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The ability to culture live pancreatic tissue slices for long periods of time would enable longitudinal studies ex vivo. Here the authors culture human and mouse pancreatic slices in a perfluorocarbon-based culture system and show stable endocrine and exocrine function for up to ten days in culture.

Summary: The transplantation of human embryonic stem cell (hESC)-derived insulin-producing β cells for the treatment of diabetes is finally approaching the clinical stage. However, even with state-of-the-art differentiation protocols, a significant percentage of undefined non-endocrine cell types are still generated. Most importantly, there is the potential for carry-over of non-differentiated cell types that may produce teratomas. We sought to modify hESCs so that their differentiated progeny could be selectively devoid of tumorigenic cells and enriched for cells of the desired phenotype (in this case, β cells). Here we report the generation of a modified hESC line harboring two suicide gene cassettes, whose expression results in cell death in the presence of specific pro-drugs. We show the efficacy of this system at enriching for β cells and eliminating tumorigenic ones both in vitro and in vivo. Our approach is innovative inasmuch as it allows for the preservation of the desired cells while eliminating those with the potential to develop teratomas. : In this article, Domínguez-Bendala and colleagues present a strategy to modify pluripotent stem cells to allow for the selective destruction of tumorigenic escapees and/or fully developed teratomas, as well as for the specific selection of differentiated insulin-producing β-like cells. Cells engineered in this manner feature a double fail-safe mechanism designed to minimize the risks associated with their clinical transplantation. Keywords: human embryonic stem cells, beta cell differentiation, suicide genes, teratoma, transplantation

Summary: Treatment of human pancreatic non-endocrine tissue with Bone Morphogenetic Protein 7 (BMP-7) leads to the formation of glucose-responsive β-like cells. Here, we show that BMP-7 acts on extrainsular cells expressing PDX1 and the BMP receptor activin-like kinase 3 (ALK3/BMPR1A). In vitro lineage tracing indicates that ALK3+ cell populations are multipotent. PDX1+/ALK3+ cells are absent from islets but prominently represented in the major pancreatic ducts and pancreatic duct glands. We identified the purinergic receptor P2Y1 (P2RY1) as a surrogate surface marker for PDX1. Sorted P2RY1+/ALK3bright+ cells form BMP-7-expandable colonies characterized by NKX6.1 and PDX1 expression. Unlike the negative fraction controls, these colonies can be differentiated into multiple pancreatic lineages upon BMP-7 withdrawal. RNA-seq further corroborates the progenitor-like nature of P2RY1+/ALK3bright+ cells and their multilineage differentiation potential. Our studies confirm the existence of progenitor cells in the adult human pancreas and suggest a specific anatomical location within the ductal and glandular networks. : Qadir et al. describe and characterize a population of multipotent, BMP-7-responsive progenitor-like cells within the human exocrine pancreas. These cells are characterized by the expression of PDX1 and ALK3, a canonical BMP receptor. Their findings shed new light on potential regenerative pathways in the human pancreas. Keywords: human pancreatic progenitor cells, ALK3, PDX1, islet regeneration, beta cell regeneration

