The zinc finger transcription factor PW1/PEG3 restrains murine beta cell cycling

Aims/hypothesis: Pw1 or paternally-expressed gene 3 (Peg3) encodes a zinc finger transcription factor that is widely expressed during mouse embryonic development and later restricted to multiple somatic stem cell lineages in the adult. The aim of the present study was to define Pw1 expression in the embryonic and adult pancreas and investigate its role in the beta cell cycle in Pw1 wild-type and mutant mice. Methods: We analysed PW1 expression by immunohistochemistry in pancreas of nonpregant and pregnant mice and following injury by partial duct ligation. Its role in the beta cell cycle was studied in vivo using a novel conditional knockout mouse and in vitro by lentivirus-mediated gene knockdown. Results: We showed that PW1 is expressed in early pancreatic progenitors at E9.5 but becomes progressively restricted to fully differentiated beta cells as they become established after birth and withdraw from the cell cycle. Notably, PW1 expression declines when beta cells are induced to proliferate and loss of PW1 function activates the beta cell cycle. Conclusions/interpretation: These results indicate that PW1 is a co-regulator of the beta cell cycle and can thus be considered a novel therapeutic target in diabetes

Semi-automated digital measurement as the method of choice for beta cell mass analysis

Pancreas injury by partial duct ligation (PDL) activates beta cell differentiation and proliferation in adult mouse pancreas but remains controversial regarding the anticipated increase in beta cell volume. Several reports unable to show beta cell volume augmentation in PDL pancreas used automated digital image analysis software. We hypothesized that fully automatic beta cell morphometry without manual micrograph artifact remediation introduces bias and therefore might be responsible for reported discrepancies and controversy. However, our present results prove that standard digital image processing with automatic thresholding is sufficiently robust albeit less sensitive and less adequate to demonstrate a significant increase in beta cell volume in PDL versus Sham-operated pancreas. We therefore conclude that other confounding factors such as quality of surgery, selection of samples based on relative abundance of the transcription factor Neurogenin 3 (Ngn3) and tissue processing give rise to inter-laboratory inconsistencies in beta cell volume quantification in PDL pancreas

(Re)generating human beta cells : status, pitfalls, and perspectives

Diabetes mellitus results from disturbed glucose homeostasis due to an absolute (type 1) or relative (type 2) deficiency of insulin, a peptide hormone almost exclusively produced by the beta cells of the endocrine pancreas in a tightly regulated manner. Current therapy only delays disease progression through insulin injection and/or oral medications that increase insulin secretion or sensitivity, decrease hepatic glucose production, or promote glucosuria. These drugs have turned diabetes into a chronic disease as they do not solve the underlying beta cell defects or entirely prevent the long-term complications of hyperglycemia. Beta cell replacement through islet transplantation is a more physiological therapeutic alternative but is severely hampered by donor shortage and immune rejection. A curative strategy should combine newer approaches to immunomodulation with beta cell replacement. Success of this approach depends on the development of practical methods for generating beta cells, either in vitro or in situ through beta cell replication or beta cell differentiation. This review provides an overview of human beta cell generation

Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3

Regulatory proteins have been identified in embryonic development of the endocrine pancreas. It is unknown whether these factors can also play a role in the formation of pancreatic endocrine cells from postnatal nonendocrine cells. The present study demonstrates that adult human pancreatic duct cells can be converted into insulin-expressing cells after ectopic, adenovirus-mediated expression of the class B basic helix-loop-helix factor neurogenin 3 (ngn3), which is a critical factor in embryogenesis of the mouse endocrine pancreas. Infection with adenovirus ngn3 (Adngn3) induced gene and/or protein expression of NeuroD/β2, Pax4, Nkx2.2, Pax6, and Nkx6.1, all known to be essential for β-cell differentiation in mouse embryos. Expression of ngn3 in adult human duct cells induced Notch ligands Dll1 and Dll4 and neuroendocrine- and β-cell–specific markers: it increased the percentage of synaptophysin- and insulin-positive cells 15-fold in ngn3-infected versus control cells. Infection with NeuroD/β2 (a downstream target of ngn3) induced similar effects. These data indicate that the Delta-Notch pathway, which controls embryonic development of the mouse endocrine pancreas, can also operate in adult human duct cells driving them to a neuroendocrine phenotype with the formation of insulin-expressing cells

Human multipotent adult progenitor cells enhance islet function and revascularisation when co-transplanted as a composite pellet in a mouse model of diabetes

