1. Volume 136, Issue 5, Pages 1750-1760.e13 (May 2009)
Notch Signaling as Gatekeeper of Rat Acinar-to-β-Cell Conversion in Vitro 
Luc Baeyens, Stefan Bonné, Tomas Bos, Ilse Rooman, Cindy Peleman, Tony Lahoutte, Michael German, Harry Heimberg, Luc Bouwens 
Gastroenterology 
Volume 136, Issue 5, Pages e13 (May 2009)
DOI: /j.gastro
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2. Figure 1
Notch pathway expression in EGF/LIF treated acinar cells. (A–E) Expression of Notch1-related genes relative to β-actin at various culture times analyzed by qRT-PCR. Ct values were between 30 and 27 (A), 30 and 27.5 (B), 29 and 25 (C), 30.5 and 28 (D), 38 and 29 (E). (n = 4). *P < .05.
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3. Figure 2
Modulation of β-cell neogenesis by Notch1-EC, Jagged1, and Delta-like ligands (Dll) 4. Percentage of cells expressing Ngn3 (A), Pdx1 (C), and insulin (E) as determined by immunocytochemistry at various time points in culture. Different conditions were compared: addition of exogenous ligands Jagged1 (black columns), Dll4 (grey columns), or Notch1-EC (extracellular domain of the receptor; blue columns) (n = 7; #P < .05 compared with controls). Micrographs illustrate the Notch1-EC condition for Ngn3 at 48 hours (B), Pdx1 at 72 hours (D), and insulin at 72 hours (F). Double staining for cytokeratin-20 (B). Scale bar, 15 μm.
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4. Figure 3
Effects of Notch modulation by RNA interference on acinar-to-β-cell conversion. (A and B) Effect of lentiviral transduction of shRNA directed against either Notch1 (A) or Hes1 (B) on the proportion of β-cells. Transduction of the cells with shNotch1 or shHes1 induced a significant increase in the proportion of β-cells (n = 4; #P = .041 compared with shScrambled condition). Treatment with the ligands Jagged1 or Dll4 prior to knockdown emphasized the effect of the RNA interference (shNotch1 or shHes1) on β-cell differentiation (n = 4; #P = .009 compared with shScrambled condition). In conditions treated with Notch1-EC, subsequent RNA interference has no additional effect (n = 4; P = .12). (C and D) Percentage of transduced cells identified by the reporter DsRed, coexpressing insulin. Transduction of the cells with shNotch1 (C) or shHes1 (D) led to a significant increase in transduced β-cells (n = 4; #P = .035 compared with shScrambled condition). Prior exposure to the ligands Jagged1 or Dll4 reduced the proportion of shScrambled transduced β-cells severely, whereas specific Notch pathway silencing (shNotch1 or shHes1) gave a clear protective effect against the action of Notch ligands (n = 4; #P = .005 compared with shScrambled condition). In conditions treated with Notch1-EC, no difference in the proportion of transduced β-cells after interference with Notch1 or Hes1 was observed when compared with control shRNA (n = 4; P = .059). (E) Evaluation of the role of Ngn3 in differentiation conditions treated with Notch1-EC. The silencing of Ngn3 completely abrogates the coexpression of the reporter eGFP with Ngn3 protein compared with controls (H) (n = 3; #P = .007).
