While regional heterogeneity in islet distribution has been well studied in rodents, less is known about human pancreatic histology. To fill gaps in our understanding, regional differences in the adult human pancreas were quantitatively analyzed including the pathogenesis of type 2 diabetes (T2D). Cadaveric pancreas specimens were collected from the head, body and tail regions of each donor, including subjects with no history of diabetes or pancreatic diseases (n = 23) as well as patients with T2D (n = 12). The study further included individuals from whom islets were isolated (n = 7) to study islet yield and function in a clinical setting of islet transplantation. The whole pancreatic sections were examined using an innovative large-scale image capture and unbiased detailed quantitative analyses of the characteristics of islets from each individual (architecture, size, shape and distribution). Islet distribution/density is similar between the head and body regions, but is >2-fold higher in the tail region. In contrast to rodents, islet cellular composition and architecture were similar throughout the pancreas and there was no difference in glucose-stimulated insulin secretion in islets isolated from different regions of the pancreas. Further studies revealed preferential loss of large islets in the head region in patients with T2D. The present study has demonstrated distinct characteristics of the human pancreas, which should provide a baseline for the future studies integrating existing research in the field and helping to advance bi-directional research between humans and preclinical models.

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.

Human islets exhibit distinct islet architecture with intermingled alpha- and beta-cells particularly in large islets. In this study, we quantitatively examined pathological changes of the pancreas in patients with type 2 diabetes (T2D). Specifically, we tested a hypothesis that changes in endocrine cell mass and composition are islet-size dependent. A large-scale analysis of cadaveric pancreatic sections from T2D patients (n = 12) and non-diabetic subjects (n = 14) was carried out combined with semi-automated analysis to quantify changes in islet architecture. The method provided the representative islet distribution in the whole pancreas section that allowed us to examine details of endocrine cell composition in individual islets. We observed a preferential loss of large islets (>60 µm in diameter) in T2D patients compared to non-diabetic subjects. Analysis of islet cell composition revealed that the beta-cell fraction in large islets was decreased in T2D patients. This change was accompanied by a reciprocal increase in alpha-cell fraction, however total alpha-cell area was decreased along with beta-cells in T2D. Delta-cell fraction and area remained unchanged. The computer-assisted quantification of morphological changes in islet structure minimizes sampling bias. Significant beta-cell loss was observed in large islets in T2D, in which alpha-cell ratio reciprocally increased. However, there was no alpha-cell expansion and the total alpha-cell area was also decreased. Changes in islet architecture were marked in large islets. Our method is widely applicable to various specimens using standard immunohistochemical analysis that may be particularly useful to study large animals including humans where large organ size precludes manual quantitation of organ morphology.

BackgroundType 1 and Type 2 diabetes mellitus (T1DM and T2DM) are caused by beta(β)-cell loss and functional impairment. Identification of mechanisms of β-cell death and therapeutic interventions to enhance β-cell survival are essential for prevention and treatment of diabetes. Oxidative stress is a common feature of both T1DM and T2DM; elevated biomarkers of oxidative stress are detected in blood, urine and tissues including pancreas of patients with DM. Islet transplantation is a promising treatment for diabetes. However, exposure to stress (chemical and mechanical) and ischemia-reperfusion during isolation and transplantation causes islet loss by generation of reactive oxygen species (ROS). Human intracellular antioxidant enzymes and related molecules are essential defenses against ROS. Antioxidant enzyme levels including superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) have been shown to be low in islet cells. However, little is known about the expression and function of antioxidant enzymes within islet cell subsets. We evaluated the expression of the key antioxidant enzymes in β- and alpha(α)-cell and accessed effects of oxidative stress, islet isolation and transplantation on β/α-cell ratio and viability in human islets.MethodsHuman pancreata from T1DM, T2DM and non-diabetic deceased donors were obtained and analyzed by confocal microscopy. Isolated islets were (I) transplanted in the renal sub-capsular space of streptozotocin-induced diabetic nude mice (in vivo bioassay), or (II) exposed to oxidative (H2O2) and nitrosative (NO donor) stress for 24 hrs in vitro. The ratio, % viability and death of β- and α-cells, and DNA damage (8OHdG) were measured.Results and conclusionsCatalase and GPX expression was much lower in β- than α-cells. The β/α-cell ratio fells significantly following islet isolation and transplantation. Exposure to oxidative stress caused a significantly lower survival and viability, with higher DNA damage in β- than α-cells. These findings identified the weakness of β-cell antioxidant capacity as a main cause of vulnerability to oxidative stress. Potential strategies to enhance β-cell antioxidant capacity might be effective in prevention/treatment of diabetes.

