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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0761
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3401-3409
Copyright © 2005 by The Endocrine Society

Combination Therapy with Epidermal Growth Factor and Gastrin Induces Neogenesis of Human Islet ß-Cells from Pancreatic Duct Cells and an Increase in Functional ß-Cell Mass

Wilma L. Suarez-Pinzon, Jonathan R. T. Lakey, Stephen J. Brand and Alex Rabinovitch

Departments of Medicine (W.L.S.-P., A.R.) and Surgery (J.R.T.L.), University of Alberta, Edmonton, Canada T6G 2S2; and Waratah Pharmaceuticals (S.J.B.), Woburn, Massachusetts 01801

Address all correspondence and requests for reprints to: Dr. Alex Rabinovitch, 430 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail: alex.rabinovitch{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pancreatic islet transplantation is a viable treatment for type 1 diabetes, but is limited by human donor tissue availability. The combination of epidermal growth factor (EGF) and gastrin induces islet ß-cell neogenesis from pancreatic exocrine duct cells in rodents. In this study we investigated whether EGF and gastrin could expand the ß-cell mass in adult human isolated islets that contain duct as well as endocrine cells. Human islet cells were cultured for 4 wk in serum-free medium (control) or in medium with EGF (0.3 µg/ml), gastrin (1.0 µg/ml), or the combination of EGF and gastrin. ß-Cell numbers were increased in cultures with EGF plus gastrin (+118%) and with EGF (+81%), but not in cultures with gastrin (–3%) or control medium (–62%). After withdrawal of EGF and gastrin and an additional 4 wk in control medium, ß-cell numbers continued to increase only in cultures previously incubated with both EGF and gastrin (+232%). EGF plus gastrin also significantly increased cytokeratin 19-positive duct cells (+678%) in the cultures. Gastrin, alone or in combination with EGF, but not EGF alone, increased the expression of pancreatic and duodenal homeobox factor-1 as well as insulin and C peptide in the cytokeratin 19-positive duct cells. Also, EGF plus gastrin significantly increased ß-cells and insulin content in human islets implanted in immunodeficient nonobese diabetic-severe combined immune deficiency mice as well as insulin secretory responses of the human islet grafts to glucose challenge. In conclusion, combination therapy with EGF and gastrin increases ß-cell mass in adult human pancreatic islets in vitro and in vivo, and this appears to result from the induction of ß-cell neogenesis from pancreatic exocrine duct cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSPLANTATION OF ISLETS isolated from human cadaver pancreases can replace insulin therapy in patients with type 1 diabetes (1), but is limited by the shortage of donor organs. This problem could be overcome if transplantable ß-cells could be generated by expansion and differentiation of adult human pancreatic progenitor cells (2, 3, 4). Islet progenitor cells reside in the pancreatic ducts of adult rodents (5, 6, 7, 8, 9, 10, 11) and humans (12, 13, 14, 15, 16), and neogenesis of ß-cells from pancreatic duct cells may be the dominant mechanism of ß-cell regeneration in humans (17, 18).

Many stimuli influence ß-cell neogenesis from pancreatic duct cells in vitro and in vivo (19). These include growth factors, such as TGF-{alpha}, epidermal growth factor (EGF), and keratinocyte growth factor (20, 21, 22, 23). Extracellular matrix also promotes ß-cell differentiation from duct cells (12, 13). In addition, gastrointestinal peptides, such as glucagon-like peptide-1 (24, 25) and gastrin (26, 27), can stimulate ß-cell neogenesis. In the rat pancreatic duct-ligated model of pancreas regeneration, gastrin enhances ß-cell neogenesis from pancreatic duct cells (26), and endogenous gastrin may be necessary for ß-cell neogenesis in this model (27). Furthermore, combined EGF and gastrin treatment was reported to increase ß-cell mass and reduce hyperglycemia in streptozotocin-diabetic rats (28) and to induce islet regeneration from pancreatic duct cells and restore normoglycemia in alloxan-diabetic mice (29). The choice of EGF and gastrin combination therapy in these studies (28, 29) was based on an earlier study in which an increase in islet mass was observed in double transgenic mice that expressed TGF-{alpha}, an EGF receptor ligand, and gastrin locally in the pancreas (30).

This study examines whether EGF and gastrin can act as ß-cell proliferative/differentiative factors in adult human pancreatic tissue in vitro and in vivo. EGF and gastrin were evaluated as ß-cell growth factors for adult human isolated islet preparations used for clinical transplantation. These islet preparations contain large numbers of exocrine duct and acinar cells in addition to endocrine cells (31). Importantly, after clinical islet transplantation, the insulin secretory response to glucose, a measure of the functional ß-cell mass, correlates significantly with the number of duct cells originally present in the islets transplanted (32). Therefore, we hypothesized that human pancreatic duct cells may serve as islet ß-cell precursors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human islets

Human pancreases were obtained, with informed consent of relatives, from brain-dead organ donors. Tissue procurement and experimental protocols were approved by the human ethics committee of University of Alberta Hospitals. Pancreases were removed from donors after in situ vascular perfusion with University of Wisconsin organ preservation solution, and islets were isolated as previously described (33, 34). Briefly, islets were isolated by intraductal controlled perfusion and digestion of the pancreas using an enzyme (Liberase human islet, Roche, Laval, Canada) and by gentle mechanical dissociation; then the islets were purified on continuous gradients of Ficoll-diatrizoic acid in a Cobe blood cell processor (model 2991, Cobe Laboratories, Lakewood, CO). Islets for these research studies were provided by University of Alberta distribution site of the Juvenile Diabetes Research Foundation Human Islet Distribution Program. The islets were incubated in 5% CO2 at 37 C for 3–5 d in RPMI 1640 culture medium containing 11 mmol/liter glucose, 2 mmol/liter L-glutamine, 0.1 mmol/liter sodium pyruvate, 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 12 mmol/liter HEPES (Invitrogen Life Technologies, Inc., Carlsbad, CA). The islets were dissociated into single cells and small clumps (≤5 cells) by incubation at 37 C for 10 min in an enzyme-free cell dissociation buffer (Invitrogen Life Technologies, Inc.), followed by syringe injection through progressively narrower gauge needles (sizes 16–22).

