Association of serum microRNAs with islet autoimmunity, disease progression and metabolic impairment in relatives at risk of type 1 diabetes.

AIMS/HYPOTHESIS: MicroRNAs (miRNAs) are key regulators of gene expression and novel biomarkers for many diseases. We investigated the hypothesis that serum levels of some miRNAs would be associated with islet autoimmunity and/or progression to type 1 diabetes. METHODS: We measured levels of 93 miRNAs most commonly detected in serum. This retrospective cohort study included 150 autoantibody-positive and 150 autoantibody-negative family-matched siblings enrolled in the TrialNet Pathway to Prevention Study. This was a young cohort (mean age = 11 years), and most autoantibody-positive relatives were at high risk because they had multiple autoantibodies, with 39/150 (26%, progressors) developing type 1 diabetes within an average 8.7 months of follow-up. We analysed miRNA levels in relation to autoantibody status, future development of diabetes and OGTT C-peptide and glucose indices of disease progression. RESULTS: Fifteen miRNAs were differentially expressed when comparing autoantibody-positive/negative siblings (range -2.5 to 1.3-fold). But receiver operating characteristic (ROC) analysis indicated low specificity and sensitivity. Seven additional miRNAs were differentially expressed among autoantibody-positive relatives according to disease progression; ROC returned significant AUC values and identified miRNA cut-off levels associated with an increased risk of disease in both cross-sectional and survival analyses. Levels of several miRNAs showed significant correlations (r values range 0.22-0.55) with OGTT outcomes. miR-21-3p, miR-29a-3p and miR-424-5p had the most robust associations. CONCLUSIONS/INTERPRETATION: Serum levels of selected miRNAs are associated with disease progression and confer additional risk of the development of type 1 diabetes in young autoantibody-positive relatives. Further studies, including longitudinal assessments, are warranted to further define miRNA biomarkers for prediction of disease risk and progression.

Immunometabolic biomarkers for partial remission in type 1 diabetes mellitus.

Shortly after diagnosis of type 1 diabetes mellitus (T1DM) and initiation of insulin therapy, many patients experience a transient partial remission (PR) phase, also known as the honeymoon phase. This phase presents a potential therapeutic opportunity due to its association with immunoregulatory and ß cell-protective mechanisms. However, the lack of biomarkers makes its characterization difficult. In this review, we cover the current literature addressing the discovery of new predictive and monitoring biomarkers that contribute to the understanding of the metabolic, epigenetic, and immunological mechanisms underlying PR. We further discuss how these peripheral biomarkers reflect attempts to arrest ß cell autoimmunity and how these can be applied in clinical practice.

SliceChip: a 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 with continuous deliver of fresh media and the ability to perform repeat serial perfusion assessments. 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. Maintenance of the physiological glucose dependent insulin secretion profile across repeat perfusion assessments of individual pancreatic slices kept under physiological oxygen levels demonstrated the culture capacity of our platform. 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 has the potential to 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.

Dynamic scRNA-seq of live human pancreatic slices reveals functional endocrine cell neogenesis through an intermediate ducto-acinar stage.

Human pancreatic plasticity is implied from multiple single-cell RNA sequencing (scRNA-seq) studies. However, these have been invariably based on static datasets from which fate trajectories can only be inferred using pseudotemporal estimations. Furthermore, the analysis of isolated islets has resulted in a drastic underrepresentation of other cell types, hindering our ability to interrogate exocrine-endocrine interactions. The long-term culture of human pancreatic slices (HPSs) has presented the field with an opportunity to dynamically track tissue plasticity at the single-cell level. Combining datasets from same-donor HPSs at different time points, with or without a known regenerative stimulus (BMP signaling), led to integrated single-cell datasets storing true temporal or treatment-dependent information. This integration revealed population shifts consistent with ductal progenitor activation, blurring of ductal/acinar boundaries, formation of ducto-acinar-endocrine differentiation axes, and detection of transitional insulin-producing cells. This study provides the first longitudinal scRNA-seq analysis of whole human pancreatic tissue, confirming its plasticity in a dynamic fashion.

Association between the Mediterranean Diet and Metabolic Syndrome with Serum Levels of miRNA in Morbid Obesity.

