Immunohistochemical analysis of the pancreas of ERTF-Pdx1-EGFP mice maintained without Dox.

<p>Sections from the pancreas of ERTF-Pdx1-EGFP mice 4, 7, and 10 weeks after Dox withdrawal (<i>n</i> = 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. *<i>P</i> < 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. **<i>P</i> < 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.</p

Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice

<div><p>A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells <i>in vivo</i>. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of <i>in vivo</i> reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes.</p></div

Detection of endocrine hormone-negative EGFP-positive islet cells.

<p>(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.</p

Cre-mediated lineage tracing approach.

<p>(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 <i>loxP</i>-flanked neo^r 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.</p

Immunohistochemical analysis of the pancreas of STZ-treated ERTF-Pdx1-EGFP mice without Dox.

<p>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 (<i>n</i> = 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 (<i>P</i> < 0.05).</p

Immunofluorescence analysis of the pancreas of RTF-Pdx1-EGFP mice given AdV-Isl1.

<p>(A)–(C) Pancreas was excised 1.5 days after AdV injection, and sections were stained with anti-CK (red), anti-phospho-STAT3 (light blue), and anti-Isl1 (green). Merged view of (A) and (B) is shown in (C) with DAPI (blue). Arrows indicate cells stained with both anti-Isl1 and anti-phospho-STAT3 antibodies. Bar = 100 µm. (D)–(F) Expression of MafA in the TC area. Sections from AdV-Isl1-injected RTF-Pdx1-EGFP mouse pancreas, excised 6 days after the injection, were stained with anti-insulin (red), anti-MafA (light blue), and anti-CK (green). Merged view of (D) and DAPI staining (blue) is shown in (E). Another part of the TC area is shown in (F) with DAPI staining. Heterogeneous staining for MafA was seen in the insulin-positive cells in the TC area. Arrows indicate insulin-positive, but MafA-negative cells. (G)–(J) Expression of MafA in the non-TC area of the same pancreatic section as (D–F). Merged view of (G) and DAPI staining is shown in (H). Merged view of (I) and DAPI staining is shown in (J). Almost all the insulin-positive islet cells were positive for MafA. Bars = 50 µm.</p

GTSF1L and GTSF2 have two N-terminal CHHC-type Zn-finger domains.

<p>(A) Domain architecture of mouse UPF0224-family proteins. The N-terminal region has two CHHC Zn-finger domains. The black lines indicate the regions used to raise rabbit antibodies against GTSF1L and GTSF2. (B) Alignment of the amino-acid residues of the mouse UPF0224-family proteins using CLUSTAL-X. The first and second CHHC Zn-finger domains are indicated in blue and red, respectively. Asterisks indicate amino-acid residues that are conserved among GTSF1, GTSF1L, and GTSF2. Pluses indicate amino-acid residues that are conserved between GTSF1L and GTSF2.</p

The inducible Pdx1 mouse model.

<p>(A) The construct of the Tet-off regulation unit for Pdx1 expression that was integrated into the <i>ROSA26</i> locus. The tetracycline transactivator gene is expressed under the control of the ROSA26 promoter. In knock-in mice (RTF-Pdx1-EGFP mice) heterozygous for the transgene, the continuous administration of Dox prevents the tTA from binding to the tetO, thereby inactivating the transcription of Pdx1 cDNA. After Dox is withdrawn, tTA binds to the tetO, transcribing the Pdx1 cDNA and EGFP cDNA. (B)–(M) Immunofluorescence analysis of Pdx1 (red) and insulin (green) (B–G) or Pdx1 (red) and cytokeratin 19 (CK) (green) (H–M) in pancreas sections from RTF-Pdx1-EGFP mice treated with Dox or untreated for 17 days. Bars = 100 µm. (N)–(O) Magnified view of the area surrounded by the dotted line in (M). Staining with anti-CK antibody (green) and DAPI (4, 6-diamidino-2-phenylindole) (blue) is shown in (O). “d” and “i” represent a duct and an islet, respectively.</p

Effects of <i>Tcl1</i> deficiency and overexpression on ES-cell differentiation.

<p>The expression of representative stem and differentiation markers was examined by real time PCR in ES cells grown in LIF(+) culture (left half of each panel) and with EB formation (right half of each panel). For EB formation, trypsinized ES cells were seeded into a bacterial grade dish, and cultured for 12 days. Values are expressed as mean ± SEM of three technical replicates. ∗<i>P</i><0.05 and ∗∗<i>P</i><0.01 by Student’s t-test. <i>Tcl1^−/−</i> (KO) vs. wild-type (WT) or <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) cells.</p

Representative genes from microarray analysis.

*<p>Genes listed are those which showed more than 2.0-fold differences in Exp. 1 and 2 and more than 1.7-fold differences in Exp. 3 between <i>Tcl1</i>-expressing and -deficient ES cells.</p>**<p>Exp. 1: <i>Tcl1^−/−</i> #4 vs. <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1.</p>***<p>Exp. 2: <i>Tcl1^−/−</i>(CAG-EGFP) #6 vs. <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #4.</p>****<p>Exp. 3: Mean values of relative gene expression in <i>Tcl1^−/−</i> #4 vs. wild-type and that in <i>Tcl1^−/−</i> #5 vs. wild-type.</p