EGF and gastrin preparations

The EGF used in this study is recombinant human EGF1–51 expressed in yeast Pichia pastoris and purified to greater than 95% by HPLC and mass spectroscopy (Waratah Pharmaceuticals, Woburn, MA); recombinant human EGF1–51 has an asparagine substitution for glutamate at position 51 and has equal biological potency to native human EGF1–53 (35). Gastrin used in this study is human gastrin-17 synthesized and purified to greater than 97% by HPLC (Star Biochemicals, Torrance CA); this gastrin-17 has a leucine substitution for methione at position 15 to prevent oxidation and is equipotent to native gastrin-17 (36). EGF and gastrin preparations were dissolved in sterile 100 mmol/liter NaCl and 50 mmol/liter NaPO4, pH 7.4, at a concentration of 3 µg/ml and were stored in aliquots at –70 C.

Islet cell cultures

Islets were isolated from five different human pancreases for the islet cell culture studies in vitro. The dissociated islet cells were washed and suspended in phenol-red-free, serum-free OptiMEM1 medium containing insulin (10 µg/ml) and transferrin (5 µg/ml) as the only protein supplements (Invitrogen Life Technologies, Inc.) plus 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (control medium). Islet cells were seeded (106 cells/2 ml/well) in control medium into six-well, flat-bottom plates for nonadherent cell suspension culture (Sarstedt, Newton, NC), and the cells were incubated in 5% CO2 at 37 C for 2, 4, 6, and 8 wk. For the first 4 wk, islet cells in triplicate wells were incubated in control medium and in media with 0.3 µg/ml EGF, 1.0 µg/ml gastrin, and 0.3 µg/ml EGF plus 1.0 µg/ml gastrin. Fresh media were replaced each week after first centrifuging the culture plates (400 x g for 10 min) to avoid cell losses. After 4 wk of incubation, islet cells were washed in control medium without EGF or gastrin, and subsequent incubations for an additional 2 and 4 wk were carried out in control medium only for all cultures. Total cell counts, cell compositions, and islet hormone contents in cells were determined before and after 2, 4, 6, and 8 wk of culture.

Studies in vivo

Islets were isolated from three different human pancreases for the islet cell studies in vivo. The dissociated islet cells were implanted under the left renal capsule of immunodeficient female nonobese diabetic-severe combined immune deficiency (NOD-scid)/Lt mice, aged 5–7 wk (The Jackson Laboratory, Bar Harbor, ME). The mice were housed and fed under specific pathogen-free conditions in a biocontainment hood and were cared for according to the guidelines of the Canadian Council on Animal Care. Islet cell implants were carried out according to a previously described procedure (37) in a biocontainment hood. In the first study, the cell composition of the human islet cells (5 x 106) was determined before implantation and 6 wk after implantation in NOD-scid mice treated with either PBS-vehicle or 30 µg/kg·d EGF plus 1000 µg/kg·d gastrin given ip daily for 6 wk. In the second study, the insulin content of the human islet cells (1 x 106) was determined before implantation and 6 wk after implantation in NOD-scid mice treated with PBS-vehicle, 30 µg/kg·d EGF plus 30 µg/kg·d gastrin, or 30 µg/kg·d EGF plus 1000 µg/kg·d gastrin given ip daily for 6 wk. In the third study, human islet cells (3 x 106) were implanted in NOD-scid mice, and the mice were treated with either PBS-vehicle or 30 µg/kg·d EGF plus 30 µg/kg·d gastrin given ip daily for 6 wk. Then the mice were fasted for 12 h and injected iv with 1.5 g/kg glucose, and blood glucose and plasma human C peptide concentrations were measured before and 10, 30, and 120 min after glucose infusion. Human islet grafts were removed from the renal subcapsular implantation site for immunohistochemical and immunocytochemical analyses and assay of insulin contents.

Immunohistochemical studies

Islet grafts were removed with a portion of underlying kidney, fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 4.5 µm. Sections were first treated with Peroxo-Block (Zymed Laboratories, South San Francisco, CA) and BEAT blocker kit (Zymed Laboratories) to block endogenous peroxidase activity. The cells were permeabilized with 3% saponin in PBS (Sigma-Aldrich Corp., Oakville, Canada). The sections were stained with a guinea pig antiinsulin antibody diluted 1:150 (DakoCytomation, Carpinteria, Ca), followed by a biotinylated goat antiguinea pig antibody diluted 1:400 (Vector Laboratories, Inc., Burlingame, CA), and then with a streptavidin-peroxidase conjugate (Zymed Laboratories), followed by a peroxidase substrate kit (3,3'-diaminobenzidine, Vector Laboratories, Inc.) that stained insulin-containing cells brown. The sections were counterstained with hematoxylin.