BACKGROUND: The Mediterranean diet (MD) could be involved in the regulation of different miRNAs related to metabolic syndrome (MS). METHODS: We analyzed the serum level of mir-let7a-5p, mir-21, mir-590, mir-107 and mir-192 in patients with morbid obesity and its association with the MD and MS. RESULTS: There is an association between the adherence to MD and higher serum levels of mir-590. Mir-590 was lower in those patients who consumed >2 commercial pastries/week. Mir-let7a was lower in those who consumed ≥1 sweetened drinks, in those who consumed ≥3 pieces of fruit/day and in those who consumed less red than white meat. A lower mir-590 and mir-let7a, and a higher mir-192 level, were found in patients who met the high-density lipoprotein cholesterol (HDL) criterion of MS. A higher mir-192 was found in those patients who met the triglyceride criterion of MS and in those with type 2 diabetes (T2DM). CONCLUSIONS: There is an association between specific serum levels of miRNAs and the amount and kind of food intake related to MD. Mir-590 was positively associated with a healthy metabolic profile and type of diet, while mir-192 was positively associated with a worse metabolic profile. These associations could be suggestive of a possible modulation of these miRNAs by food.

MicroRNA signature of the human developing pancreas.

BACKGROUND: MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. RESULTS: The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga), was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase algorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. CONCLUSIONS: We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in parallel by other microRNAs as well. This study may further the understanding of gene expression regulation in the human developing pancreas.

Pancreas tissue slices from organ donors enable in situ analysis of type 1 diabetes pathogenesis.

In type 1 diabetes (T1D), autoimmune destruction of pancreatic ß cells leads to insulin deficiency and loss of glycemic control. However, knowledge about human pancreas pathophysiology in T1D remains incomplete. To address this limitation, we established a pancreas tissue slice platform of donor organs with and without diabetes, facilitating the first live cell studies of human pancreas in T1D pathogenesis to our knowledge. We show that pancreas tissue slices from organ donors allow thorough assessment of processes critical for disease development, including insulin secretion, ß cell physiology, endocrine cell morphology, and immune infiltration within the same donor organ. Using this approach, we compared detailed pathophysiological profiles for 4 pancreata from donors with T1D with 19 nondiabetic control donors. We demonstrate that ß cell loss, ß cell dysfunction, alterations of ß cell physiology, and islet infiltration contributed differently to individual cases of T1D, allowing insight into pathophysiology and heterogeneity of T1D pathogenesis. Thus, our study demonstrates that organ donor pancreas tissue slices represent a promising and potentially novel approach in the search for successful prevention and reversal strategies of T1D.

MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas.

MicroRNAs (miRNA) are small non-coding RNAs that inhibit gene expression through binding to complementary messenger RNA sequences. miRNAs have been predicted to target genes important for pancreas development, proper endocrine cell function and metabolism. We previously described that miRNA-7 (miR-7) was the most abundant and differentially expressed islet miRNA, with 200-fold higher expression in mature human islets than in acinar tissue. Here we have analyzed the temporal and spatial expression of miR-7 in human fetal pancreas from 8 to 22 weeks of gestational age (wga). Human fetal (8-22wga) and adult pancreases were processed for immunohistochemistry, in situ hybridization, and quantitative RT-PCR of miRNA and mRNA. miR-7 was expressed in the human developing pancreas from around 9wga and reached its maximum expression levels between 14 and 18wga, coinciding with the exponential increase of the pancreatic endocrine hormones. Throughout development miR-7 expression was preferentially localized to endocrine cells and its expression persisted in the adult pancreas. The present study provides a detailed analysis of the spatiotemporal expression of miR-7 in developing human pancreas. The specific localization of miR-7 expression to fetal and adult endocrine cells indicates a potential role for miR-7 in endocrine cell differentiation and/or function. Future functional studies of a potential role for miR-7 function in islet cell differentiation and physiology are likely to identify novel targets for the treatment of diabetes and will lead to the development of improved protocols for generating insulin-producing cells for cell replacement therapy.

A Double Fail-Safe Approach to Prevent Tumorigenesis and Select Pancreatic ß Cells from Human Embryonic Stem Cells.

The transplantation of human embryonic stem cell (hESC)-derived insulin-producing ß 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, ß 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 ß 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.

Quantitative differential expression analysis reveals miR-7 as major islet microRNA.

MicroRNAs (miRNAs) are non-coding gene products that regulate gene expression through specific binding to target mRNAs. Cell-specific patterns of miRNAs are associated with the acquisition and maintenance of a given phenotype, such as endocrine pancreas (islets). We hypothesized that a subset of miRNAs could be differentially expressed in the islets. Using miRNA microarray technology and quantitative RT-PCR we identified a subset of miRNAs that are the most differentially expressed islet miRNAs (ratio islet/acinar>150-fold), miR-7 being the most abundant. A similarly high ratio for miR-7 was observed in human islets. The ratio islet/acinar for miR-375, a previously described islet miRNA, was <10 and is 2.5x more abundant in the islets than miR-7. Therefore, we conclude that miR-7 is the most abundant endocrine miRNA in islets while miR-375 is the most abundant intra-islet miRNA. Our results may offer new insights into regulatory pathways of islet gene expression.

