fig 2.
Immunohistochemical analysis of the pancreas of ERTF-Pdx1-EGFP mice.
(A-H) Pancreas sections from ERTF-Pdx1-EGFP mice were stained for amylase (red), Pdx1 (green), insulin (purple), and DNA (blue). Strong ectopic expression of Pdx1 in the acinar cells was induced when Dox was withheld from the drinking water for more than 6 weeks. Only the islets were positive for Pdx1 in Dox (+) control pancreas sections as shown in (A) and (E). Bar = 50 μm. (I-P) Pancreas sections from ERTF-Pdx1-EGFP mice were stained for CK19 (red), Pdx1 (green), insulin (purple), and DNA (blue). Strong ectopic expression of Pdx1 in the CK19-positive duct cells was observed when Dox was withheld from the drinking water for more than 10 weeks. “d” represents duct cells. Bar = 50 μm. (Q) Pancreas sections from ERTF-Pdx1-EGFP mice 7 weeks after Dox withdrawal were stained for CK19 (red), and EGFP (green). Arrows indicate EGFP/CK19 double-positive cells. “d” represents duct cells. Bar = 25 μm. (R) Pancreas sections from ERTF-Pdx1-EGFP mice without Dox treatment for 10 weeks were stained for insulin (red), EGFP (green), HNF1β (purple), and DNA (blue). Arrows indicate EGFP/HNF1β double-positive cells, which were detected from around 6 weeks after Dox withdrawal and were probably derived from acinar cells, suggesting that acinar-to-ductal transdifferentiation occurred. Arrows indicate double-positive cells. “d” represents duct cells. Bar = 25 μm.
Fig 3.
Immunohistochemical analysis of the acinar-to-β-cell transdifferentiation/reprogramming.
(A-C) Sections from the pancreas of ERTF-Pdx1-EGFP mice 3, 6, and 10 weeks after Dox withdrawal were stained for insulin (red), EGFP (green), and DNA (blue). The presence of EGFP-positive cells expressing insulin suggests the occurrence of acinar-to-β-cell transdifferentiation/reprogramming. No EGFP-positive cells were seen when Dox treatment was continued (A). Bars = 50 μm. (D) Pancreas sections were obtained from ERTF-Pdx1-EGFP mice 10 weeks after Dox withdrawal and stained for insulin (red), EGFP (green), and DNA (blue). Bar = 200 μm. (E-G) Magnified views of the dotted line-box in (D) are shown. Staining of E-cadherin (light blue) was included in (G). Bars = 50 μm. A substantial number of EGFP-positive islet cells were insulin-positive, and these double-positive cells were evenly distributed in the islets. These changes in islets were seen all over the pancreas (D).
Fig 4.
Immunohistochemical analysis of the pancreas of ERTF-Pdx1-EGFP mice maintained without Dox.
Sections from the pancreas of ERTF-Pdx1-EGFP mice 4, 7, and 10 weeks after Dox withdrawal (n = 3, 3, and 4, respectively) were stained for insulin, EGFP, and DNA. (A) The percentage of EGFP-positive cells among insulin-positive islet cells was calculated for each mouse. The total number of insulin-positive cells examined was 4968, 6690, and 9555 at 4, 7, and 10 weeks after Dox withdrawal, respectively. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05. (B) EGFP expression in islet-like clusters containing fewer than 10 insulin-positive cells was examined for the pancreas of these mice. The percentage of islet-like clusters containing insulin-producing cells with only EGFP-negative (open bars), EGFP-positive and negative (grey bars), and only EGFP-positive (black bars) cells was determined. (C-E) Immunohistochemical analysis of the pancreas of ERTF-Pdx1-EGFP mice 10 weeks after Dox withdrawal. Sections were stained for insulin (red) and EGFP (green). Right panel (E) is a merged view of (C) and (D). Arrow shows an islet-like cluster in which all the insulin-positive cells were also EGFP-positive. Arrowhead shows EGFP-positive insulin-producing cells in an islet-like cluster. Bar = 50 μm. (F-I) Increased BrdU-positive cells in the islets of ERTF-Pdx1-EGFP mice 6 weeks after Dox withdrawal. Pancreas sections were stained for BrdU (red), DNA (blue), and EGFP (green). Merged image of (G) with insulin staining (blue) is shown in (H). Bar = 25 μm. The percentage of BrdU-positive cells among EGFP-negative and -positive insulin-positive islet cells was determined (I). In the islets, BrdU-positive cells were significantly more enriched in EGFP/insulin double-positive cells than in insulin-positive EGFP-negative cells. The total number of insulin-positive islet cells examined was 1514, 1762, 2233, and 3375 each from four ERTF-Pdx1-EGFP mice. **P < 0.01. (J-L) Pancreas sections from ERTF-Pdx1-EGFP mice 10 weeks after Dox withdrawal were stained for glucagon (red) and EGFP (green). Serial section to that used in (C-E) was used. Right panel (L) is a merged view of (J) and (K). No EGFP/glucagon double-positive cells were observed. Bar = 50 μm.
