Development of Bioartificial Pancreas/Pancreas Organoids

The pancreas is a complex and highly specialized organ, whose main function is to maintain glucose homeostasis in the body. This homeostasis is regulated by the endocrine compartment of the pancreas. Multiple potential molecular and metabolic perturbations occurring in these cells may result in a wide spectrum of disorders. The study of the pancreas to dissect the underlying mechanisms of pathology has been challenging, owing to the lack of adequate biological models. This caveat has been recently addressed by the generation of organ-like structures (“organoids”) and artificial constructs that aim at mimicking the cytoarchitecture of the pancreas. Advances in our understanding of how the pancreas forms during embryonic development have provided us with insights on how to direct differentiation toward the formation of endocrine cells. In vitro differentiated endocrine cells, for instance, may be used to treat pancreatic disorders such as insulin-dependent diabetes and as a screening tool for potential therapies. This approach has progressively displaced the use of islets isolated from cadaveric donors, a material of scarce availability and broad donor-related variability. In fact, human embryonic stem cell (hESc)-derived islets are currently in clinical trials for type 1 diabetes. This short review discusses the current state of the art in the generation of renewable sources of pancreatic endocrine cells, with a special emphasis in future therapeutic applications

SliceChip: Benchtop Fluidic Platform for Organotypic Culture and Serial Assessment of Human and Rodent Pancreatic Slices

Enzymatically isolated pancreatic islets are the most commonly used ex vivo testbeds for diabetes research. Recently, precision-cut living slices of human pancreas are emerging as an exciting alternative because they maintain the complex architecture of the endocrine and exocrine tissues, and do not suffer from the mechanical and chemical stress of enzymatic isolation. We report a fluidic pancreatic SliceChip platform with dynamic environmental controls that generates a warm, oxygenated, and bubble-free fluidic pathway across singular immobilized slices and continuously delivers fresh nutrients while removing harmful digestive enzyme. A degasser ensures the system remains bubble-free while systemic pressurization with compressed oxygen ensures slice medium remains adequately oxygenated. Computational modeling of perfusion and oxygen dynamics within SliceChip guide the system’s physiomimetic culture conditions. Serial assessments of pancreatic slice insulin secretion in response to glucose stimulations demonstrated the effectiveness of our system in preserving pancreatic islet function under physiological oxygen levels over several days. Fluorescent images acquired every 4 hours of transgenic murine pancreatic slices were reliably stable and recoverable over a 5-day period due to the inclusion of a 3D-printed bioinert metallic anchor that maintained slice position within the SliceChip. Our slice on a chip platform should expand the useability of human pancreatic slices for diabetes pathogenesis and the development of new therapeutic approaches, while also enabling organotypic culture and assessment of other tissue slices such as brain and patient tumors

Intra-Amniotic Soluble Endoglin Impairs Lung Development in Neonatal Rats

Soluble endoglin (sENG) is increased in the amniotic fluid of women with preeclampsia and chorioamnionitis. Preterm infants born to women with these disorders have an increased risk of aberrant lung development. Whether this increased risk is secondary to elevated sENG levels is unclear. The objective of this study was to determine whether intrauterine exposure to an adenovirus overexpressing sENG impairs neonatal lung angiogenesis by modulating lung endothelial nitric oxide synthase (eNOS) signaling. Pregnant Sprague-Dawley rats were randomly assigned to receive ultrasound-guided intra-amniotic injections of adenovirus overexpressing sENG (Ad-sENG) or control adenovirus (Ad-control) on embryonic day 17. After this exposure, rat pups were maintained in normoxia and evaluated on postnatal day 14. Intra-amniotic Ad-sENG decreased lung vascular endothelial growth factor receptor 2 and eNOS expression in rat pups. This was accompanied by a marked decrease in lung angiogenesis and alveolarization. Ad-sENG-exposed pups also had an increase in right ventricular systolic pressure, weight ratio of right ventricle to left ventricle plus septum, and pulmonary vascular remodeling. In addition, exposure of human pulmonary artery endothelial cells to recombinant sENG reduced endothelial tube formation and protein levels of eNOS, phosphorylated eNOS, and phosphorylated Smad1/5. Together, our findings demonstrate that intrauterine exposure to an adenovirus overexpressing sENG disrupts lung development by impairing Smad1/5-eNOS signaling. We speculate that sENG-mediated dysregulation of Smad1/5-eNOS signaling contributes to impaired lung development and potentially primes the developing lung for further postnatal insults. Further studies exploring the relationship between amniotic fluid sENG levels and preterm respiratory outcomes will be necessary