CD40-CD40 ligand interaction activates proinflammatory pathways in pancreatic islets.

Pancreatic islet transplantation is becoming an alternative to insulin therapy in patients suffering from brittle type 1 diabetes. A major obstacle to the procedure is the early graft loss caused by nonspecific inflammation at the site of implantation. We recently discovered that CD40, a member of tumor necrosis factor (TNF) receptor family, is expressed in pancreatic beta-cells. CD40 expression in nonhematopoietic cells is generally associated with inflammation. Therefore, we investigated the potential proinflammatory role of CD40 in human and nonhuman primate islets. Islet beta-cells responded to CD40L interaction by secreting interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein (MIP)-1beta, the latter a chemokine first reported to be produced by islets. Induction of IL-8 and MIP-1beta was confirmed at the transcriptional level by quantitative RT-PCR. MIP-1beta expression in beta-cells was verified by double-immunofluorescence staining. CD40-CD40L interaction activates extracellular signal-regulated kinase 1/2 and nuclear factor-kappaB pathways in insulinoma NIT-1 cells, and inhibitors of either pathway suppress cytokine/chemokine production in islets. Moreover, ligation of CD40 receptor upregulates intercellular adhesion molecule-1, associated with inflammation, at both transcriptional and translational levels. Our results in vitro indicate that the CD40 receptor expressed by beta-cells could be activated in vivo, inducing proinflammatory responses contributing to early islet graft loss after transplantation.

Protein transduction: a novel approach to induce in vitro pancreatic differentiation.

It is widely believed that human embryonic stem (huES) cells may represent a valid alternative to donor pancreata as a source of islets for transplantation. Much is known about the transcription factors whose sequential activation results in the generation of islets during pancreatic development. This knowledge has been used to articulate the theoretical possibility that such process might be recapitulated in vitro from stem cells. However, our understanding of the extracellular signals that prompt the developing pancreas to follow this sequence of molecular events is very limited. Also, the simplicity of in vitro systems makes it difficult, if not impossible, to mimic the complex signaling pattern observed in living embryos. Protein transduction (PT) technology may provide researchers with a new powerful tool to sequentially induce stem cell differentiation, entirely bypassing the need for unraveling the signaling pattern that drives the process in vivo. Here we discuss this novel application of the flourishing PT technology, which may revolutionize the way we direct stem cells along any specific lineage.

The Human Endocrine Pancreas: New Insights on Replacement and Regeneration.

Islet transplantation is an effective cell therapy for type 1 diabetes (T1D) but its clinical application is limited due to shortage of donors. After a decade-long period of exploration of potential alternative cell sources, the field has only recently zeroed in on two of them as the most likely to replace islets. These are pluripotent stem cells (PSCs) (through directed differentiation) and pancreatic non-endocrine cells (through directed differentiation or reprogramming). Here we review progress in both areas, including the initiation of Phase I/II clinical trials using human embryonic stem cell (hESc)-derived progenitors, advances in hESc differentiation in vitro, novel insights on the developmental plasticity of the pancreas, and groundbreaking new approaches to induce ß cell conversion from the non-endocrine compartment without genetic manipulation.

Elevation of cytotoxic lymphocyte gene expression is predictive of islet allograft rejection in nonhuman primates.

Hyperglycemia and increased insulin requirements are indicators of ongoing islet allograft rejection, but there are no methods to predict or confirm rejection. Elevation of cytotoxic lymphocyte (CL) gene expression in peripheral blood (PB) has been correlated with renal allograft rejection in humans, but no published study has assessed the utility of monitoring these markers as predictors of rejection before the onset of clinical symptoms. We have established quantitative real-time PCR methods to determine the levels of mRNA transcripts for the CL genes granzyme B (GB), perforin, and fas ligand in blood samples from rhesus and cynomolgus monkeys. Four rhesus monkeys with long-term islet allograft function were studied. Antirejection (anti-CD154) therapy was discontinued, and weekly PB samples were obtained to determine whether the levels of mRNA transcripts for CL genes correlated with and/or were predictive of islet allograft rejection, defined as a loss of C-peptide production. For all monkeys, elevation of CL gene expression preceded rejection by 83--197 days, with GB as the best predictor. Elevated mRNA levels were sustained for 2--2.5 months in three of four animals and 1 month in the other, thus suggesting that the testing of these parameters may have practical applications in clinical islet cell transplantation.

Delivery of TAT/PTD-fused proteins/peptides to islets via pancreatic duct.

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-beta-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-beta-galactosidase peptide conjugated to FITC was not compartment selective, as both cytoplasmic and nucleic cellular compartments were positively stained. Furthermore, TAT-beta-galactosidase peptide delivery was highly effective, as even cells located in the inner core region of the islets were transduced. Finally, transduced TAT-beta-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.

