Over the last two decades, increased availability of human pancreatic tissues has allowed for major expansions in our understanding of islet biology in health and disease. Indeed, studies of fixed and frozen pancreatic tissues, as well as efforts using viable isolated islets obtained from organ donors, have provided significant insights toward our understanding of diabetes. However, the procedures associated with islet isolation result in distressed cells that have been removed from any surrounding influence. The pancreas tissue slice technology was developed as an in situ approach to overcome certain limitations associated with studies on isolated islets or fixed tissue. In this Perspective, we discuss the value of this novel platform and review how pancreas tissue slices, within a short time, have been integrated in numerous studies of rodent and human islet research. We show that pancreas tissue slices allow for investigations in a less perturbed organ tissue environment, ranging from cellular processes, over peri-islet modulations, to tissue interactions. Finally, we discuss the considerations and limitations of this technology in its future applications. We believe the pancreas tissue slices will help bridge the gap between studies on isolated islets and cells to the systemic conditions by providing new insight into physiological and pathophysiological processes at the organ level.

Insulin-producing β-cells in pancreatic islets are regulated by systemic cues and, locally, by adjacent islet hormone-producing ‘non-β-cells’ (namely α-cells, δ-cells and γ-cells). Yet whether the non-β-cells are required for accurate insulin secretion is unclear. Here, we studied mice in which adult islets are exclusively composed of β-cells and human pseudoislets containing only primary β-cells. Mice lacking non-β-cells had optimal blood glucose regulation, enhanced glucose tolerance, insulin sensitivity and restricted body weight gain under a high-fat diet. The insulin secretion dynamics in islets composed of only β-cells was comparable to that in intact islets. Similarly, human β-cell pseudoislets retained the glucose-regulated mitochondrial respiration, insulin secretion and exendin-4 responses of entire islets. The findings indicate that non-β-cells are dispensable for blood glucose homeostasis and β-cell function. These results support efforts aimed at developing diabetes treatments by generating β-like clusters devoid of non-β-cells, such as from pluripotent stem cells differentiated in vitro or by reprograming non-β-cells into insulin producers in situ. Pancreatic islet β-cells can regulate insulin secretion in vivo, even in the absence of non-β-cells.

Histopathological heterogeneity in the human pancreas is well documented; however, functional evidence at the tissue level is scarce. Herein, we investigate in situ glucose -stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in donors without diabetes (ND; n = 15), positive for one islet autoantibody (1AAb+; n = 7), and with type 1 diabetes (T1D; <14 months duration, n = 5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features are comparable across regions in ND. In T1D, insulin secretion and beta -cell volume are significantly reduced within all regions, while glucagon and enzymes are unaltered. Beta -cell volume is lower despite normal insulin secretion in 1AAb+, resulting in increased volume -adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ are consistent across the PH, PB, and PT. This study supports low inter -regional variation in pancreas slice function and, potentially, increased metabolic demand in 1AAb+.

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional β-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of β-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as β-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal β-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

Histopathological heterogeneity in human pancreas has been well documented; however, functional evidence at the tissue level is scarce. Herein we investigated in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in no diabetes (ND, n=15), single islet autoantibody-positive (1AAb+, n=7), and type 1 diabetes donors (T1D, <14 months duration, n=5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features were comparable across the regions in ND. In T1D, insulin secretion and beta-cell volume were significantly reduced within all regions, while glucagon and enzymes were unaltered. Beta-cell volume was lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ were consistent across PH, PB and PT. This study supports low inter-regional variation in pancreas slice function and potentially, increased metabolic demand in 1AAb+.