Antisense miR-7 Impairs Insulin Expression in Developing Pancreas and in Cultured Pancreatic Buds

MicroRNAs regulate gene expression by inhibiting translation or inducing target mRNA degradation. MicroRNAs regulate organ differentiation and embryonic development, including pancreatic specification and islet function. We showed previously that miR-7 is highly expressed in human pancreatic fetal and adult endocrine cells. Here we determined the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic days e10.5 and e11.5, then began to increase at e13.5 through e14.5, and eventually decreased by e18. In situ hybridization and immunostaining analysis showed that miR-7 colocalizes with endocrine marker Isl1, suggesting that miR-7 is expressed preferentially in endocrine cells. Whole-mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall downregulation of insulin production, decreased β-cell numbers, and glucose intolerance in the postnatal period. This phenomenon is specific for miR-7 and possibly due to a systemic effect on pancreatic development. On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds leads to β-cell death and generation of β-cells expressing less insulin than those in MO control. Therefore, in addition to the potential indirect effects on pancreatic differentiation derived from its systemic downregulation, the knockdown of miR-7 appears to have a β-cell-specific effect as well. These findings suggest that modulation of miR-7 expression could be utilized in the development of stem cell therapies to cure diabetes. </jats:p

251-LB: Tracking Beta-Cell Regeneration in Human Pancreatic Slices using Adenovirus Transduction

Introduction: The study of pancreatic regeneration would benefit greatly from the design and validation of robust human-based models. Human Pancreatic Slices (HPSs) are thin organotypic sections of live pancreatic tissue. The sectioning method preserves the overall histological structure of the organ, maintaining the integrity of the extracellular matrix and the natural interaction between the endocrine and exocrine compartments, as well as the local neural, vascular and immune milieu. Conditions for the long-term culture of HPSs, recently reported by our team, have enabled the real-time analysis of beta-cell neogenesis using adenoviral (AV) co-transduction of a red-green reporter and an insulin tracer in human pancreatic slices. However, the ability of these cells to respond to glucose was not established at that time. Methods: To determine whether new INS+ cells respond to glucose, we have designed an AV in which INS-dependent recombination leads to the expression of a blue marker (moxBFP) and a Calcium Imaging Reporter (gcAMP6s, green) whose intensity is proportional to glucose-dependent INS secretion. This allowed us to monitor glucose-stimulated calcium influx in the newly created cells. To track the generation of new beta cells with an even higher degree of resolution we subsequently placed the transduced slices in a custom-made microfluid chip. Conclusions: This setting further allows for longitudinal functional analyses in precise and highly controlled experimental conditions. Our ability to study regeneration in a clinically meaningful model represents a groundbreaking advance that may fast-track the screening and preclinical development of therapeutic agents

Neonatal intermittent hypoxia persistently impairs lung vascular development and induces long-term lung mitochondrial DNA damage

