Reprogramming of human pancreatic exocrine cells to ß-like cells.

Rodent acinar cells exhibit a remarkable plasticity as they can transdifferentiate to duct-, hepatocyte- and islet ß-like cells. We evaluated whether exocrine cells from adult human pancreas can similarly respond to proendocrine stimuli. Exocrine cells from adult human pancreas were transduced directly with lentiviruses expressing activated MAPK (mitogen-activated protein kinase) and STAT3 (signal transducer and activator of transcription 3) and cultured as monolayers or as 3D structures. Expression of STAT3 and MAPK in human exocrine cells activated expression of the proendocrine factor neurogenin 3 in 50% to 80% of transduced exocrine cells. However, the number of insulin-positive cells increased only in the exocrine cells grown initially in suspension before 3D culture. Lineage tracing identified human acinar cells as the source of Ngn3- and insulin-expressing cells. Long-term engraftment into immunocompromised mice increased the efficiency of reprogramming to insulin-positive cells. Our data demonstrate that exocrine cells from human pancreas can be reprogrammed to transplantable insulin-producing cells that acquire functionality. Given the large number of exocrine cells in a donor pancreas, this approach presents a novel strategy to expand cell therapy in type 1 diabetes.

Hedgehog signals inhibit postnatal beta cell neogenesis from adult rat exocrine pancreas in vitro.

AIMS/HYPOTHESIS: Transdifferentiation of pancreatic exocrine cells into insulin-producing beta cells may represent an important alternative to islets required for diabetes cell therapy. Rat pancreatic acinar cells are known to transdifferentiate into functional beta cells, with recapitulation of several pancreas developmental features. Considering the inhibitory functions of hedgehog signalling in early and mid-stage pancreatic development, we questioned whether it also operates in transdifferentiating acinar cells and whether its modulation would influence postnatal beta cell neogenesis in vitro. METHODS: Rat exocrine cells were precultured in suspension for 4 days and then incubated with EGF and leukaemia inhibitory factor (LIF) for 72 h. The hedgehog signalling pathway was modulated during this, and its effects analysed by RT-PCR, immunocytochemistry and western blot. RESULTS: Our data indicate induction of Dhh and Ihh, but not Shh, expression during acinar cell culture, resulting in activation of hedgehog targets (Ptc1, Gli1). Exposure of the metaplastic cells to EGF and LIF induced beta cell differentiation without affecting endogenous hedgehog activity. Whereas blocking endogenous hedgehog only slightly increased beta cell neogenesis, exposure to embryoid body-conditioned medium activated hedgehog signalling as well as other pathways such as Notch, resulting in severe blockade of beta cell neogenesis. Interestingly, this effect was partially rescued by treatment with the hedgehog inhibitor, 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)-cyclopamine (KAAD-cyclopamine), alone. CONCLUSIONS/INTERPRETATION: We report here Dhh/Ihh-dependent activation of hedgehog targets during pancreatic exocrine cell metaplasia in vitro and a persistent inhibitory function of hedgehog signalling in a model of postnatal beta cell differentiation.

Epidermal growth factor (EGF)-receptor signalling is needed for murine beta cell mass expansion in response to high-fat diet and pregnancy but not after pancreatic duct ligation.

AIMS/HYPOTHESIS: Epidermal growth factor receptor (EGFR) signalling is essential for the proper fetal development of pancreatic islets and in the postnatal formation of an adequate beta cell mass. In this study we investigated the role of EGFR signalling in the physiological states of beta cell mass expansion in adults during metabolic syndrome and pregnancy, as well as in regeneration after pancreatic duct ligation. METHODS: Heterozygous Pdx1-EGFR-dominant-negative (E1-DN) mice, which have a kinase-negative EGFR under the Pdx1 promoter, and wild-type mice were both subjected to a high-fat diet, pregnancy and pancreatic duct ligation. RESULTS: The beta cell mass of wild-type mice fed the high-fat diet increased by 70% and the mice remained normoglycaemic; the E1-DN mice became diabetic and failed to show any compensatory beta cell mass expansion. Similarly, pregnant wild-type mice had four times more proliferating beta cells and a 75% increase in beta cell mass at mid-gestation, in contrast to the pregnant E1-DN mice, which did not show any significant beta cell compensation and were hyperglycaemic in an intraperitoneal glucose tolerance test. However, after pancreatic duct ligation, both the wild-type and E1-DN mice showed similar expression of Ngn3 (also known as Neurog3) and beta cell proliferation increased to a similar level in the ligated part of pancreas. CONCLUSIONS/INTERPRETATIONS: EGFR signalling is essential in beta cell mass expansion during a high-fat diet and pregnancy where replication is the primary mechanism for compensatory beta cell mass expansion. In contrast, EGFR signalling appears not to be crucial to increased beta cell proliferation after pancreatic duct ligation.