The pancreatic islet is mainly composed of beta-, alpha- and delta-cells with small numbers of pancreatic polypeptide (PP) and epsilon cells. It is known that there is a region in the head of the pancreas that is rich in PP-cells. In the present study, we examined the distribution of PP-cells, and assessed the influence of the PP-cell rich region to quantify the total islet mass. Pancreatic tissues were collected from donors with no history of diabetes or pancreatic diseases (n = 12). A stereological approach with a computer-assisted large-scale analysis of whole pancreatic sections was applied to quantify the entire distribution of endocrine cells within a given section. The initial whole pancreas analysis showed that a PP-cell rich region was largely restricted to the uncinate process with a clear boundary. The distinct distribution of PP-cells includes irregularly shaped clusters composed solely of PP-cells. Furthermore, in the PP-cell rich region, beta- and alpha-cell mass is significantly reduced compared to surrounding PP-cell poor regions. The results suggest that the analysis of the head region should distinguish the PP-cell rich region, which is best examined separately. This study presents an important implication for the regional selection and interpretation of the results.

Islet isolation and purification using a continuous density gradient may reduce the volume of tissue necessary for implantation into patients, therefore minimizing the risks associated with intraportal infusion in islet transplantation. On the other hand, the purification procedure might result in a decreased number of islets recovered due to various stresses such as exposure to cytokine/chemokine. While a Ficoll-based density gradient has been widely used in purification for clinical trials, purification with Iodixanol (OptiPrep) has been recently reported in islet transplant series with successful clinical outcomes. The aim of the current study was to compare the effects of the purification method using OptiPrep-based and Ficoll-based density gradients. Human islet isolations were performed using a modified automated method. After the digestion phase, pre-purification digests were divided into two groups and purified using a semiautomated cell processor with either a continuous Ficoll or OptiPrep-based density gradient. The quantity, purity, viability and cellular composition of islet preparations from each group were assessed. Cytokine/chemokine and tissue factor production from islet preparations after 48h culture were also measured. Although islet purity, post-purification IEQ, islet recovery rate, FDA/PI and fractional β-cell viability were comparable, β-cell mass after 48h culture significantly improved in OptiPrep group when compared to the Ficoll group. The production of cytokine/chemokine including IL-1β, TNF-α, IFN-γ, IL-6, IL-8, MIP-1β, MCP-1 and RANTES but not tissue factor from the OptiPrep group was significantly lower during 48h culture after isolation. Each preparation contained the similar number of ductal cells and macrophages. Endotoxin level in both gradient medium was also comparable. The purification method using OptiPrep gradient media significantly reduced cytokine/chemokine production but not tissue factor from human islet preparations and improved β-cell survival during pre-transplant culture. Our results suggest that the purification method using OptiPrep gradient media may be of assistance in increasing successful islet transplantation.

β-Cell replacement therapy is a promising field of research that is currently evaluating new sources of cells for clinical use. Pancreatic epithelial cells are potent candidates for β-cell engineering, but their large-scale expansion has not been evidenced yet. Here we describe the efficient expansion and β-cell differentiation of purified human pancreatic duct cells (DCs). When cultured in endothelial growth-promoting media, purified CA19-9(+) cells proliferated extensively and achieved up to 22 population doublings over nine passages. While proliferating, human pancreatic duct-derived cells (HDDCs) downregulated most DC markers, but they retained low CK19 and SOX9 gene expression. HDDCs acquired mesenchymal features but differed from fibroblasts or pancreatic stromal cells. Coexpression of duct and mesenchymal markers suggested that HDDCs were derived from DCs via a partial epithelial-to-mesenchymal transition (EMT). This was supported by the blockade of HDDC appearance in CA19-9(+) cell cultures after incubation with the EMT inhibitor A83-01. After a differentiation protocol mimicking pancreatic development, HDDC populations contained about 2% of immature insulin-producing cells and showed glucose-unresponsive insulin secretion. Downregulation of the mesenchymal phenotype improved β-cell gene expression profile of differentiated HDDCs without affecting insulin protein expression and secretion. We show that pancreatic ducts represent a new source for engineering large amounts of β-like-cells with potential for treating diabetes.

We demonstrated, for the first time, the ability to bind immune regulatory cells to target cells with preservation of their viability and function and protective activity against immune attack. If successfully tested in an animal model, local delivery of immunoprotective Tregs on the surface of transplanted pancreatic islets may be an alternative or improvement to the currently used immunosuppression.