Immunocytochemical studies

Islet grafts were dissociated into single cells using the cell dissociation buffer and the methods used to dissociate isolated pancreatic islets. Cells from islet grafts and from culture studies in vitro were washed five times in PBS buffer, pH 7.4, then seeded onto glass slides (104 cells/sample, two samples per slide, 16 slides/experimental condition) coated with 3-aminopropyltriethoxysilane (Sigma-Aldrich Corp., St. Louis, MO). Cells attached to slides were fixed with 4% paraformaldehyde in PBS for 1 h at 25 C, washed twice with PBS, and kept frozen at –86 C until immunostained. Slides were thawed, and cells were treated with 1% paraformaldehyde in PBS at 4 C for 10 min. One cell sample on each slide was incubated with a specific antibody to the cell type to be identified, and the second cell sample was incubated with an isotype control antibody. Insulin-positive cells were identified by incubation with a guinea pig antiinsulin antibody diluted 1:150 (DakoCytomation), followed by a biotinylated goat antiguinea pig antibody diluted 1:400 (Vector Laboratories, Inc.), and then a streptavidin alkaline phosphatase conjugate (Zymed Laboratories) and substrate kit [5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (BCIP/NBT), Vector Laboratories, Inc.] that stained insulin-positive cells navy blue. C Peptide-positive cells on another slide were identified by incubation with a mouse antihuman C peptide antibody diluted 1:150 (Cedarlane Laboratories, Hornby, Canada), followed by a biotinylated goat antimouse antibody diluted 1:75 (DakoCytomation), and then a streptavidin alkaline phosphatase conjugate (Zymed Laboratories) and substrate kit (BCIP/NBT, Vector Laboratories, Inc.) that stained C peptide-positive cells navy blue. Glucagon-positive cells on another slide were identified by incubation with a rabbit antihuman glucagon antibody diluted 1:10 (Biomeda, Foster City, CA), followed by a biotinylated goat antirabbit antibody diluted 1:75 (Vector Laboratories, Inc.), then a streptavidin alkaline phosphatase conjugate (Zymed Laboratories) and substrate kit (BCIP/NBT, Vector Laboratories, Inc.) that stained glucagon-positive cells navy blue. To identify cytokeratin 19-positive duct cells and amylase-positive cells, cells on other slides were first treated with Peroxo-Block (Zymed Laboratories) and BEAT blocker kit (Zymed Laboratories) to block endogenous peroxidase activity. The cells were then permeabilized with 3% saponin in PBS. Cytokeratin 19 (CK19)-positive duct cells were identified by incubation with a mouse antihuman CK19 antibody diluted 1:15 (DakoCytomation), followed by a biotinylated goat antimouse antibody diluted 1:75 (DakoCytomation), and then a streptavidin peroxidase conjugate (Zymed Laboratories) and substrate kit (3-amino-9-ethyl-carbazole, Zymed Laboratories) that stained CK19-positive duct cells red. Amylase-positive acinar cells on another slide were identified by incubation with a rabbit anti-{alpha}-amylase antibody diluted 1:50 (Biomeda), followed by a biotinylated goat antirabbit antibody diluted 1:75 (Vector Laboratories, Inc.), and then a streptavidin peroxidase conjugate (Zymed Laboratories) and substrate kit (3-amino-9-ethyl-carbazole, Zymed Laboratories) that stained amylase-positive cells red. To identify CK19-positive cells expressing pancreatic and duodenal homeobox factor-1 (PDX-1), the cells were first incubated with a rabbit anti-PDX-1 antibody diluted 1:1000 (provided by Dr. Helena Edlund, University of Umea, Umea, Sweden), followed by a biotinylated goat antirabbit antibody diluted 1:75 (Vector Laboratories, Inc.), and then a streptavidin alkaline phosphatase conjugate (Zymed Laboratories) and substrate kit (BCIP/NBT, Vector Laboratories, Inc.) that stained PDX-1-positive cells navy blue; then the cells were treated to block endogenous peroxidase activity, permeabilized with saponin, and stained for CK19 using the peroxidase enzyme detection method described above. Double-stained cells (PDX-1+CK19+) were stained navy blue in the nucleus (PDX-1+) and red in the cytoplasm (CK19+). To identify CK19-positive cells that expressed insulin and C peptide, cells were first stained for insulin or C peptide using the alkaline phosphatase enzyme detection methods described above, then endogenous peroxidase activity was blocked, and cells were permeabilized with saponin and stained for CK19 using the peroxidase enzyme detection method described above. Double-stained cells (insulin+CK19+ and C peptide+CK19+) were stained navy blue (insulin+ or C peptide+) and red (CK19+) in the cytoplasm. Cells that coexpressed insulin and C peptide (insulin+C peptide+) were stained for insulin (navy blue) using the alkaline phosphatase enzyme detection method; then endogenous peroxidase was blocked, and cells were stained for C peptide (red) using the peroxidase enzyme detection method. Finally, the slides were treated with a mounting medium (Crystal Mount, Biomeda) and sealed with coverslips. A total of 3000 cells in each of duplicate samples were scored blindly by two independent observers using oil immersion x100 magnification light microscopy. No cell staining was observed in samples incubated with isotype control primary antibodies and the respective secondary antibodies.

Apoptosis assay

Cells in apoptosis were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling using a cell death detection kit (Roche). Cells with DNA strand breaks (apoptotic cells) were identified by red nuclear staining.

Insulin, glucagon, and C peptide assays

Insulin and glucagon were extracted from cells by incubation in acidified ethanol (75% ethanol, 1.5% 12 mmol/liter HCl, and 23.5% H2O) for 18 h at 4 C. Ethanol extracts of cells were diluted in assay buffers. Insulin was measured using an electrochemiluminescence immunoassay together with the Elecsys 1010/2010/Modular Analytics E170 immunoassay analyzer (Roche, Mannheim, Germany). Glucagon was measured by RIA (Diagnostic Products Corp., Los Angeles, CA). Human C peptide in plasma of NOD-scid mice with human islet grafts was measured by RIA with no cross-reactivity to mouse C peptide (Linco Research, Inc., St. Charles, MO).

Statistics

All data were analyzed using one-way ANOVA, followed by Bonferroni multiple comparisons test, except for the study of plasma human C peptide responses of human islet grafts, where data were analyzed using Student’s unpaired t test. Differences between group means were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Composition of islet cells cultured with EGF and gastrin