Fig 5.
Detection of endocrine hormone-negative EGFP-positive islet cells.
(A-E) PECAM-1 staining of endocrine hormone-negative EGFP-positive islet cells. Pancreas sections from ERTF-Pdx1-EGFP mice 10 weeks after Dox withdrawal were stained with the anti-GFP antibody (green), anti-PECAM-1 antibody (light blue), a mixture of antibodies against the endocrine hormones insulin, glucagon, PP, and somatostatin (red), and DAPI (blue). Single staining views and merged views are shown. Arrowhead indicates an endocrine hormone-negative EGFP-positive islet cell, which was also negative for PECAM-1, suggesting that it was not from the endothelial lineage. Arrows indicate endocrine hormone-negative EGFP-negative islet cells. These cells were positive for PECAM-1, suggesting that they were from the endothelial lineage. Bar = 50 μm. (F-J) Pancreas sections from ERTF-Pdx1-EGFP mice with Dox (F) and without Dox for 10 weeks (G-J) were stained with the anti-GFP antibody (green) (H, J), anti-Nkx6.1 antibody (light blue), a mixture of antibodies against the endocrine hormones insulin, glucagon, PP, and somatostatin (red), and DAPI (blue). Arrows indicate endocrine hormone-negative EGFP-positive Nkx6.1 positive islet cells. No endocrine hormone-negative Nkx6.1 positive islet cells were seen when Dox treatment was continued (F). Bar = 50 μm.
Fig 6.
Effect of Pdx1 expression in ERTF-Pdx1-EGFP mice with STZ-induced diabetes.
(A) Time course. ERTF-Pdx1-EGFP mice were given STZ 4 weeks after Dox withdrawal. Six weeks after STZ injection, the ipGTT was carried out and their pancreata were used for immunostaining analyses. (B-C) Non-fasting blood glucose levels were measured every three or ten days after STZ injection. Improvement of non-fasting blood glucose levels was seen in the ERTF-Pdx1-EGFP mice maintained without Dox (- Dox; n = 6). In contrast, sustained high blood glucose levels were seen in the Dox (+) ERTF-Pdx1-EGFP mice (+ Dox; n = 6). Note that the maximum glucose level of detection was 600 mg/dl. (D) The ipGTT was performed 6 weeks after STZ injection. The ERTF-Pdx1-EGFP mice maintained without Dox showed improved glucose tolerance (n = 6) compared with the Dox (+) ERTF-Pdx1-EGFP mice (n = 6). Data are shown as mean ± S.E. **P < 0.01 by Student’s t-test.
Fig 7.
Immunohistochemical analysis of the pancreas of STZ-treated ERTF-Pdx1-EGFP mice without Dox.
ERTF-Pdx1-EGFP mice were given STZ 4 weeks after Dox withdrawal. Six weeks after STZ injection, their pancreata were used for immunostaining analyses. (A) Immunostaining for insulin (red) and EGFP (green). Bar = 200 μm. (B-G) Magnified views of the islet regions including the dotted line-box in (A). Bars = 25 μm. Merged images including DAPI staining are also shown in (D) and (G). (H) Insulin-positive cells were rarely seen in the STZ-treated wild-type controls. (I) The percentage of EGFP-positive cells among insulin-positive islet cells was determined for each islet of the pancreas from three STZ-treated and three STZ-untreated ERTF-Pdx1-EGFP mice, 10 weeks after Dox withdrawal (n = 18–40 for each mouse). Each bar represents the mean for a single animal (19%, 28%, 24%, 63%, 60%, and 69%, respectively). The proportion of EGFP-positive cells among insulin-positive islet cells was significantly higher in the STZ-treated animals than in the STZ-untreated controls (P < 0.05).