A genomic-based approach identifies FXYD domain containing ion transport regulator 2 (FXYD2)gammaa as a pancreatic beta cell-specific biomarker.

AIMS/HYPOTHESIS: Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers. METHODS: We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays. RESULTS: After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na(+)-K(+)-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2gammaa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2gammaa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2gammaa and loss of insulin-positive cells. CONCLUSIONS/INTERPRETATION: We propose human FXYD2gammaa as a novel beta cell-specific biomarker.

Can beta-cells be derived from exocrine pancreas?

A major goal of research aiming at improving islet cell replacement therapy is to find the most suitable progenitor cell type from which functional beta-cells can be generated in large numbers. Many possibilities have been raised, including beta-cells themselves, embryonic or adult stem cells and reprogramming of other cell types. Some of these progenitor types may be active or reside in a dormant state in adults in vivo, while others can be rather considered to be products of tissue engineering in vitro. Starting from the available pancreas organs from cadaveric donors, an attractive possibility is to reprogram acinar exocrine cells into beta-cells. Indeed, acinar cells isolated from adult rats display a pronounced plasticity in culture. After an initial step of dedifferentiation, they can be redirected to the beta-cell phenotype by adding agonists of the JAK2/STAT3 signalling pathway to the medium (epidermal growth factor and leukaemia inhibitory factor). The acinar cells that undergo exocrine-to-endocrine transdifferentiation first need to re-express neurogenin-3 and then need to escape inhibition by Notch signalling. The insulin-expressing cells that are generated in this way are glucose-regulated and can normalize glycaemia after transplantation into diabetic immunocompromised mice. It will now be important to translate these findings to human cells.

Hedgehog signals in pancreatic differentiation from embryonic stem cells: revisiting the neglected.

Recent demonstrations of insulin expression by progenies of mouse and human embryonic stem (ES) cells have attracted interest in setting up these cells as alternative sources of beta-cells needed in diabetes cell therapy. It is widely acknowledged that information gathered in the field of developmental biology as applied to the pancreas is of relevance for designing in vitro differentiation strategies. However, looking back at the protocols used so far, it appears that the natural route toward the pancreas, which goes via the definitive endoderm, was usually bypassed. As a consequence Hedgehog signaling, the earliest inhibitor of pancreas initiation from the endoderm, was generally not considered. A recall of the status of this pathway during ES cell differentiation appears necessary, especially in the light of findings that Activin A treatment of mouse and human ES cells coax them into definitive endoderm, a lineage showing wide Hedgehog ligands expression with the potential to hinder pancreatic programming.

Ngn3 expression during postnatal in vitro beta cell neogenesis induced by the JAK/STAT pathway.

The basic helix-loop-helix protein Neurogenin3 specifies precursor cells of the endocrine pancreas during embryonic development, and is thought to be absent postnatally. We have studied Ngn3 expression during in vitro generation of beta-cells from adult rat exocrine pancreas tissue treated with epidermal growth factor and leukaemia inhibitory factor. This treatment induced a transient expression of both Ngn3 and its upstream activator hepatocyte nuclear factor 6. Inhibition of EGF and LIF signalling by pharmacological antagonists of the JAK2/STAT3 pathway, or knockdown of Ngn3 by RNA interference prevented the generation of new insulin-positive cells. This study demonstrates that in vitro growth factor stimulation can induce recapitulation of an embryonic endocrine differentiation pathway in adult dedifferentiated exocrine cells. This could prove to be important for understanding the mechanism of beta-cell regeneration and for therapeutic ex vivo neogenesis of beta cells.

Expression and function of leukaemia inhibitory factor and its receptor in normal and regenerating rat pancreas.