The cell composition of islets isolated from adult human pancreases was determined by immunocytochemical staining. This revealed that the islets contained 7 ± 2% glucagon+ {alpha}-cells, 23 ± 3% insulin+ ß-cells, 5 ± 1% CK19+ duct cells, and 33 ± 2% amylase+ acinar cells. Thus, before culture, of 1.0 x 106 total islet cells seeded/well, 0.70 ± 0.17 x 105 were {alpha}-cells, 2.34 ± 0.25 x 105 were ß-cells, 0.52 ± 0.07 x 105 were CK19+ duct cells, and 3.34 ± 0.22 x 105 were acinar cells (Fig. 1Go). After 4 wk of culture in serum-free control medium, all cell types decreased in number. EGF alone increased the numbers of all cell types during the first 4 wk of culture. Gastrin alone prevented decreases in the numbers of insulin+ cells and increased CK19+ cells after 2 and 4 wk of culture. The combination of EGF and gastrin increased the numbers of glucagon+ cells, insulin+ cells, and CK19+ cells after 2 and 4 wk of culture. After the initial 4-wk incubation with EGF or gastrin alone or in combination, cell cultures were incubated for an additional 2 and 4 wk in serum-free control medium without EGF or gastrin (Fig. 1Go). In cultures previously incubated with EGF and gastrin, insulin+ cells continued to increase in number despite removal of EGF and gastrin, resulting in a 3-fold increase in insulin+ cells after 8 wk (7.77 ± 0.22 vs. 2.34 ± 0.25 x 105 cells at the start of culture; P < 0.001). In contrast, insulin+ cells decreased in number in cultures treated with EGF alone when EGF was withdrawn. Glucagon+ cells continued to increase in number after withdrawal of EGF or EGF plus gastrin. CK19+ duct cells also continued to increase in number after withdrawal of EGF or EGF plus gastrin, and after 8 wk CK19+ cells were increased 6-fold by EGF (3.41 ± 0.22 x 105 cells) and 8-fold by EGF plus gastrin (4.05 ± 0.23 x 105 cells) compared with 0.52 ± 0.07 x 105 CK19+ cells at the start of the cultures (P < 0.001). Cell viability was maintained best by EGF alone or in combination with gastrin. Thus, cell apoptosis after 8 wk was 3.0 ± 0.4% for EGF cultures and 6.8 ± 1.0% for EGF plus gastrin cultures compared with 15.1 ± 1.9% for gastrin cultures and 17.8 ± 2.5% for cells in control cultures.



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  		FIG. 1. Composition of human islet cells incubated in control culture medium and media with 0.3 µg/ml EGF, 1.0 µg/ml gastrin, and EGF plus gastrin for 4 wk (shaded area), followed by 4 wk in control medium only for all cultures. Islet cells (1 x 106) were seeded into individual wells (0 wk of culture). The numbers of {alpha}-cells (glucagon+), ß-cells (insulin+), acinar cells (amylase+), and duct cells (CK19+) were determined by immunocytochemical staining. The mean ± SE are shown for islets isolated from five human pancreases.

 
Endocrine hormone contents of islet cells cultured with EGF and gastrin

EGF alone significantly increased the insulin content of islet cell cultures after 8 wk, and the combination of EGF and gastrin increased the insulin content 2-fold (Table 1Go), which was somewhat less than the 3-fold increase in the number of insulin+ cells observed with this combination therapy (Fig. 1Go). Also, EGF plus gastrin increased the glucagon content of islets 1.5-fold (Table 1Go), and this was somewhat less than the 2.5-fold increase in glucagon+ cells observed with this combination therapy (Fig. 1Go).


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  		TABLE 1. Endocrine hormone contents of human islet cells cultured with EGF and gastrin

 
Expression of PDX-1 and insulin in CK19+ duct cells

EGF plus gastrin increased CK19+ duct cells in parallel with insulin+ cells during 8 wk of culture (Fig. 1Go). To determine whether CK19+ duct cells might give rise to ß-cells, CK19+ cells were also stained for expression of the ß-cell differentiation transcription factor PDX-1 and for insulin (Fig. 2Go). Many CK19+ cells were found after culture with EGF (Fig. 2BGo) or EGF plus gastrin (Fig. 2DGo), and fewer were found after culture with gastrin alone (Fig. 2CGo), as also shown in Fig. 1Go. Gastrin, however, induced PDX-1 expression in CK19+ cells (Fig. 2CGo), whereas EGF did not (Fig. 2BGo), but EGF did potentiate gastrin’s induction of PDX-1 expression in CK19+ cells (Fig. 2DGo). The immunocytochemical effects of EGF and gastrin shown in Fig. 2Go are quantified in Fig. 3Go. EGF alone induced only small and transient increases in PDX-1 (Fig. 3AGo) and a later small increase in insulin (Fig. 3BGo) in CK19+ cells. In contrast, gastrin induced PDX-1 expression in 61 ± 6% of CK19+ cells after 2 wk of culture, and this persisted after gastrin was removed (Fig. 3AGo). Similarly, gastrin induced insulin expression in 45 ± 6% of CK19+ cells after 2 wk of culture, and this was similar 4 wk after gastrin was removed (Fig. 3BGo). The combination of EGF and gastrin induced PDX-1 in 82 ± 2% of CK19+ cells at 2 wk, and this persisted after EGF and gastrin were removed (Fig. 3AGo). Similarly, EGF plus gastrin induced insulin expression in 62 ± 9% of CK19+ cells at 2 wk, and this persisted after EGF and gastrin were removed (Fig. 3BGo).



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  		FIG. 2. Photomicrographs of human islet cells incubated in control culture medium for 8 wk (A, E, and I); in medium with 0.3 µg/ml EGF for 4 wk, then in control medium for 4 wk (B, F, and J); in medium with 1.0 µg/ml gastrin for 4 wk, then in control medium for 4 wk (C, G, and K); and in medium with EGF plus gastrin for 4 wk, then in control medium for 4 wk (D, H, and L). Two-color immunocytochemical staining was used to identify CK19+ pancreatic duct cells (stained red) that express PDX-1 (A–D). PDX-1+CK19+ cells are stained navy blue in the nucleus (PDX-1+) and red in the cytoplasm (CK19+; C and D). CK19+ duct cells that express insulin are stained both navy blue (insulin+) and red (CK19+) in the cytoplasm (E–H). CK19+ duct cells that express C peptide are stained both navy blue (C peptide+) and red (CK19+) in the cytoplasm (J–L). Original magnification: x100; insets, x300.

 


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  		FIG. 3. Expression of the ß-cell transcription factor, PDX-1 (A), and insulin (B) in CK19+ pancreatic duct cells. Human islet cells were incubated in control culture medium and in media with 0.3 µg/ml EGF, 1.0 µg/ml gastrin, and EGF plus gastrin for 4 wk (shaded area), followed by 4 wk in control medium only for all cultures. The percentages of CK19+ duct cells expressing PDX-1 (PDX-1+CK19+) and insulin (insulin+CK19+) were determined by two-color immunocytochemical staining. The mean ± SE are shown for islets isolated from five human pancreases.