AIMS/HYPOTHESIS: Hypoxia in the initial days after islet transplantation leads to considerable loss of islet mass and contributes to disappointing outcomes in the clinical setting. The aim of the present study was to investigate whether co-transplantation of human non-endothelial bone marrow-derived multipotent adult progenitor cells (MAPCs), which are non-immunogenic and can secrete angiogenic growth factors during the initial days after implantation, could improve islet engraftment and survival.METHODS: Islets (150) were co-transplanted, with or without human MAPCs (2.5 × 105) as separate or composite pellets, under the kidney capsule of syngeneic alloxan-induced diabetic C57BL/6 mice. Blood glucose levels were frequently monitored and IPGTTs were carried out. Grafts and serum were harvested at 2 and 5 weeks after transplantation to assess outcome.RESULTS: Human MAPCs produced high amounts of angiogenic growth factors, including vascular endothelial growth factor, in vitro and in vivo, as demonstrated by the induction of neo-angiogenesis in the chorioallantoic membrane assay. Islet-human MAPC co-transplantation as a composite pellet significantly improved the outcome of islet transplantation as measured by the initial glycaemic control, diabetes reversal rate, glucose tolerance and serum C-peptide concentration compared with the outcome following transplantation of islets alone. Histologically, a higher blood vessel area and density in addition to a higher vessel/islet ratio were detected in recipients of islet-human MAPC composites.CONCLUSIONS/INTERPRETATION: The present data suggest that co-transplantation of mouse pancreatic islets with human MAPCs, which secrete high amounts of angiogenic growth factors, enhance islet graft revascularisation and subsequently improve islet graft function

Original Contribution Increased oxygen radical formation and mitochondrial dysfunction mediate beta cell apoptosis under conditions of AMP-activated protein kinase stimulation

Abstract AMP-activated protein kinase influences cellular metabolism, glucose-regulated gene expression, and insulin secretion of pancreatic beta cells. Its sustained activation by culture at low glucose concentrations or in the presence of 5-aminoimidazole-4-carboxamide riboside (AICAR) was shown to trigger apoptosis in beta cells. This study shows that both low glucose-and AICAR-induced apoptosis are associated with increased formation of mitochondrial superoxide-derived radicals and decreased mitochondrial activity. Mitochondrial dysfunction was reflected by an increased oxidized state of the mitochondrial flavins (FMN/FAD) but not of NAD(P)H. It was accompanied by suppression of glucose oxidation and glucose-induced insulin secretion, while palmitate oxidation appeared unaffected. When the cellular accumulation of superoxide-derived radicals was quenched by the ROS scavengers vitamin E, N-acetylcysteine, or the SOD-mimetic compound MnTBAP, apoptosis was significantly inhibited. Both low glucose and AICAR also elevated the expression of BH3-domain-only Bcl-2 antagonists, and induced caspase-3 activation, causing caspase-dependent truncation of Bcl-2. Overexpression of recombinant human Bcl-2 prevented caspase-3 activation, endogenous Bcl-2 processing, and apoptosis, but did not attenuate oxygen radical formation, AMPK activation, or JNK phosphorylation. We conclude that apoptosis by prolonged AMPK activation in beta cells results from enhanced production of mitochondria-derived oxygen radicals and onset of the intrinsic mitochondrial apoptosis pathway, followed by caspase activation and Bcl-2 cleavage which may amplify the death signal

Suppression of Epithelial to Mesenchymal Transitioning (EMT) Enhances Ex Vivo Reprogramming of Human Exocrine Pancreatic Tissue towards Functional Insulin Producing β-Like Cells

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer

Generation of Functional Beta-Like Cells from Human Exocrine Pancreas

<div><p>Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin⁺ cells. The resultant β-like cells expressed insulin protein levels at ~15–30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.</p></div

Expansion of Adult Human Pancreatic Tissue Yields Organoids Harboring Progenitor Cells with Endocrine Differentiation Potential.

Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential

A Specific CNOT1 Mutation Results in a Novel Syndrome of Pancreatic Agenesis and Holoprosencephaly through Impaired Pancreatic and Neurological Development.

We report a recurrent CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.IB is funded by Wellcome (WT206194). ATH and SE are the recipients of a Wellcome Trust Senior Investigator award and ATH is employed as a core member of staff within the NIHR funded Exeter Clinical Research Facility and is an NIHR senior investigator. EDF was a Naomi Berrie Fellow in Diabetes Research during the study. SEF has a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number: 105636/Z/14/Z). CCW holds a Wellcome Trust Intermediate Clinical Fellowship (Grant Number: 105914/Z/14/Z). HH is funded by the Research Foundation-Flanders (FWO), the VUB Research Council and Stichting Diabetes Onderzoek Nederland