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The chemical behavior of various oligoenes 2 has been studied. The catalytic hydrogenation of diene 3 yielded monoene 4. Triene 7 was hydrogenated to diene 8, monoene 9 and saturated hydrocarbon 10. Bromine addition to 3 and 7 yielded the dibromides 17 and 18, respectively, i.e., the oligoene system has been attacked at its terminal olefinic carbon atoms. Analogously, the higher vinylogs 19 and 20 yielded the 1,8- and 1,10-bromine adduts 23 and 24, respectively, when less than 1 equivalent of bromine was employed. Treatment of tetraene 19 with excess bromine provided tetrabromide 25. In epoxidation reactions, both with meta-chloroperbenzoic acid (MCPBA) and dimethyldioxirane (DMDO) two model oligoenes were studied: triene 7 and tetraene 19. Whereas 7 furnished the rearrangement product 31 with MCPBA, it yielded the symmetrical epoxide 32 with DMDO. Analogously, 19 was converted to mono-epoxide 33 with MCPBA and to 34 with DMDO. Diels–Alder addition of 7 with N-phenyltriazolinedione (PTAD) did not take place. Extension of the conjugated π-system to the next higher vinylog, 19, caused NPTD-addition to the symmetrical adduct 37 in good yield. Comparable results were observed on adding NPTD (equivalent amount) to pentaene 20 and hexaene 21. Using 36 in excess provided the 2:1-adduct 40 from 21 and led to a complex mixture of adducts from heptaene 22. With tetracyanoethylene (TCNE) as the dienophile, tetraolefin 19 yielded the symmetrical adduct 43, although the reaction temperature had to be increased. Pentaene 20 and hexaene 21 led to corresponding results, adducts 44 and 45 being produced in acceptable yields. With nonaene 42 and TCNE the 2:1-adduct 48 was generated according to its spectroscopic data. Exploratory photochemical studies were carried out with tetraene 19 as the model compound. On irradiation this reacted with oxygen to the stable endo-peroxide 52.

Cellular stress, combined with dysfunctional, inadequate mitochondrial phosphorylation, produces an excessive amount of reactive oxygen species (ROS) and an increased level of ROS in cells, which leads to oxidation and subsequent cellular damage. Because of its cell damaging action, an association between anomalous ROS production and disease such as Type 1 (T1D) and Type 2 (T2D) diabetes, as well as their complications, has been well established. However, there is a lack of understanding about genome-driven responses to ROS-mediated cellular stress. Over the last decade, multiple studies have suggested a link between oxidative stress and microRNAs (miRNAs). The miRNAs are small non-coding RNAs that mostly suppress expression of the target gene by interaction with its 3’untranslated region (3′UTR). In this paper, we review the recent progress in the field, focusing on the association between miRNAs and oxidative stress during the progression of diabetes.

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

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.

Nonspecific inflammation in the transplant microenvironment results in β-cell dysfunction and death influencing negatively graft outcome. MicroRNA (miRNA) expression and gene target regulation in transplanted islets are not yet well characterized. We evaluated the impact of inflammation on miRNA expression in transplanted rat islets. Islets exposed in vitro to proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation both in vivo and in vitro. Induction of miRNAs was dependent on NF-κB, a pathway linked with cytokine-mediated islet cell death. RT-PCR confirmed expression of 8 miRNAs. The association between these miRNAs and mRNA target-predicting algorithms in genome-wide RNA studies of β-cell inflammation identified 238 potential miRNA gene targets. Several genes were ontologically associated with regulation of insulin signaling and secretion, diabetes, and islet physiology. One of the most activated miRNAs was miR-21. Overexpression of miR-21 in insulin-secreting MIN6 cells downregulated endogenous expression of the tumor suppressor Pdcd4 and of Pclo, a Ca2+ sensor protein involved in insulin secretion. Bioinformatics identified both as potential targets. The integrated analysis of miRNA and mRNA expression profiles revealed potential targets that may identify molecular targets for therapeutic interventions.

Recombinant proteins are an important tool for research and therapeutic applications. Therapeutic proteins have been delivered to several cell types and tissues and might be used to improve the outcome of the cell transplantation. Recombinant proteins are propagated in bacteria, which will contaminate them with the lypopolysacharide endotoxin found in the outer bacterial membrane. Endotoxin could interfere with in vitro biological assays and is the major pathological factor, which must be removed or inactivated before in vivo administration. Here we describe a one-step protocol in which the endotoxin activity on recombinant proteins is remarkably reduced by transient exposure to acidic conditions. Maximum endotoxin deactivation occurs at acidic pH below their respective isoelectric point (pI). This method does not require additional protein purification or separation of the protein from the endotoxin fraction. The endotoxin level was measured both in vitro and in vivo. For in vitro assessment we have utilized Limulus Amebocyte Lysate method for in vivo the pyrogenic test. We have tested the above-mentioned method with five different recombinant proteins, including a monoclonal antibody clone 5c8 against CD154 produced by hybridomas. More than 99% of endotoxin was deactivated in all of the proteins; the recovery of the protein after deactivation varied between maximum 72.9% and minimum 46.8%. The anti-CD154 clone 5c8 activity remained unchanged as verified by the measurement of binding capability to activated lymphocytes. Furthermore, the effectiveness of this method was not significantly altered by urea, commonly used in protein purification. This procedure provides a simple and cost-efficient way to reduce the endotoxin activity in antibodies and recombinant proteins.