Adults born preterm have an increased risk of pulmonary vascular disease. Extreme preterm infants often require supplemental oxygen but they also exhibit frequent intermittent hypoxemic episodes (IH). Here, we test the hypothesis that neonatal IH induces lung endothelial cell mitochondrial DNA (mitDNA) damage and contributes to long-term pulmonary vascular disease and pulmonary hypertension (PH). Newborn C57BL/6J mice were assigned to the following groups:) normoxia,) hyperoxia (O65%),) normoxia cycling with IH (O21% + O10%), and) hyperoxia cycling with IH (O65% + O10%) for 3 wk. IH episodes were initiated on. Lung angiogenesis, PH, and mitDNA lesions were assessed at 3 wk and 3 mo. In vitro, the effect of IH on tubule formation and mitDNA lesions was evaluated in human pulmonary microvascular endothelial cells (HPMECs). Data were analyzed by ANOVA. In vitro, IH exposure reduced tubule formation and increased mitDNA lesions in HPMECs. This was most marked in HPMECs exposed to hyperoxia cycling with IH. In vivo, neonatal IH increased lung mitDNA lesions, impaired angiogenesis, and induced PH in 3-wk-old mice. These findings were pronounced in mice exposed to hyperoxia cycling with IH. At 3 mo follow-up, mice exposed to neonatal IH had persistently increased lung mitDNA lesions and impaired lung angiogenesis, even without concomitant hyperoxia exposure. Neonatal IH induces lung endothelial cell mitDNA damage and causes persistent impairment in lung angiogenesis. These findings provide important mechanistic insight into the pathogenesis of pulmonary vascular disease in preterm survivors.Our current study demonstrates that neonatal intermittent hypoxia (IH) alters lung endothelial cell function, induces mitochondrial DNA lesions, and impairs lung vascular growth into adulthood. Moreover, when superimposed on hyperoxia, neonatal IH induces a severe lung vascular phenotype that is seen in preterm infants with PH. These findings suggest that neonatal IH contributes to PH in adults born preterm and importantly, that mitochondrial protection strategies may mitigate these deleterious effects

c-Myc Drives inflammation of the maternal-fetal interface, and neonatal lung remodeling induced by intra-amniotic inflammation

Background: Intra-amniotic inflammation (IAI) is associated with increased risk of preterm birth and bronchopulmonary dysplasia (BPD), but the mechanisms by which IAI leads to preterm birth and BPD are poorly understood, and there are no effective therapies for preterm birth and BPD. The transcription factor c-Myc regulates various biological processes like cell growth, apoptosis, and inflammation. We hypothesized that c-Myc modulates inflammation at the maternal-fetal interface, and neonatal lung remodeling. The objectives of our study were 1) to determine the kinetics of c-Myc in the placenta, fetal membranes and neonatal lungs exposed to IAI, and 2) to determine the role of c-Myc in modulating inflammation at the maternal-fetal interface, and neonatal lung remodeling induced by IAI.Methods: Pregnant Sprague-Dawley rats were randomized into three groups: 1) Intra-amniotic saline injections only (control), 2) Intra-amniotic lipopolysaccharide (LPS) injections only, and 3) Intra-amniotic LPS injections with c-Myc inhibitor 10058-F4. c-Myc expression, markers of inflammation, angiogenesis, immunohistochemistry, and transcriptomic analyses were performed on placenta and fetal membranes, and neonatal lungs to determine kinetics of c-Myc expression in response to IAI, and effects of prenatal systemic c-Myc inhibition on lung remodeling at postnatal day 14.Results: c-Myc was upregulated in the placenta, fetal membranes, and neonatal lungs exposed to IAI. IAI caused neutrophil infiltration and neutrophil extracellular trap (NET) formation in the placenta and fetal membranes, and neonatal lung remodeling with pulmonary hypertension consistent with a BPD phenotype. Prenatal inhibition of c-Myc with 10058-F4 in IAI decreased neutrophil infiltration and NET formation, and improved neonatal lung remodeling induced by LPS, with improved alveolarization, increased angiogenesis, and decreased pulmonary vascular remodeling.Discussion: In a rat model of IAI, c-Myc regulates neutrophil recruitment and NET formation in the placenta and fetal membranes. c-Myc also participates in neonatal lung remodeling induced by IAI. Further studies are needed to investigate c-Myc as a potential therapeutic target for IAI and IAI-associated BPD