 
Insulin-stained cells coexpress C peptide

To confirm that insulin immunoreactivity in cells represented insulin produced in the cells and not insulin taken up from the culture medium, cells were double immunostained for insulin and C peptide, which is produced in the cell from proinsulin in equimolar amounts with insulin. More than 90% of insulin-stained cells in cultures that had been incubated with EGF, gastrin, and EGF plus gastrin also stained positive for C peptide (Fig. 4AGo). Also, after incubation with EGF alone, 13.1 ± 3.8% of CK19+ cells coexpressed insulin (insulin+CK19+ cells; Figs. 4BGo and 2FGo); similarly, 11.7 ± 3.5% of CK19+ cells coexpressed C peptide (C peptide+CK19+ cells; Figs. 4CGo and 2JGo). After incubation with gastrin alone, 41.7 ± 2.9% of CK19+ cells coexpressed insulin (insulin+CK19+ cells; Figs. 4BGo and 2GGo); similarly, 39.2 ± 2.9% of CK19+ cells coexpressed C peptide (C peptide+CK19+ cells; Figs. 4CGo and 2KGo). After incubation with EGF plus gastrin, 64.1 ± 9.4% of CK19+ cells coexpressed insulin (insulin+CK19+ cells; Figs. 4BGo and 2HGo); similarly, 60.9 ± 7.9% of CK19+ cells coexpressed C peptide (C peptide+CK19+ cells; Figs. 4CGo and 2LGo).



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  		FIG. 4. Coexpression of C peptide and insulin (C peptide+insulin+; A) and levels of expression of insulin in CK19+ duct cells (insulin+CK19+; B) and C peptide in CK19+ duct cells (C peptide+CK19+; C) were determined by two-color immunocytochemical staining. Human islet cells were incubated in control culture medium (con) and in media with 0.3 µg/ml EGF, 1.0 µg/ml gastrin, and EGF plus gastrin for 4 wk, then in control medium for 4 wk for all cultures. The mean ± SE are shown for islets isolated from five human pancreases.

 
Effects of EGF and gastrin on human islet cells in vivo

Human pancreatic islets were isolated and dissociated as in the culture studies in vitro, then the islet cells (5 x 106) were implanted under the kidney capsule in immunodeficient NOD-scid mice. The mice were treated with either vehicle (PBS) or EGF plus gastrin for 6 wk. Immunohistochemical examination of human islet graft sections showed more insulin-containing cells in the islet grafts from NOD-scid mice treated with EGF plus gastrin (Fig. 5BGo) than in mice treated with vehicle (Fig. 5AGo). Also, immunocytochemical analysis of cells dissociated from the grafts revealed more insulin-stained cells in islet grafts of mice treated with EGF plus gastrin (Fig. 5DGo) than in mice treated with vehicle (Fig. 5CGo). Glucagon+ cells, insulin+ cells, and CK19+ cells were significantly increased in human islet grafts in mice treated with EGF plus gastrin compared with islet grafts in mice treated with vehicle (Fig. 6Go). These findings in vivo (Fig. 6Go) are similar to those in vitro (Fig. 1Go); i.e. combined EGF plus gastrin treatment increased glucagon+ {alpha}-cells, insulin+ ß-cells, and CK19+ duct cells in human islets. Also, insulin content was significantly increased in islet grafts of mice treated with 30 µg/kg·d EGF plus 1000 µg/kg·d gastrin and were slightly, but not significantly, increased in islet grafts of mice treated with 30 µg/kg·d EGF and 30 µg/kg·d gastrin compared with islet grafts in mice treated with vehicle (Fig. 7Go). Insulin secretory responses of the human islet grafts after glucose challenge were tested in mice treated with 30 µg/kg·d EGF and 30 µg/kg·d gastrin. Insulin was measured by immunoassay of human C peptide using an RIA that did not recognize mouse C peptide. Insulin secretory responses of human islet cells implanted in EGF- plus gastrin-treated mice (plasma human C peptide levels in mice) were significantly greater 10 and 120 min after iv glucose administration than the responses of human islets implanted in vehicle-treated mice (Fig. 8BGo). Blood glucose levels were slightly, but not significantly, lower in EGF- plus gastrin-treated mice (Fig. 8AGo).



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  		FIG. 5. Photomicrographs of human islet cells implanted under the renal capsule of immunodeficient NOD-scid mice treated for 6 wk with vehicle (A and C) and 30 µg/kg·d EGF and 1000 µg/kg·d gastrin (B and D). Islet graft sections show more insulin-containing cells (stained brown) in grafts from mice treated with EGF plus gastrin (B) than in grafts from mice treated with vehicle (A). Immunocytochemical analysis of cells dissociated from the islet grafts show more insulin+ cells (stained dark blue) in grafts from mice treated with EGF plus gastrin (D) than in grafts from mice treated with vehicle (C).

 


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  		FIG. 6. Human islet cells were implanted under the renal capsule of immunodeficient NOD-scid mice. Islet cell types were identified by immunocytochemical staining before implantation and 6 wk later (post-implant) in mice treated with PBS (vehicle) or 30 µg/kg·d EGF plus 1000 µg/kg·d gastrin given ip daily for 6 wk. The mean ± SE are shown for islet cells from one human donor pancreas in triplicate before and after implantation in three mice, each treated with vehicle or EGF plus gastrin. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. before implantation). §, P < 0.05; {dagger}, P < 0.01; {ddagger}, P < 0.001 (vs. after implantation, vehicle).

 


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  		FIG. 7. Human islet cells were implanted under the renal capsule of immunodeficient NOD-scid mice. Insulin contents in cells were measured before implantation and 6 wk later (post-implant) in mice treated with vehicle (0 EGF and 0 gastrin), 30 µg/kg·d EGF plus 30 µg/kg·d gastrin, and 30 µg/kg·d EGF plus 1000 µg/kg·d gastrin given ip daily for 6 wk. The mean ± SE are shown for islet cells from one human donor pancreas in quadruplicate before and after implantation in four mice for each treatment group shown. *, P < 0.01 (vs. before implantation). {dagger}, P < 0.05 (vs. after implantation, vehicle).