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5. Figure 4
Newly generated β-cells are of acinar origin as demonstrated by lectin tracing. (A and B) WGA label specificity and efficiency in the pancreas 72 hours after intraparenchymal injection of the lectin. WGA labels specifically acinar cells with an efficiency of 59.7% ± 0.3% (n = 4) and is not found in any other pancreatic cell type (A). Immunostaining for amylase (red) reveals coexpression with the lectin label (green) (B). Scale bar, 100 μm. (C and D) WGA label specificity and efficiency evaluated after isolation of the pancreatic acini. WGA label was observed in 62.8% ± 2.2% of the acinar cells and was absent in other pancreatic cell types (C). Staining for the acinar transcription factor Mist1 (red) shows that the WGA lectin (green) is only present in acinar cells (D). Scale bar, 15 μm. (E and F) WGA label specificity and efficiency evaluated after suspension culture (96 hours) to induce acinar dedifferentiation, followed by the β-cell differentiation treatment on monolayer cultures (72 hours). Cells have lost their acinar phenotype and gained a duct-like morphology (characterized by expression of cytokeratin 20). 63.5% ± 2.3% (n = 4) Of the cytokeratin-positive fraction contained the lectin label, and, after proendocrine treatment, 62.1% ± 1.7% (n = 4) of the β-cells contained WGA (E). Staining for the ductal marker cytokeratin 20 (blue) and β-cell marker insulin (red) showed a coexpression with the acinar cell tracer WGA (green) (F). Scale bar, 15 μm.
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6. Figure 5
In vitro maturity profile of newly formed β-cells. (A) Percentage of insulin expressing cells that coexpress other β-cell markers analyzed by immunocytochemistry. Comparison among in vitro generated β-cells, engrafted β-cells, and mature islet β-cells. New β-cells (white columns) express Pdx1 and C-peptide at the same level as islet β-cells (black columns) (n = 4; P = .32), but a significantly lower proportion of the β-cells expressed other maturity markers such as Glut2, IAPP, Chromogranin A, and MafA (n = 4; #P = .006 compablack with islet β-cells). After transplantation of the cells, the engrafted β-cells (gray columns) matured to the level of islet β-cells (n = 4; P = .45). (B) Comparison of the cellular insulin content of new β-cells (white columns) and islet β-cells (black columns). No significant difference between both groups was detected (n = 3; P = .28). (C) Secreted insulin as a percentage of total cellular insulin after stimulation with 20 mmol/L glucose. Comparison between new β-cells (white columns) and islet β-cells (black columns); a lower capacity for glucose-induced insulin secretion is seen in new β-cells as compared with mature β-cells (n = 3; #P = .023).
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7. Figure 6
Beta cells from acinar origin are capable to revert hyperglycaemia upon transplantation in diabetic animals. (A) Immunodeficient mice were injected with alloxan at day −2 to induce β-cell destruction and hyperglycemia. Upon transplantation of acinar-derived β-cells generated by our differentiation protocol with Notch1-EC (solid line) under the kidney capsule on day 0 (1 × 10 E5 new β-cells per animal), normoglycemia was restored (yellow area). Nephrectomy of the graft-bearing kidney on day 31 resulted in acute reversal to the diabetic state, proving that the grafted cells had restored normoglycemia. Mice transplanted with control grafts (no proendocrine treatment in vitro) remained hyperglycemic (dashed line) (n = 4; #P = .037). (B) Lentivirus-mediated Luciferase-labeling of the transplanted cells allowed for noninvasive imaging and revealed the formation of grafts with a stable mass when they were generated by our differentiation protocol with Notch1-EC (solid line). Control grafts displayed immediate deterioration in the intensity of the luminescent signal. By day 4, the signal was almost completely abrogated, indicative for loss of the graft (dashed line) (n = 4; #P = .031). (C) Visualization of the luminescent signal in a mouse engrafted with cells generated by our differentiation protocol with Notch1-EC. Luminescent signals correspond to the ectopic site of implantation and display stable signal intensity until the graft was removed.
