Identification of Insulin‐Producing Cells Derived from Embryonic Stem Cells by Zinc‐Chelating Dithizone

Shiroi, Akira; Yoshikawa, Masahide; Yokota, Hiroshi; Fukui, Hiroshi; Ishizaka, Shigeaki; Tatsumi, Kouko; Takahashi, Yoshiko

doi:10.1634/stemcells.20-4-284

Cited by 196 publications

(123 citation statements)

References 45 publications

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“…By Ficoll gradient centrifugation, the pancreatic acinar cell-enriched exocrine fraction was recovered as a pellet. Although the pellet contained only a small amount of native pancreatic ␤ cells (Ϸ0.5% of total cells recovered), we further removed native ␤ cells by staining the pancreatic islets and their fragments with dithizone (DTZ) (34,35), followed by handpicking under a dissecting microscope. After this procedure, the frequency of insulin-positive cells was Ϸ0.01%, as assessed by immunostaining and quantitative realtime RT-PCR for insulin genes.…”

Section: Resultsmentioning

confidence: 99%

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells

Minami

Okuno

Miyawaki

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

240

211

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Although several studies have suggested that insulin-secreting cells can be generated in vitro from cells residing in adult exocrine pancreas, neither the origin of these cells nor their precise insulin secretory properties was obtained. We show here that insulinsecreting cells can be derived from adult mouse pancreatic exocrine cells by suspension culture in the presence of EGF and nicotinamide. The frequency of insulin-positive cells was only 0.01% in the initial preparation and increased to Ϸ5% in the culture conditions. Analysis by the Cre͞loxP-based direct cell lineage tracing system indicates that these newly made cells originate from amylase͞ elastase-expressing pancreatic acinar cells. Insulin secretion is stimulated by glucose, sulfonylurea, and carbachol, and potentiation by glucagon-like peptide-1 also occurs. Insulin-containing secretory granules are present in these cells. In addition, we found that the enzymatic dissociation of pancreatic acini itself leads to activation of EGF signaling, and that inhibition of EGF receptor kinase blocks the transdifferentiation. These data demonstrate that pancreatic acinar cells can transdifferentiate into insulin-secreting cells with secretory properties similar to those of native pancreatic ␤ cells, and that activation of EGF signaling is required in such transdifferentiation.

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Section: Resultsmentioning

confidence: 99%

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells

Minami

Okuno

Miyawaki

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

240

211

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“…Recent efforts have been directed at the generation of insulin-producing cells using the ES-EB culture system [25,[39][40][41][42][43][44] to provide islet cells for transplantation and cure of type I diabetes. However, previous ES cell culture studies [25,[39][40][41][42][43][44] did not address nor specify the extent of endoderm and pancreatic differentiation in their cultures.…”

Section: Discussionmentioning

confidence: 99%

Committing Embryonic Stem Cells to Early Endocrine Pancreas In Vitro

et al. 2004

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Type I diabetes is marked by a deficiency of endocrine β cells in the pancreatic islets of Langerhans. Daily injection of insulin is the current treatment for the disease. Because insulin injection cannot match the precise timing and dosing of physiological secretion of insulin by islets in response to hyperglycemia, severe side effects develop over time. Transplantation of islets represents a potential cure; however, limitations on the availability of cadaveric organs restrict this procedure to only a small percentage of patients. Pancreatic endocrine stem cells exist in the developing embryonic pancreas. The isolation, expansion, and differentiation of pancreatic endocrine stem cells would provide an unlimited source of islet cells for transplantation and treatment for type I diabetes. A potential source for endocrine stem cells is embryonic stem (ES) cells. Because of their unlimited proliferation and differentiation potentials, ES cells are considered an important source for cell therapies targeted to several diseases, including diabetes.There are two nonallelic insulin genes (insulin I and II) expressed in multiple sites in mice during development [1][2][3][4][5][6][7] Abstract A panel of genetic markers was used to assess the in vitro commitment of murine embryonic stem (ES) cells toward the endoderm-derived pancreas and to distinguish insulin-expressing cells of this lineage from other lineagessuch as neuron, liver, and yolk sac. There are two nonallelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominately insulin II, small amounts of insulin I, and no glucagon. We found that ES-derived embryoid bodies cultured in the presence of stage-specific concentrations of monothioglycerol and 15% fetal calf serum, followed by serum-free conditions, give rise to a population that expresses insulin I, insulin II, pdx-1 (a pancreas marker), and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin particles, and its de novo production was confirmed by staining for C-peptide. Most, but not all, of the insulin + or C-peptide + cells coexpress glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of β-cell specification and differentiation factors activin βB, nicotinamide, and exendin-4 to later-stage culture increased insulin-positive cells to 2.73% of the total population, compared with the control culture, which gave rise to less than 1% insulin-staining cells. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.

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“…The response to 200 μmol/l tolbutamide was also determined. Beta cells were identified after imaging by staining with dithizone [18].…”

Section: Methodsmentioning

confidence: 99%

Pancreatic deletion of insulin receptor substrate 2 reduces beta and alpha cell mass and impairs glucose homeostasis in mice

et al. 2007

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Aims/hypothesis Insulin signalling pathways regulate pancreatic beta cell function. Conditional gene targeting using the Cre/loxP system has demonstrated that mice lacking insulin receptor substrate 2 (IRS2) in the beta cell have reduced beta cell mass. However, these studies have been complicated by hypothalamic deletion when the RIPCre (B6.Cg-tg(Ins2-cre) 25Mgn/J) transgenic mouse (expressing Cre recombinase under the control of the rat insulin II promoter) is used to delete floxed alleles in insulin-expressing cells. These features have led to marked insulin resistance making the beta cellautonomous role of IRS2 difficult to determine. To establish the effect of deleting Irs2 only in the pancreas, we generated PIrs2KO mice in which Cre recombinase expression was driven by the promoter of the pancreatic and duodenal homeobox factor 1 (Pdx1, also known as Ipf1) gene. Materials and methodsIn vivo glucose homeostasis was examined in PIrs2KO mice using glucose tolerance and glucose-stimulated insulin secretion tests. Endocrine cell mass was determined by morphometric analysis. Islet function was examined in static cultures and by performing calcium imaging in Fluo3am-loaded beta cells. Islet gene expression was determined by RT-PCR. Results The PIrs2KO mice displayed glucose intolerance and impaired glucose-stimulated insulin secretion in vivo. Pancreatic insulin and glucagon content and beta and alpha cell mass were reduced. Glucose-stimulated insulin secretion and calcium mobilisation were attenuated in PIrs2KO islets. Expression of the Glut2 gene (also known as Slc2a2) was also reduced in PIrs2KO mice. Conclusions/interpretation These studies suggest that IRS2-dependent signalling in pancreatic islets is required not only for the maintenance of normal beta and alpha cell mass but is also involved in the regulation of insulin secretion.

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Identification of Insulin‐Producing Cells Derived from Embryonic Stem Cells by Zinc‐Chelating Dithizone

Cited by 196 publications

References 45 publications

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells

Committing Embryonic Stem Cells to Early Endocrine Pancreas In Vitro

Pancreatic deletion of insulin receptor substrate 2 reduces beta and alpha cell mass and impairs glucose homeostasis in mice

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