 


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  		FIG. 8. Human islet cells were implanted under the renal capsule of immunodeficient NOD-scid mice, and the mice were treated with vehicle or 30 µg/kg·d EGF plus 30 µg/kg·d gastrin given ip daily for 6 wk. The mice were then fasted for 12 h and injected iv with 1.5 g/kg glucose. Blood glucose (A) and plasma human C peptide (B) concentrations were measured before (0 min) and 10, 30, and 120 min after glucose injection. The mean ± SE are shown for six vehicle-treated mice and four EGF- plus gastrin-treated mice implanted with islet cells from one human donor pancreas. *, P < 0.05; **, P < 0.01 (vs. vehicle).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Successful transplantation of human pancreatic islets provides the prospect of a cure for type 1 diabetes; however, the lack of sufficient donor pancreases greatly limits the widespread use of this approach. This study shows that the combination of EGF and gastrin as the only added growth factors can substantially increase (3-fold) ß-cell numbers in adult human pancreatic tissue cultured in vitro. Furthermore, the increase in ß-cells persisted even after EGF and gastrin were withdrawn from the cultures. In contrast, in medium not supplemented with EGF plus gastrin, insulin-expressing cells decreased to only 12% of the starting numbers over the same duration of culture. This increase in insulin-positive cells appears to result from activation of ß-cell neogenesis from pancreatic duct cells present in isolated islets. This is shown by the parallel increase in duct cells, identified by the ductal marker, CK19, and their expression of the ß-cell differentiation proteins, PDX-1 and insulin. The combination of EGF and gastrin induced C peptide as well as insulin expression in CK19-positive duct cells, thereby confirming that insulin immunostaining in CK19-positive cells represented insulin produced in the duct cells and not insulin absorbed from the culture medium. Also, insulin and C peptide immunoreactivity marked the same cell population. Interestingly, EGF plus gastrin increased the insulin content in cells 2-fold, whereas insulin-positive cells were increased 3-fold. This difference could represent the expansion of differentiating CK19-positive cells, approximately one third of which expressed insulin at 8 wk of culture. These less differentiated ß-cells may express lower levels of insulin than more differentiated ß-cells.

To determine whether EGF plus gastrin induced differentiation of ß-cells that were functional, we examined the insulin secretory responses of human islet cells implanted in immunodeficient NOD-scid mice treated with EGF and gastrin. First, we found that EGF plus gastrin therapy in vivo increased both CK19-positive cells and insulin-positive cells in human islet grafts, similar to the findings in vitro. Second, insulin contents in human islet grafts were increased by EGF plus gastrin. Third, insulin secretory responses to glucose by human islet grafts were significantly increased in EGF- plus gastrin-treated mice. These studies in vitro and in vivo indicate that combination therapy with EGF and gastrin can induce an increase in the functional mass of adult human islet ß-cells.

An increase in the ß-cell mass stimulated by EGF plus gastrin confirms and extends to the adult human pancreas the original findings in double-transgenic mice that expressed TGF-{alpha}, an EGF receptor ligand, and gastrin locally in the pancreas. In that study (30), mice expressing TGF-{alpha} alone developed an increased pancreatic duct cell mass, but no increase in ß-cell mass. Mice expressing only gastrin showed no increase in either duct or islet mass. Double-transgenic mice expressing both TGF-{alpha} and gastrin, however, exhibited a decrease in duct cells and an increase in ß-cell mass (30). Also, our present finding that EGF and gastrin combination therapy induced ß-cell neogenesis from duct cells in adult human pancreatic tissue is concordant with recent reports that this peptide combination increased ß-cell mass and reduced hyperglycemia in streptozotocin-diabetic rats (28), and induced islet regeneration from duct cells and restored normoglycemia in alloxan-diabetic mice (29).

ß-Cells have been generated in vitro by culture of both adult rodent (9, 10) and human (12, 13, 14) pancreatic tissue enriched for duct cells. In one study, adult human pancreatic cultures formed clusters and cysts containing cells mostly positive for CK19-positive duct cells along with cells immunoreactive for insulin and other islet hormones (12). In other studies, adult human pancreatic cultures contained proliferating CK19+ duct cells that reexpressed PDX-1 (13) and differentiated into endocrine cells (14). Taken together, these studies suggest that duct cells in the adult human pancreas may serve as endocrine cell precursors. This concept is supported by a recent report that 2 yr after clinical islet transplantation, the acute insulin response to glucose, a measure of the functional ß-cell mass, correlates significantly with the number of CK19-positive pancreatic duct cells originally present in the islets transplanted (32).

The nature of the ß-cell precursors in the adult mammalian pancreas is controversial (38). A recent study suggested that differentiated ß-cells, rather than duct or other putative precursor cells, may represent the most robust source of new ß-cells in adult mice (39). This cell lineage analysis in transgenic mice used insulin expression to mark ß-cells; however, this technique also may mark putative precursor cells that express insulin. Another recent study used clonal isolation techniques to identify and expand islet endocrine cell precursors in the adult mouse pancreas, and ß-cell precursors were found in both islets and pancreatic ducts (40).

Although the relative importance of replication and neogenesis pathways for new ß-cells in adult rats and mice is controversial, current evidence favors the neogenetic pathway in humans. For example, studies of the human fetal pancreas transplanted into immunodeficient mice have suggested that increases in functional ß-cell mass resulted more from differentiation of precursor cells than from proliferation of preexistent ß-cells (41, 42). Also, ß-cell neogenesis from duct cells is the major contributor to the increased ß-cell mass in adult humans with obesity (18). This is concordant with the finding that extraislet insulin-positive cells associated with pancreatic ductules are frequent not only in the human fetus and newborn, but also in the adult human pancreas (43). In addition, neoformation of ß-cells from centroacinar and duct cells could still be detected in autopsy specimens from pancreases of patients with recent-onset type 1 diabetes (44).

In the present study, we found that EGF and gastrin had complementary, but different, actions in stimulating ß-cell neogenesis in the adult human pancreas. EGF stimulated the proliferation of CK19-positive duct cells, whereas gastrin induced the expression of the transcription factor, PDX-1, in these duct cells and their differentiation into insulin-positive ß-cells. These findings are consonant with the different roles that EGF and gastrin have in islet development. EGF receptor ligands are expressed in the developing pancreas before gastrin (45). EGF receptor signaling stimulates proliferation and branching morphogenesis of fetal pancreatic ducts; this process is impaired, and islet cell differentiation is delayed in mice lacking EGF receptors (46). Gastrin expression is activated during the secondary transition phase when the developing pancreas changes from protodifferentiated ducts to the fully differentiated exocrine and endocrine pancreas (47).