Introduction: Pancreatic islet transplantation is a safe and minimally invasive treatment for insulin-dependent diabetes but its availability is limited due to immunosuppression requirements and donor shortage. Addressing these limitations, our group developed immune-shielding strategies for islet macro-encapsulation [1] and exploring pig islets (adult or neonatal) as alternative cell sources [2]. In our macro-encapsulation setting, it is essential to use intact uniform islets with high purity and ideal size to reduce oxygen demand, maximize applicable density, and obtain optimal diffusion characteristics. This can be achieved by the Pseudo-islets (PIs) system. However, unlike other species, porcine islets fail to form PIs due to differences in basement membrane compositions [3]. Here, we managed to overcome this issue and establish a reproducible protocol of PIs generation from neonatal pig islets and characterize their morphology and functionality. Method: - Pseudo-islets generation: Islets were dissociated into single cells by Accumax. Then seeded into a microwell plate at different seeding density to generate different sizes. After 7 days, the PIs were harvested for analysis. - Static GSIS: islets were pre-incubated in 5mM glucose for 2 hrs, then in 5 mM glucose for 1hr (basal) then in 11mM glucose for 1 hr (Stimulatory). Total insulin content was extracted by RIPA buffer. Samples were analyzed using the Cisbio insulin kit and Synergy neo2 plate reader. - Flow Cytometry: islets were dissociated into single cells by accumax, stained with zombie dye to exclude dead cells, then fixed and permeabilized by Cytoperm/cytofix then blocked then stained against insulin, glucagon and somatostatin for 1hr. Samples analyzed by LSR Fortessa. - Immunostaining islets were fixed and permeabilized by Cytoperm/Cytofix overnight, blocked by blocking buffer then stained overnight against insulin, glucagon and somatostatin. Images were acquired by Zeiss LSM 960. Results: Our protocol enables reproducible production of size homogenous Pseudo-islets (Fig.1). In comparison to native islets, the pseudo-islets formation does not alter the ratios of the major cellular subtypes (Fig. 2A) nor their architecture (Fig. 2B). Their functionality (assessed by static GSIS) were also comparable. (Fig. 3A and 3B). Conclusions: We successfully established a reproducible PIs generation protocol from porcine source. The generated PIs have similar cellular subtypes and they are functionally comparable to the native islets.References: [1] Ludwig, B. et al. “Transplantation of human islets without immunosuppression”.2013. [2] Ludwig, B. et al. “ Favorable outcome of experimental islet xenotransplantation without immunosuppression in a nonhuman primate model of diabetes” 2017. [3] Wang et al, “Characterization of Integrin Expression in Islets Isolated from Hamster, Canine, Porcine, and Human Pancreas” 1999.

Introduction: Xenotransplantation of porcine islets is a promising approach for the treatment of patients with insulin-deficient diabetes mellitus. The optimization of strategies to improve islet engraftment and long-term survival is an active field of preclinical research. Ca2+ influx into beta cells is an important driver of insulin secretion. Therefore, monitoring of Ca2+ kinetics in beta cells provides an important readout for the maturation of neonatal porcine islets (NPIs) in vitro and of NPI grafts in vivo. Methods: The coding sequence for the ultra-sensitive protein calcium sensor GCaMP6, codon optimized for expression in mammalian cells, was put under the control of a ubiquitous CAG promoter. Porcine primary kidney cells were nucleofected with the CAG-GCaMP6 expression construct and after 5 days cells showing GFP fluorescence due to spontaneous Ca2+ transients were isolated by flow cytometry. Somatic cell nuclear transfer of these cells resulted in one pregnancy and birth of 2 male piglets, thereof 1 stillborn. Validation of transgene expression was first performed on a tail sample of the living founder pig, and later on tissue samples of his offspring. Pancreas tissue of his neonatal offspring was used for NPI isolation and NPI transplantation into the anterior chamber of the eye (ACE) in mice for NPI graft maturation. Ca2+ kinetics was assessed during live cell imaging on NPIs and on the explanted graft 8 weeks after transplantation upon a glucose-stimulated insulin secretion (GSIS) test. Results: Strong ubiquitous expression of the CAG-GCaMP6 transgene both in the tail sample of the founder pig as well as in all tissue samples such as pancreas was proven by GFP immunohistochemistry. A strong ubiquitous GCaMP6 expression in NPIs was detected. Upon potassium chloride challenge in in vitro GSIS, most cells of NPIs showed highest fluorescence response. However, only few cells exhibited a glucose-induced Ca2+-induced response represented by a wave of increased measured fluorescence intensity in in vitro matured NPIs indicating few contents on mature beta cells in these NPIs. NPIs engrafted in the mouse AEC did not show a Ca2+ reporter response upon glucose challenge, due to inhibitory effect of isoflurane anesthetic on glucose-induces ATP and Ca2+ increase. In contrast, multiple pulsatile Ca2+ reporter responses in numerous cells were measured upon glucose stimulus of AEC NPI grafts ex vivo, with highest response upon final potassium chloride stimulus (Fig. 1). Immunohistochemical staining of the AEC NPI grafts confirmed a high proportion of beta cells in the grafts. Conclusion: Live cell imaging of Ca2+ kinetics in GCaMP expressing NPIs represents a novel tool to evaluate the beta cell maturation stages both in vitro and in vivo.The study was supported by the Deutsche Forschungsgemeinschaft (TRR127) and by the German Federal Ministry of Education and Research (BMBF) to the German Centre for Diabetes Research (DZD e.V.) (Grant No. 82DZD00802).

Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.

Introduction: In recent years, numerous improvements in islet isolation strategies and the introduction of a tailor-made immunosuppressive and anti-inflammatory therapy regimen have positively influenced the clinical outcome of human pancreatic islet transplantation. In spite of these successes, however, considerable restrictions remain, which make a broader application of this therapeutic approach difficult. In order to circumvent the restriction caused by a lack of donor organs, the development of a macroencapsulation device with xenogeneic islets would be ideal, which offers sufficient immune isolation while maintaining the regulated islet function. Key factors for optimal islet material in the encapsulation setting are to provide intact islet clusters with high purity and homogeneity and a minimal size distribution in order to reduce oxygen demand, maximize applicable density and optimize diffusion characteristics. Therefore, the focus of this work is the optimization and standardization of the islet material by using so-called pseudo-islets. Method: With the help of a new type of spherical culture technology (Sphericalplate® 5D from KUGELMEIERS®) we were able to generate pseudo-islets with a defined size.For this purpose, islets were dissociated into single cells and seeded in densities of 300, 600, 900, 1200 or 1500 cells / microwell to identify the optimal cluster size. To this end, we used our established quality control assessment to compare pseudo-islets to native control NICCs. Based on long-term considerations of safety, efficacy, and cost-effectiveness, we investigated porcine neonatal islet like cell clusters (NICCs) as an alternative cell source to adult pig islets. Results: In our study, we were able to generate homogeneous, standardized pseudo-islets from porcine NICCs with a scalable size between 73–132 µm.The size of the pseudo-islets formed could be defined by the number of individual cells seeded per microwell. Pseudo-NICCs showed improved vitality compared to native control islets. In addition, we observed that control and pseudo-islets react dynamically to glucose and begin to secrete insulin after a glucose challenge. The cell composition of the islets was determined by triple immunostaining against insulin, glucagon and somatostatin and showed a comparable proportion of endocrine cells in porcine pseudo-NICCs compared to native NICCs. After transplantation into the anterior chamber of the eye, pseudo-NICCs showed correct growth and less cell loss compared to the control. Conclusion: We were able to show the potential of this culture technology to produce scalable and homogeneous islet preparations. Particularly in the encapsulation environment, where the rate of oxygen consumption and diffusion properties are essential for function and maximizing density, the creation of pseudo-islets with optimal dimensions may have significant beneficial effects on islet survival after transplantation and could help to overcome nutrient diffusion limitations.

Noninvasive in vivo imaging techniques are attractive tools to longitudinally study various aspects of islet of Langerhans physiology and pathophysiology. Unfortunately, most imaging modalities currently applicable for clinical use do not allow the comprehensive investigation of islet cell biology due to limitations in resolution and/or sensitivity, while high-resolution imaging technologies like laser scanning microscopy (LSM) lack the penetration depth to assess islets of Langerhans within the pancreas. Significant progress in this area was made by the combination of LSM with the anterior chamber of the mouse eye platform, utilizing the cornea as a natural body window to study cell physiology of transplanted islets of Langerhans. We here describe the transplantation and longitudinal in vivo imaging of islets of Langerhans in the anterior chamber of the mouse eye as a versatile tool to study different features of islet physiology in health and disease.

In type 1 diabetes (T1D), autoimmune destruction of pancreatic β 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, β 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 β cell loss, β cell dysfunction, alterations of β 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.

GLP-1Rimaging with radiolabelled exendin has proven to be a powerful tool to quantifybeta-cell mass (BCM) in vivo. AsGLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose levels on GLP-1R-mediated exendinuptake in both murine and human islets and its implications for BCMquantification. Periods of hyperglycemiasignificantly reduced exendin uptake in murine and human islets, which wasparalleled by a reduction in GLP-1R expression. Detailed mapping of the traceruptake and insulin and GLP-1R expression conclusively demonstrated that theobserved reduction in tracer uptake directly correlates to GLP-1R expressionlevels. Importantly, the linear correlation between tracer uptake and beta-cellarea was maintained in spite of the reduced GLP-1R expression levels.Subsequent normalization of blood glucose levels restored absolute traceruptake and GLP-1R expression in beta-cells and the observed loss in isletvolume was halted. This manuscriptemphasizes the potency of nuclear imaging techniques to monitor receptorregulation non-invasively. Our findings have significantimplications for clinical practice, indicating that blood glucose levels should be near-normalized for at least three weeksprior to GLP-1R agonist treatment or quantitativeradiolabeled exendin imaging for BCM analysis.