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8. Figure 7
Phenotypical analysis of the engrafted cells. (A–D) The phenotype of engrafted β-cells was evaluated by immunohistochemistry after 36 days. Double staining using anti-insulin (green) and anti-Pdx1 (red) (A), anti-C-peptide (red) (B), anti-Glut2 (red) (C), or anti-MafA (red) (D) shows that almost all cells coexpress both markers, which demonstrates in vivo maturation. Scale bar, 10 μm. (E) Double staining using anti-insulin (green) and anti-glucagon (red) revealed the presence of very few α-cells within the transplanted graft. Scale bar, 10 μm. (F) Upon removal of the graft, the maturity of the β-cells was evaluated by immunohistochemistry. Double staining using anti-insulin (green) and anti-IAPP (red) showed that almost all cells coexpressed both markers. In contrast to their in vitro phenotype, the acinar-derived β-cells underwent an in vivo maturation process postimplantation. Scale bar, 10 μm.
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9. Supplementary Figure 1
Effect of modulating Notch signaling on the exocrine cell population. (A) Expression pattern (relative to endogenous actin) of the ductal marker cytokeratin-20 (CK20) in dedifferentiated acinar cells cultured in the presence of EGF and LIF (1), or additionally exposed to Jagged1 (2), Dll4 (3) or Notch1-EC (4). A significant increase in CK20 expression is seen following Notch stimulation (#P < 0.05; n = 4), whereas inhibiting Notch signaling decreases CK20 expression (*P < 0.05; n = 4) compared to EGF/LIF treated controls. (B) Protein expression pattern of cytokeratin-20 (CK20) in EGF and LIF treated cultures (1) and cultures additionally exposed to Jagged1 (2), Dll4 (3) or Notch1-EC (4). Actin is used as internal loading control.
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10. Supplementary Figure 2
Effect of modulating Notch signaling on Notch signaling. Gene transcript analysis by real-time RT-PCR of Notch1 (A) or its downstream target Hes1 (B). Relative fold change in Notch1 or Hes1 transcript levels in the presence of EGF and LIF (1), or in conditions additionally exposed to Jagged1 (2), Dll4 (3) or Notch1-EC (4). A significant increase in both Notch1 and Hes1 mRNA expression is seen following Notch stimulation (#P < 0.01; n = 5), whereas inhibiting Notch signaling decreases Notch1 and Hes1 mRNA expression (*P < 0.05; n = 5) compared to EGF/LIF treated controls. Error bars represent mean ± s.e.m.
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11. Supplementary Figure 3
Transduction efficiency of the different silencing viruses. (A and B) Proportion of cells transduced after infection with lentivirus containing shRNA directed against either Notch1 (A) or Hes1 (B). No significant differences in transduction efficiency was noted between the different viruses (n = 3; P > 0.05 compared to shScrambled condition). Additional treatment with the ligands Jagged1 or Dll4 or with the extracellular part of the Notch1 receptor (Notch1-EC) induced no difference in the proportion of transduced cells (n = 3; P > 0.05). (C) Evaluation of the transduction efficiency after infection with lentiviruses Le-shScrambled-eGFP and Le-shNgn3-eGFP in conditions treated with EGF, LIF and Notch1-EC. No significant differences between both conditions were observed (n = 3; P > 0.05).
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12. Supplementary Figure 4
Lentivirus-mediated RNA interference. (A–C) Illustration of co-expression of the beta cell marker insulin and DsRed reporter protein in conditions transduced with Le-shNotch1-DsRed (A), Le-shHes1-DsRed (B) or Le-Scrambled-DsRed (C) and treated with the differentiation factors. More insulin/reporter co-expression was observed after specific silencing of the Notch pathway (A and B) compared to controls (C). Scale bar = 10 μm. (D–E) Immunocytochemical analysis revealed the presence of Ngn3/reporter co-expressing cells after 48h of pro-endocrine treatment in the shScrambled condition (D), contrasted by the complete absence of these cells after specific Ngn3 silencing (E). Scale bar = 10 μm.