BMP-7 Induces Adult Human Pancreatic Exocrine-to-Endocrine Conversion.

The exocrine pancreas can give rise to endocrine insulin-producing cells upon ectopic expression of key transcription factors. However, the need for genetic manipulation remains a translational hurdle for diabetes therapy. Here we report the conversion of adult human nonendocrine pancreatic tissue into endocrine cell types by exposure to bone morphogenetic protein 7. The use of this U.S. Food and Drug Administration-approved agent, without any genetic manipulation, results in the neogenesis of clusters that exhibit high insulin content and glucose responsiveness both in vitro and in vivo. In vitro lineage tracing confirmed that BMP-7-induced insulin-expressing cells arise mainly from extrainsular PDX-1(+), carbonic anhydrase II(-) (mature ductal), elastase 3a (acinar)(-) , and insulin(-) subpopulations. The nongenetic conversion of human pancreatic exocrine cells to endocrine cells is novel and represents a safer and simpler alternative to genetic reprogramming.

Real-time sequence-specific primer polymerase chain reaction amplification of HLA class II alleles: a novel approach to analyze microchimerism.

The careful assessment of microchimerism is essential to investigate the effects of donor bone marrow-derived cells in transplantation. We have developed a protocol to assess microchimerism based on the HLA mismatch between the recipient and the donor. Our approach combines real-time polymerase chain reaction (PCR) with sequence-specific primer PCR (SSP-PCR) to selectively amplify and measure the abundance of donor HLA alleles in DNA samples extracted from the recipient after transplant. To optimize and validate the reliability of this method at different levels of microchimerism, we tested serial dilutions of donor DNA into recipient DNA. We demonstrate that donor alleles can be readily detected and reliably measured at concentrations as low as 0.1%. This method is simple and rapid and could find practical application in the assessment of microchimerism in patients receiving organ or cellular transplants in conjunction with donor bone marrow cells infusion.

Quantification of ribozyme target RNA using real-time PCR.

An important part of the ribozyme efficiency-screening process is to have a fast and accurate way to measure steady-state levels of the target RNA. Here, we describe the use of real-time polymerase chain reaction (PCR) for quantification of ribozyme target transcripts. In contrast to classical quantitative PCR, real-time PCR does not require extensive manipulation or generation of relatively complex reagents, thus reducing the risk of contamination. PCR products generated by Taq polymerase in the presence of SYBR Green dye I can be monitored each cycle by collecting fluorescence signals emitted only as the double-stranded DNA is formed. The temperature at which the fluorescent data used for quantification are collected is based on the melting-curve analysis of the amplified product. After constructing a standard curve by plotting the log of the standards' copy number vs their fractional cycle number, the copy number of the unknown samples is automatically determined by interpolation of this curve. However it is very important to validate the melting curve profile with standard gel electrophoresis, particularly while setting up the technique. Real-time PCR is fast and reproducible. Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 h. The coefficient of variance is 15% in the range of 104-106 gene copies.

MicroRNAs in islet immunobiology and transplantation.

The ultimate goal of diabetes therapy is the restoration of physiologic metabolic control. For type 1 diabetes, research efforts are focused on the prevention or early intervention to halt the autoimmune process and preserve ß cell function. Replacement of pancreatic ß cells via islet transplantation reestablishes physiologic ß cell function in patients with diabetes. Emerging research shows that microRNAs (miRNAs), noncoding small RNA molecules produced by a newly discovered class of genes, negatively regulate gene expression. MiRNAs recognize and bind to partially complementary sequences of target messenger RNA (mRNA), regulating mRNA translation and affecting gene expression. Correlation between miRNA signatures and genome-wide RNA expression allows identification of multiple miRNA-mRNA pairs in biological processes. Because miRNAs target functionally related genes, they represent an exciting and indispensable approach for biomarkers and drug discovery. We are studying the role of miRNA in the context of islet immunobiology. Our research aims at understanding the mechanisms underlying pancreatic ß cell loss and developing clinically relevant approaches for preservation and restoration of ß cell function to treat insulin-dependent diabetes. Herein, we discuss some of our recent efforts related to the study of miRNA in islet inflammation and islet engraftment. Our working hypothesis is that modulation of the expression of specific microRNAs in the transplant microenvironment will be of assistance in enhancing islet engraftment and promoting long-term function.

MicroRNA expression in alpha and beta cells of human pancreatic islets.

microRNAs (miRNAs) play an important role in pancreatic development and adult ß-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 ß-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 ß-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 ß-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 ß-cells (ß-miRNAs). Bioinformatic analysis identified potential targets of ß-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 ß-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 ß-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.