The clinical implications of our findings are that EGF plus gastrin may be used to increase the ß-cell mass of isolated human islets or other duct cell-enriched pancreatic fractions in vitro before transplantation. Alternatively, EGF plus gastrin may be administered to the islet transplant recipient after islet transplantation to induce ß-cell neogenesis in the graft, as we have shown for human islet cells implanted in NOD-scid mice. A more widespread application of the present findings might be to treat type 1 diabetic subjects with EGF plus gastrin in an attempt induce ß-cell neogenesis from the patient’s own pancreatic duct cells, providing that autoimmune responses to the regenerated ß-cells can be prevented.


   Footnotes
 
This work was supported by grants from the Juvenile Diabetes Research Foundation International (1-2003-246), Waratah Pharmaceuticals (Woburn, MA), Transition Therapeutics (Toronto, Canada), and the Muttart Diabetes Research and Training Center, University of Alberta (Edmonton, Canada).

First Published Online March 15, 2005

Abbreviations: BCIP/NBT, 5-Bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium; CK19, cytokeratin 19; EGF, epidermal growth factor; NOD-scid, nonobese diabetic-severe combined immune deficiency; PDX-1, pancreatic and duodenal homeobox factor-1.

Received April 23, 2004.

Accepted March 7, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ryan EA, Lakey JRT, Paty BW, Imes S, Korbutt GS, Bigam D, Rajotte RV, Kneteman NM, Shapiro AMJ 2002 Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 51:2148–2157[Abstract/Free Full Text]
  2. Berna G, Leon-Quinto T, Ensenat-Waser R, Montanya E, Martin F, Soria B 2001 Stem cells and diabetes. Biomed Pharmacother 55:206–212[Medline]
  3. Serup P, Madsen OD, Mandrup-Poulsen T 2001 Science, medicine and the future: islet and stem cell transplantation for treating diabetes. Br Med J 322:29–32[Free Full Text]
  4. Lechner A, Habener JF 2003 Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus. Am J Physiol 284:E259–E266
  5. Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE 1993 A second pathway for regeneration of adult exocrine and endocrine pancreas: a possible recapitulation of embryonic development. Diabetes 42:1715–1720[Abstract]
  6. Gu D, Sarvetnick N 1993 Epithelial cell proliferation and islet neogenesis in IFN-{gamma} transgenic mice. Development 118:33–46[Abstract]
  7. Vinik A, Pittenger G, Rafaeloff R, Rosenberg L, Duguid WP 1996 Determinants of pancreatic islet cell mass: a balance between neogenesis and senescence/apoptosis. Diabet Rev 4:235–263
  8. Paris M, Bernard-Kargar C, Berthault MF, Bouwens L, Ktorza A 2003 Specific and combined effects of insulin and glucose on functional pancreatic ß-cell mass in vivo in adult rats. Endocrinology 144:2717–2727[Abstract/Free Full Text]
  9. Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG 2000 Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med 6:278–282[CrossRef][Medline]
  10. Rooman I, Heremans Y, Heimberg H, Bouwens L 2000 Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro. Diabetologia 43:907–914[CrossRef][Medline]
  11. Bertelli E, Regoli M, Orazioli D, Bendayan M 2001 Association between islets of Langerhans and pancreatic ductal system in adult rat. Where endocrine and exocrine meet together? Diabetologia 44:575–584[CrossRef][Medline]
  12. Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, O’Neil JJ 2000 In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA 97:7999–8004[Abstract/Free Full Text]
  13. Gmyr V, Kerr-Conte J, Belaich S, Vandewalle B, Leteurtre E, Vantyghem MC, Lecomte-Houcke M, Proye C, Lefebvre J, Pattou F 2000 Adult human cytokeratin 19-positive cells reexpress insulin promoter factor 1 in vitro: further evidence for pluripotent pancreatic stem cells in humans. Diabetes 49:1671–1680[Abstract]
  14. Gao R, Ustinov J, Pulkkinen M-A, Lundin K, Korsgren I, Otonkoski T 2003 Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture. Diabetes 52:2007–2015[Abstract/Free Full Text]
  15. Beattie GM, Itkin-Ansari P, Cirulli V, Leibowitz G, Lopez AD, Bossie S, Mally MI, Levine F, Hayek A 1999 Sustained proliferation of Pdx-1+ cells derived from human islets. Diabetes 48:1013–1019[Abstract]
  16. Bogdani M, Lefebvre V, Buelens N, Buck T, Pipeleers-Marichal M, In’t Veld P, Pipeleers D 2003 Formation of insulin-positive cells in implants of human pancreatic duct cell preparations from young donors. Diabetologia 46:830–838[CrossRef][Medline]
  17. Tyrberg B, Ustinov J, Otonkoski T, Andersson A 2001 Stimulated endocrine cell proliferation and differentiation in transplanted human pancreatic islets: effects of the ob gene and compensatory growth of the implantation organ. Diabetes 50:301–307[Abstract/Free Full Text]
  18. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC 2003 ß-Cell deficit and increased ß-cell apoptosis in humans with type 2 diabetes. Diabetes 52:102–110[Abstract/Free Full Text]
  19. Bouwens L, Kloppel G 1996 Islet neogenesis in the pancreas. Virchows Arch 427:553–560[Medline]
  20. Song SY, Gannon M, Washington MK, Scoggins CR, Meszoely IM, Goldenring JR, Marino CR, Sandgren EP, Coffey Jr RJ, Wright CV, Leach SD 1999 Expansion of PDX-1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor {alpha}. Gastroenterology 117:1416–1426[CrossRef][Medline]
  21. Yamamoto K, Miyagawa J, Waguri M, Sasada R, Igarashi K, Li M, Nammo T, Moriwaki M, Imagawa A, Yamagata K, Nakajima H, Namba M, Tochino Y, Hanafusa T, Matsuzawa Y 2000 Recombinant human betacellulin promotes the neogenesis of ß-cells and ameliorates glucose intolerance in mice with diabetes induced by selective alloxan perfusion. Diabetes 49:2021–2027[Abstract]