AIMS/HYPOTHESIS: It was recently reported that culturing adult exocrine cells in the presence of epidermal growth factor and leukaemia inhibitory factor (LIF) resulted in their transdifferentiation into endocrine beta cells. The aim of this study was to examine the expression and function of LIF in the pancreas. MATERIALS AND METHODS: We studied the expression of LIF and its receptor components, LIF-receptor-beta and gp130, by immunohistochemistry, western blotting and RT-PCR in normal rat pancreas, pancreas with duct ligation-induced islet neogenesis, and in pancreatic cell cultures. Isolated duct fragments were cultured in the presence of LIF and a janus kinase 2 (JAK2) inhibitor. RESULTS: LIF was detected by immunohistochemistry, western blot and RT-PCR in the ducts of the normal pancreas. Both LIF-receptor-beta and gp130 were detected by RT-PCR in the pancreas. Immunostaining revealed gp130 exclusively in the ducts and centro-acinar cells. After duct ligation-induced tissue injury, upregulation of LIF and its receptor occurred in rat pancreas. Metaplastic exocrine cells also started to express LIF and this was increased after alloxan treatment. Signalling via LIF-receptor-beta/gp130 involves the JAK/signal transducer and activator of transcription (STAT) pathway. LIF induced increased activation of STAT3 in pancreatic cells. In isolated duct fragments, addition of LIF resulted in a significant increase in duct cell proliferation, while a specific inhibitor of the JAK/STAT signalling pathway inhibited proliferation. CONCLUSION/INTERPRETATION: Our observations show that LIF and its receptor are expressed in cells from pancreatic ducts. The cytokine plays a role in pancreatic physiology, controls duct cell proliferation and is involved in repair processes following pancreatic injury.

Rejection of atrial sensing artifacts by a pacing lead with short tip-to-ring spacing.

AIMS: The ability of a new pacing lead design, with a 10 mm tip-to-ring spacing, to facilitate rejection of sensed far field R-waves and myopotentials was evaluated. METHODS AND RESULTS: Measurements were performed in 66 patients. The occurrence of far field R-wave sensing and myopotential sensing was determined by means of the surface ECG and the ECG markers provided by the pacemaker. At an atrial sensitivity of 0.25 mV and an atrial blanking of 50 ms far field R-wave sensing was observed in 12 patients (18.2%) and at an atrial sensitivity of 1.0 mV no far-field R-wave sensing was observed. Myopotentials were sensed in 3 patients. In all patients the measured P-wave amplitude was at least twice the estimated amplitude of the far field R-wave at an atrial blanking of 50 ms. CONCLUSION: The results from this study show that a small tip-to-ring spacing allows for programming of a high atrial sensitivity and short atrial blanking with an acceptably low risk for atrial artifact sensing.

In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells.

AIMS/HYPOTHESIS: Transplantation of insulin-producing beta cells from donors can cure diabetes, but they are available in insufficient quantities. In this study, we investigated the possibility of generating insulin-producing cells from adult rat exocrine cells cultured in the presence of growth factors. METHODS: Rat exocrine pancreatic cells were isolated and treated in vitro with epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Analysis was performed by immunocytochemistry, DNA measurement and radioimmunoassay. Cells were transplanted to alloxan-treated (70 mg/kg) nude mice and glycaemia was monitored for 21 days. Nephrectomy was performed on day 15. RESULTS: In a 3-day culture period, addition of LIF plus EGF to the medium resulted in an 11-fold increase of the beta cell mass. This could not be attributed to the very low mitotic activity of contaminating beta cells. Furthermore, when contaminating beta cells were initially destroyed with alloxan, this effect was even more pronounced. The newly formed cells secreted insulin in response to glucose and were immunoreactive for C-peptide-I, Pdx-1 and GLUT-2, which are characteristics of mature beta cells. Electron microscopy showed that they also contained insulin-immunoreactive secretory granules. Some insulin-positive cells were immunoreactive for amylase and cytokeratin-20, or were binucleated, which are characteristics of exocrine cells. The cells were able to restore normoglycaemia when transplanted to alloxan-diabetic mice, and hyperglycaemia recurred upon removal of the graft. CONCLUSIONS/INTERPRETATION: Our study shows that functional beta cells can be generated from exocrine tissue by transdifferentiation and thereby may offer a new perspective for beta cell therapy.

Combined gastrin and epidermal growth factor treatment induces islet regeneration and restores normoglycaemia in C57Bl6/J mice treated with alloxan.