Delivering cytoprotective proteins/peptides into pancreata prior to islet isolation through protein transduction (PT) is a novel strategy to enhance the yield of viable transplantable islets. Previous work has shown that the protein transduction domain PTD-5 efficiently transduced islets via the pancreatic duct. TAT/PTD is a well-characterized PTD with the capability to cross even the hemato–encephalic barrier. In this study, we investigated the utilization of the 11-aa TAT protein transduction domain (TAT/PTD) to deliver peptides or proteins of different sizes ranging from 1.2 to 120 kDa, as the TAT/PTD and TAT/PTD-BH4 peptide, or the TAT/PTD–β-galactosidase fusion protein, into islets through the pancreatic duct. Using flow cytometry analysis we found that TAT/PTD derivatives transduced practically 100% of the islet cell population. Moreover, confocal laser scanning microscopy in live, nonfixed islets confirmed these results assessing transduction of TAT/PTD molecules into intact nondisaggregated islets. TAT–β-galactosidase peptide conjugated to FITC was not compartment selective, as both cytoplasmic and nucleic cellular compartments were positively stained. Furthermore, TAT–β-galactosidase peptide delivery was highly effective, as even cells located in the inner core region of the islets were transduced. Finally, transduced TAT–β-galactosidase fusion protein was biologically active after islet isolation and manipulation, and islet insulin secretion capability was not compromised by peptide transduction. These findings suggest that the transduction of chimeric TAT/PTD proteins can represent an efficient tool of molecular delivery independent of the size, to enhance or modify a specific phenotype at the nuclei or cytoplasmic level.

OBJECTIVE:: The purpose of the present study was to determine the effect of insulin therapy on hepatic function, structure, and hepatic mRNA and protein cytokine expression during the hypermetabolic cascade post burn.SUMMARY BACKGROUND DATA:: Liver function and morphology are crucial for survival of patients suffering from trauma, operations, or infections. Insulin decreased mortality and prevented the incidence of multiorgan failure in critically ill patients.METHODS:: Rats received a thermal injury and were randomly divided into the insulin or control group. Our outcome measures encompassed the effect of insulin on hepatic proteins, hepatic pro- and anti-inflammatory cytokines mRNA and proteins, hepatocyte proliferation, including Bcl-2 and hepatocyte apoptosis, with caspases-3 and caspases-9.RESULTS:: Insulin significantly improved hepatic protein synthesis by increasing albumin and decreasing c-reactive protein and fat (P < 0.05). Insulin decreased the hepatic inflammatory response signal cascade by decreasing hepatic pro-inflammatory cytokines mRNA and proteins IL-1β and tumor necrosis factor at pretranslational levels. Insulin increased hepatic cytokine mRNA and protein expression of IL-2 and IL-10 at a pretranslational level when compared with controls (P < 0.05). Insulin increased hepatocyte proliferation along with Bcl-2 concentration, while decreasing hepatocyte apoptosis along with decreased caspases-3 and -9 concentration, thus improving liver morphology (P < 0.05).CONCLUSIONS:: Our data provide insight that insulin attenuates the inflammatory response by decreasing the pro-inflammatory and increasing the anti-inflammatory cascade, thus restoring hepatic homeostasis, which has been shown to be critical for organ function and survival of critically ill patients. [ABSTRACT FROM AUTHOR]