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13. Supplementary Figure 5
Wheat Germ Agglutinin specificity after injection and isolation. (A) WGA label specificity and efficiency in the pancreas 72h after intraparenchymal micro-injection of the lectin. Co-localization of the fluorescent lectin with beta cells was never observed in this condition (n = 4). Immunostaining for insulin (red) reveals the expression of the lectin label (green) is separated from the beta cell population. Scale bar = 100 μm. (B) WGA label specificity and efficiency evaluated after partial dissociation and isolation of the pancreatic acini. WGA label was observed in the acinar cells, but not in ductal or centro-acinar cells. Staining for the duct/centro-acinar phenotypical marker cytokeratin 20 (CK20) (red) indicated that the WGA lectin (green) was only present in acinar cells. Scale bar = 25 μm. (C) After dissociation of the pancreas, pancreatic islets were isolated, dissociated and the beta cells were purified using FACS. Fluorescence-activated cell sorting revealed a subpopulation (2.4% of the total cell population) that displayed lectin positivity based on fluorescein intensity. (D) Immunocytochemical analysis of this subpopulation using staining for the beta cell marker insulin (red) showed no co-expression of beta cells with the acinar cell tracer WGA (green). Scale bar = 15 μm (E) WGA label localisation after partial dissociation and isolation of the pancreatic acini. WGA label (green) is localized in the cytoplasm pf the acinar cells, as demonstrated by E-cadherin staining (red). Scale bar = 15 μm. (F) Freshly isolated acinar cells labeled with WGA (red) were mixed with isolated beta cells labeled with ConA (green). Scale bar = 20 μm. (G) After alloxan and pro-endocrine treatment of the mixed acinar and beta cell cultures, only the acinar WGA label (red) could be detected in the insulin-expressing cells (blue) present in the monolayer culture. Scale bar = 10 μm.
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14. Supplementary Figure 6
Luminescent intensity of the pre-implantation graft and animal body weight during in vivo experiments. (A) The body weight of animals transplanted with a control graft (dotted line) did not differ significantly from animals transplanted with a treated graft (full line) at any time point (n = 4; P = 0.12). (B) No significant difference was observed between control grafts and grafts pre-treated with EGF, LIF and Notch1-EC (indicated as ELrN) prior to implantation under the kidney capsule, when comparing luminescent signal intensity (expressed as Relative Luminescent Units) (n = 4; P > 0.05). Cells untransduced with the luciferase overexpression construct displayed only minimal luminescence.
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15. Supplementary Figure 7
Phenotypical maturity of the graft. (A) Staining using anti-Luciferase (green) confirmed Luciferase expression in about 30% of the insulin-positive cells (red). Scale bar = 10 μm. (B) Histological analysis of control pancreas (B1) or pancreas from alloxan-treated, engrafted mice (B2). Insulin-positive cells were nearly totally absent after alloxan- treatment. Scale bar = 500 μm. (C–D) Upon removal of the graft, the maturity of the beta cells was evaluated by immunohistochemistry. Double staining using anti-chromograninA (red) (C) or anti-synaptophysin (red) (D) showed that almost all insulin-expressing cells (green) co-expressed both markers. In contrast to their in vitro phenotype, the acinar-derived beta cells underwent an in vivo maturation process post-implantation. Scale bar = 10 μm.
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16. Supplementary Figure 8
Electron microscopy of acinar-derived beta cells. (A) Acinar-derived beta cells (BC) 72h after initiating the pro-endocrine treatment in vitro. Scale bar = 5 μm. (B) Acinar-derived beta cells (BC) 31 days after engraftment of these cells under the kidney capsule of diabetic nude mice. KTE: Kidney Tubular Epithelium; EC: Endothelial cell. Scale bar = 5 μm. (C) Detailed picture of the secretory granules of acinar-derived beta cells in vitro (panel A). In this condition, the granules exhibit an atypical appearance indicative of the immaturity of these cells. Scale bar = 200 nm. (D) Detailed picture of the secretory granules of acinar-derived beta cells after transplantation under the kidney capsule (panel B). The secretory granules show the typical characteristics of mature insulin granules, with a dense core surrounded by a halo. Scale bar = 200 nm.
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