AIMS/HYPOTHESIS: Increasing beta-cell mass and/or function could restore glucose homeostasis in diabetes mellitus. Hitherto, trophic factors for beta-cell regeneration after toxic events have been difficult to identify. We evaluated the application of gastrin and epidermal growth factor after alloxan-induced pancreatic beta-cell damage. METHODS: After alloxan treatment (70 mg/kg), mice were implanted with Alzet osmotic minipumps releasing gastrin and epidermal growth factor for one week. We monitored glycaemia, did histological analyses of the pancreata and quantified pancreatic beta-cell mass and insulin content. RESULTS: Alloxan treatment alone resulted in a persisting hyperglycaemic state. Combined gastrin and epidermal growth factor treatment restored normoglycaemia in 3 days, an effect which seemed permanent. Glucose tolerance tests showed normal glucose responsiveness. Gastrin on its own and epidermal growth factor on its own did not alleviate hyperglycaemia. Islet mass, islet density and pancreatic insulin content were higher in mice treated with gastrin and epidermal growth factor than in untreated mice with persisting hyperglycaemia. In normoglycaemic control mice treatment with gastrin and epidermal growth factor did not affect these parameters. We detected transitional cytokeratin-positive ductal to endocrine insulin-expressing cells and noted increased ductal but not beta-cell proliferation. CONCLUSIONS/INTERPRETATION: Our results show that combined treatment with gastrin and epidermal growth factor can induce sufficient regeneration of a functional islet mass to restore glucose homeostasis.

Netrin-1 expression in fetal and regenerating rat pancreas and its effect on the migration of human pancreatic duct and porcine islet precursor cells.

AIMS/HYPOTHESIS: We investigated the expression and function of netrin-1, a diffusible laminin-like protein known to regulate neuronal-cell migration in the pancreas. We questioned whether this factor regulates migration of pancreatic epithelial cells and whether this could be involved in islet neogenesis. METHODS: We studied fetal and adult rat pancreas wherein duct ligation induced islet neogenesis. Netrin-1 expression was analysed by RT-PCR, western blot and immunohistochemistry. In vitro cell migration was measured with a human pancreatic duct cell line (CAPAN-2) and with fetal porcine islet cells. We also studied the expression of two netrin-receptors, neogenin and deleted in colorectal cancer. RESULTS: We found a transient expression of netrin-1 mRNA and protein in fetal pancreas from E15 to E18, and in adult pancreas after duct ligation. In normal adult pancreas there was very little netrin-1 expression. Netrin-1 expression was observed both in endocrine and exocrine cells. At the immunohistochemical level, it was expressed by islet cells during tissue regeneration. We could show that netrin-1 increases the migration of fetal islet cells and of a ductal cell line, mainly via a chemokinetic effect. From the two well-established netrin receptors, DCC and neogenin, we only found neogenin to be expressed in the pancreas. Neogenin expression coincided with the period of netrin-1 up-regulation. CONCLUSION/INTERPRETATION: Netrin-1 is involved in pancreatic morphogenesis and tissue remodelling and plays a role in the regulation of duct-cell and fetal-islet cell migration. This can be of importance in islet regeneration, where migration of islet precursors takes place.

Mitogenic effect of gastrin and expression of gastrin receptors in duct-like cells of rat pancreas.

BACKGROUND & AIMS: Ductular metaplastic cells are observed during pancreas injury. Growth control by gastrin and expression of gastrin/cholecystokinin (CCK) B receptors were evaluated in these cells. METHODS: Acinoductal transdifferentiation was induced in vitro by culturing of acinar cells, and ductular metaplasia was obtained in vivo by ligation of the pancreatic ducts. Mitogenic effects of gastrin I on ductal complexes in vivo and of tetragastrin, pentagastrin, and gastrin I and II, with or without the CCK-B receptor antagonist L-365,260, on duct-like cells in vitro were analyzed by 5-bromo-2'-deoxyuridine labeling. Immunocytochemistry, Western blotting, and reverse-transcription polymerase chain reaction were applied for detection of the CCK-B receptor. RESULTS: Gastrin analogues induced a mitogenic stimulus in the duct-like cells in vitro and in ductal complexes in duct-ligated rat pancreas. Immunocytochemistry showed expression of CCK-B receptors in these models and in fetal but not normal adult exocrine pancreas. Additionally, up-regulation of CCK-B receptors during ductular metaplasia was shown by Western blotting and reverse-transcription polymerase chain reaction. CONCLUSIONS: Duct-like pancreatic epithelial cells in vitro and ductal complexes in vivo express gastrin/CCK-B receptors and proliferate in response to gastrin.

Resolution recovery for list-mode reconstruction in SPECT.