  22. Cras-Meneur C, Elghazi L, Czernichow P, Scharfmann R 2001 Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differentiation. Diabetes 50:1571–1579[Abstract/Free Full Text]
  23. Krakowski ML, Kritzik MR, Jones EM, Krachl T, Lee J, Arnush M, Gu D, Mroczkowski B, Sarvetnick N 1999 Transgenic expression of epidermal growth factor and keratinocyte growth factor in ß-cells results in substantial morphological changes. J Endocrinol 162:167–175[Abstract]
  24. Xu G, Stoffers DA, Habener JF, Bonner-Weir S 1999 Exendin-4 stimulates both ß-cell replication and neogenesis, resulting in increased ß-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48:2270–2276[Abstract]
  25. Tourrel C, Bailbè D, Meile MJ, Kergoat M, Portha B 2001 Glucagon-like peptide-1 and exendin-4 stimulate ß-cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes 50:1562–1570[Abstract/Free Full Text]
  26. Rooman I, Lardon J, Bouwens L 2002 Gastrin stimulates ß-cell neogenesis and increases islet mass from transdifferentiated but not from normal exocrine pancreatic tissue. Diabetes 51:686–690[Abstract/Free Full Text]
  27. Rooman I, Bouwens L 2002 Islet neogenesis in the regeneration model of rat pancreatic duct ligation requires endogenous gastrin action via CCK2 receptors. Diabetologia 45(Suppl 2):A26
  28. Brand SJ, Tagerud S, Lambert P, Magil SG, Tartarkiewicz K, Doiron K, Yan Y 2002 Pharmacological treatment of chronic diabetes by stimulating pancreatic ß-cell regeneration with systemic co-administration of EGF and gastrin. Pharmacol Toxicol 91:414–420[CrossRef][Medline]
  29. Rooman I, Bouwens L 2004 Combined gastrin and epidermal growth factor treatment induces islet regeneration and restores normoglycemia in C57BL6/J mice treated with alloxan. Diabetologia 47:259–265[CrossRef][Medline]
  30. Wang TC, Bonner-Weir S, Oates PS, Chulak M, Simon B, Merlino GT, Schmidt EV, Brand SJ 1993 Pancreatic gastrin stimulates islet differentiation of transforming growth factor {alpha}-induced ductular precursor cells. J Clin Invest 92:1349–1356
  31. Shapiro AMJ, Lakey JRT, Ryan EA, Korbutt GS, Toth EL, Warnock GL, Kneteman NM, Rajotte RV 2000 Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238[Abstract/Free Full Text]
  32. Street CN, Lakey JRT, Shapiro AMJ, Imes S, Rajotte RV, Ryan EA, Lyon JG, Kin T, Avila J, Tsujimura T, Korbutt GS 2004 Islet graft assessment in the Edmonton protocol. Implications for predicting long-term clinical outcome. Diabetes 53:3107–3114[Abstract/Free Full Text]
  33. Lakey JRT, Warnock GL, Shapiro AMJ, Korbutt GS, Ao Z, Kneteman NM, Rajotte RV 1999 Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. Cell Transplant 8:285–292[Medline]
  34. Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW 1988 Automated method for isolation of human pancreatic islets. Diabetes 37:413–420[Abstract]
  35. Calnan DP, Fagbemi A, Berlanga-Acosta J, Marchbank T, Sizer T, Lakhoo K, Edwards AD, Playford RJ 2000 Potency and stability of C terminal truncated human epidermal growth factor. Gut 47:622–627[Abstract/Free Full Text]
  36. Petersen B, Christinsen J, Rehfeld JF 1981 Acid potency and elimination of the 15-leucine gastrin-17 analogue in man. Scand J Gastroenterol 16:437–440
  37. Wang T, Singh B, Warnock G, Rajotte RV 1992 Prevention of recurrence of IDDM in islet-transplanted diabetic NOD mice by adjuvant immunotherapy. Diabetes 41:114–117[Abstract]
  38. Halban PA 2004 Cellular sources of new pancreatic ß-cells and therapeutic implications for regenerative medicine. Nat Cell Biol 6:1021–1025[CrossRef][Medline]
  39. Dor Y, Brown J, Martinez OI, Melton D 2004 Adult pancreatic ß-cells are formed by self-replication rather than stem-cell differentiation. Nature 429:41–46[CrossRef][Medline]
  40. Seaberg RM, Smukler SR, Kieffer TJ, Enikolopov G, Asghar Z, Wheeler MB, Korbutt G, van der Kooy D 2004 Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages. Nat Biotechnol 22:1115–1124[CrossRef][Medline]
  41. Beattie GM, Otonkoski T, Lopez AD, Hayek A 1997 Functional ß-cell mass after transplantation of human fetal pancreatic cells: differentiation or proliferation? Diabetes 46:244–248[Abstract]
  42. Si Z, Tuch BE, Walsh DA 2001 Development of human fetal pancreas after transplantation into SCID mice. Cells Tissues Organs 168:147–157[CrossRef][Medline]
  43. Bouwens L, Pipeleers DG 1998 Extra-insular ß cells associated with ductules are frequent in adult human pancreas. Diabetologia 41:629–633[CrossRef][Medline]
  44. Gepts W, DeMey J 1978 Islet cell survival determined by morphology: an immunocytochemical study of the islets of Langerhans in juvenile diabetes mellitus. Diabetes 27(Suppl 1):251–261
  45. Wang RN, Rehfeld JF, Nielsen FC, Kloppel G 1997 Expression of gastrin and transforming growth factor-{alpha} during duct to islet cell differentiation in the pancreas of duct-ligated adult rats. Diabetologia 40:887–893[CrossRef][Medline]
  46. Miettinen PJ, Huotari M-A, Koivisto T, Ustinov J, Palgi J, Rasilainen S, Lehtonen E, Keski-Oji J, Otonkoski T 2000 Impaired migration and delayed differentiation of pancreatic islet cells in mice lacking EGF-receptors. Development 127:2617–2627[Abstract]
  47. Brand SJ, Fuller PJ 1988 Differential gastrin gene expression in rat gastrointestinal tract and pancreas during neonatal development. J Biol Chem 263:5341–5347[Abstract/Free Full Text]



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