Type 2 diabetes is characterized by peripheral insulin resistance and insufficient insulin release from pancreatic islet β cells. However, the role and sequence of β cell dysfunction and mass loss for reduced insulin levels in type 2 diabetes pathogenesis are unclear. Here, we exploit freshly explanted pancreas specimens from metabolically phenotyped surgical patients using an in situ tissue slice technology. This approach allows assessment of β cell volume and function within pancreas samples of metabolically stratified individuals. We show that, in tissue of pre-diabetic, impaired glucose-tolerant subjects, β cell volume is unchanged, but function significantly deteriorates, exhibiting increased basal release and loss of first-phase insulin secretion. In individuals with type 2 diabetes, function within the sustained β cell volume further declines. These results indicate that dysfunction of persisting β cells is a key factor in the early development and progression of type 2 diabetes, representing a major target for diabetes prevention and therapy.

Immunofluorescent staining is a widespread tool in basic science to understand organ morphology and (patho-) physiology. The analysis of imaging data is often performed manually, limiting throughput and introducing human bias. Quantitative analysis is particularly challenging for organs with complex structure such as the kidney. In this study we present an approach for automatic quantification of fluorescent markers and histochemical stainings in whole organ sections using open source software. We validate our novel method in multiple typical challenges of basic kidney research and demonstrate its general relevance and applicability to other complex solid organs for a variety of different markers and stainings. Our newly developed software tool "AQUISTO", applied as a standard in primary data analysis, facilitates efficient large scale evaluation of cellular populations in various types of histological samples. Thereby it contributes to the characterization and understanding of (patho-) physiological processes.

BACKGROUND:Plasma insulin levels are predominantly the product of the morphological mass of insulin producing beta cells in the pancreatic islets of Langerhans and the functional status of each of these beta cells. Thus, deficiency in either beta cell mass or function, or both, can lead to insufficient levels of insulin, resulting in hyperglycemia and diabetes. Nonetheless, the precise contribution of beta cell mass and function to the pathogenesis of diabetes as well as the underlying mechanisms are still unclear. In the past, this was largely due to the restricted number of technologies suitable for studying the scarcely accessible human beta cells. However, in recent years, a number of new platforms have been established to expand the available techniques and to facilitate deeper insight into the role of human beta cell mass and function as cause for diabetes and as potential treatment targets.SCOPE OF REVIEW:This review discusses the current knowledge about contribution of human beta cell mass and function to different stages of type 1 and type 2 diabetes pathogenesis. Furthermore, it highlights standard and newly developed technological platforms for the study of human beta cell biology, which can be used to increase our understanding of beta cell mass and function in human glucose homeostasis.MAJOR CONCLUSIONS:In contrast to early disease models, recent studies suggest that in type 1 and type 2 diabetes impairment of beta cell function is an early feature of disease pathogenesis while a substantial decrease in beta cell mass occurs more closely to clinical manifestation. This suggests that, in addition to beta cell mass replacement for late stage therapies, the development of novel strategies for protection and recovery of beta cell function could be most promising for successful diabetes treatment and prevention. The use of today's developing and wide range of technologies and platforms for the study of human beta cells will allow for a more detailed investigation of the underlying mechanisms and will facilitate development of treatment approaches to specifically target human beta cell mass and function.

Islet-cell hormone release is modulated by signals from endothelial and endocrine cells within the islet. However, models of intraislet vascularization and paracrine cell signaling are mostly based on the rodent pancreas. We assessed the architecture and endocrine cell interaction of the vascular network in unperturbed human islets in situ and their potential to re-establish their endogenous vascular network after transplantation in vivo. We prepared slices of fresh pancreas tissue obtained from nondiabetic patients undergoing partial pancreatectomy. In addition, we transplanted human donor islets into the anterior chamber of the mouse eye. Next, we performed three-dimensional in situ and in vivo imaging of islet cell and vessel architecture at cellular resolution and compared our findings with mouse and porcine islets. Our data reveal a significantly different vascular architecture with decreased vessel diameter, reduced vessel branching, and shortened total vessel network in human compared with mouse islets. Together with the distinct cellular arrangement in human islets, this limits β to endothelial cell interactions, facilitates connection of α and β cells, and promotes the formation of independent β-cell clusters within islets. Furthermore, our results show that the endogenous vascular network of islets is significantly altered after transplantation in a donor age-related mechanism. Thus, our study provides insight into the vascular architecture and cellular arrangement of human islets with apparent consequences for intercellular islet signaling. Moreover, our findings suggest that human islet engraftment after transplantation can be improved by using alternative, less mature islet-cell sources.