The purpose of the study was to evaluate the resolution recovery in the list-mode iterative reconstruction algorithm (LMIRA) for SPECT. In this study we compare the performance of the proposed method with other iterative resolution recovery methods for different noise levels. We developed an iterative reconstruction method which uses list-mode data instead of binned data. The new algorithm makes use of a more accurate model of the collimator structure. We compared the SPECT list-mode reconstruction with MLEM, OSEM and RBI, all including resolution recovery. For the evaluation we used Gaussian shaped sources with different FWHM at three different locations and three noise levels. For these distributions we calculated the reconstructed images for a different number of iterations. The absolute error for the reconstructed images was used to evaluate the performance. The performance of all four methods is comparable for the sources located in the centre of the field of view. For the sources located out of the centre, the error of the list-mode method is significantly lower than that of the other methods. Splitting the system model into a separate object-dependent and detector-dependent module gives us a flexible reconstruction method. With this we can very easily adapt the resolution recovery to different collimator types.

Role of pancreatic beta-cells in the process of beta-cell death.

Studies on the pathogenesis of type 1 diabetes have mainly focused on the role of the immune system in the destruction of pancreatic beta-cells. Lack of data on the cellular and molecular events at the beta-cell level is caused by the inaccessibility of these cells during development of the disease. Indirect information has been collected from isolated rodent and human islet cell preparations that were exposed to cytotoxic conditions. This article reviews in vitro experiments that investigated the role of beta-cells in the process of beta-cell death. beta-Cells rapidly die in necrosis because of toxic levels of oxidizing radicals or of nitric oxide; they progressively become apoptotic after prolonged culture at low glucose or with proinflammatory cytokines. Their susceptibility to necrosis or apoptosis varies with their functional state and thus with the environmental conditions. A change in cellular phenotype can alter its recognition of potentially cytotoxic agents and its defense mechanisms against cell death. These observations support the view that beta-cells are not necessarily passive victims of a cytotoxic process but can actively participate in a process of beta-cell death. Their role will be influenced by neighboring non-beta-cells, which can make the islet internal milieu more protective or toxic for the beta-cells. We consider duct cells as potentially important contributors to this local process.

PET imaging using gamma cameras.

This paper will review the recent advances and future developments in the field of coincidence imaging of positron emitters with a conventional Anger-type gamma camera. FDG imaging has shown high clinical importance in cardiology, neurology and especially oncology. Since access to full ring PET is mainly limited to university hospitals, there have been new developments allowing PET imaging on the standard Anger gamma camera. First the principles of coincidence imaging on a gamma camera will be reviewed. We will discuss the limitations of this technique, and the techniques used to partly overcome these limitations. The different configurations of the gamma camera operating in coincidence mode are pointed out. Different corrections for image degrading effects and reconstruction methods are evaluated in the final part.

Iterative reconstruction algorithms in nuclear medicine.

Iterative reconstruction algorithms produce accurate images without streak artifacts as in filtered backprojection. They allow improved incorporation of important corrections for image degrading effects, such as attenuation, scatter and depth-dependent resolution. Only some corrections, which are important for accurate reconstruction in positron emission tomography and single photon emission computed tomography, can be applied to the data before filtered backprojection. The main limitation for introducing iterative algorithms in nuclear medicine has been computation time, which is much longer for iterative techniques than for filtered backprojection. Modern algorithms make use of acceleration techniques to speed up the reconstruction. These acceleration techniques and the development in computer processors have introduced iterative reconstruction in daily nuclear medicine routine. We give an overview of the most important iterative techniques and discuss the different corrections that can be incorporated to improve the image quality.

Image-correction techniques in SPECT.

This overview takes a look at different correction techniques for Single Photon Emission Computed Tomography (SPECT). We discuss the influence of the detection system followed by the scatter and attenuation caused by the object of investigation. When possible we describe how the correction methods for the different physical effects can be incorporated in the reconstruction method, being either filtered backprojection or iterative reconstruction.

MRI guided segmentation and quantification of SPECT images of the basal ganglia: a phantom study.

Due to the limited resolution of single-photon emission computed tomography (SPECT) imaging devices, tissue interfaces are not well defined in the reconstructed image, even though resolution recovery techniques may be used during reconstruction. Therefore, segmentation of a particular region and quantification of the tracer uptake in that region is critical due to spillover effects, when based on the SPECT image only. In this study, we present two methods for quantification of tracer uptake in a SPECT image, defined by a matched high resolution structural magnetic resonance image. We show preliminary results of both techniques, when applied for quantifying regional uptake in the different compartments of a phantom simulating the basal ganglia. These results indicate that the quantification method, which takes into account the blurring by the SPECT imaging device, promises to be perform better in the presence of background activity.