Improvement effects of transplanting pancreatic islet that previously incubated with biomaterials on the diabetic nephropathy in STZ- diabetic rats.

BACKGROUND: Islet transplantation is an effective treatment for diabetes or even its complications. Aim of this study is to investigate efficacy of biomaterial treated islet transplantation on treating diabetic nephropathy. METHODS: Male rats were randomly divided into 6 groups; Control, diabetic control, diabetic transplanted with untreated islets, with platelet rich plasma treated islets, with pancreatic islets homogenate treated islets, or with these biomaterials combination treated islets. Islets cultured with biomaterials and transplanted to diabetic rats. After 60 days, biochemical, oxidative stress, and stereological parameters were assessed. RESULTS: Serum albumin and BUN concentration, decreased and increased respectively, Oxidative stress of kidney impaired, kidney weight, volume of kidney, cortex, medulla, glomerulus, proximal and distal tubules, collecting ducts, vessels, inflammatory, necrotic and fibrotic tissue in diabetic group increased compared to control group (p < 0.001). In treated groups, especially pancreatic islets homogenate treated islets transplanting animals, there was significant changes in kidney weight, and volume of kidney, proximal and distal tubules, Henle's loop and collecting ducts compared with diabetic group (p = 0.013 to p < 0.001). Combination treated islets animals showed significant increase in vessel volume compared to diabetic group (p < 0.001). Necrotic and fibrotic tissue significantly decreased in islets treated than untreated islet animals, it was higher in pancreatic islets homogenate, and combination treated islets groups (p = 0.001). CONCLUSIONS: Biomaterials treated islets transplanting could improve diabetic nephropathy. Improvement of oxidative stress followed by controlling glucose level, and effects of growth factors presenting in biomaterials can be considered as capable underlying mechanism of ameliorating inflammatory, necrotic and fibrotic tissue volume.

Revisiting pancreatic islet isolation in murine models: A practical and effective technical protocol.

The endocrine pancreas is composed of clusters of cell groups called pancreatic islets. These cells are responsible for the synthesis and secretion of hormones crucial for glycemic homeostasis, such as insulin and glucagon. Therefore, these cells were the targets of many studies. One method to study and/or understand endocrine pancreatic physiology is the isolation of these islets and stimulation of hormone production using different concentrations of glucose, agonists, and/or antagonists of specific secretagogues and mimicking the stimulation of hormonal synthesis and secretion. Many researchers studied pancreatic physiology in murine models due to their ease of maintenance and rapid development. However, the isolation of pancreatic islets involves meticulous processes that may vary between rodent species. The present study describes a simple and effective technical protocol for isolating intact islets from mice and rats for use as a practical guide for researchers. The method involves digestion of the acinar parenchyma by intraductal collagenase. Isolated islets are suitable for in vitro endocrine secretion analyses, microscopy techniques, and biochemical analyses.

Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

The MODY-associated KCNK16 L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults.

The gain-of-function mutation in the TALK-1 K+ channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion. The KCNK16 gene encoding TALK-1 is the most abundant and ß-cell-restricted K+ channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.

Distinct cellular immune responses in children en route to type 1 diabetes with different first-appearing autoantibodies.

Previous studies have revealed heterogeneity in the progression to clinical type 1 diabetes in children who develop islet-specific antibodies either to insulin (IAA) or glutamic acid decarboxylase (GADA) as the first autoantibodies. Here, we test the hypothesis that children who later develop clinical disease have different early immune responses, depending on the type of the first autoantibody to appear (GADA-first or IAA-first). We use mass cytometry for deep immune profiling of peripheral blood mononuclear cell samples longitudinally collected from children who later progressed to clinical disease (IAA-first, GADA-first, ≥2 autoantibodies first groups) and matched for age, sex, and HLA controls who did not, as part of the Type 1 Diabetes Prediction and Prevention study. We identify differences in immune cell composition of children who later develop disease depending on the type of autoantibodies that appear first. Notably, we observe an increase in CD161 expression in natural killer cells of children with ≥2 autoantibodies and validate this in an independent cohort. The results highlight the importance of endotype-specific analyses and are likely to contribute to our understanding of pathogenic mechanisms underlying type 1 diabetes development.

Multiscale and multimodal imaging for three-dimensional vascular and histomorphological organ structure analysis of the pancreas.

Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering.

Acinar to ß-like cell conversion through inhibition of focal adhesion kinase.

Insufficient functional ß-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing ß-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore ß-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.

AnnoSpat annotates cell types and quantifies cellular arrangements from spatial proteomics.

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs for atlas-scale datasets like Human Pancreas Analysis Program (HPAP), we develop AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX shows the higher performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulates known islet pathobiology and shows differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.

From the Clinical to the Bench: Exploring the Insulin Modulation Effects of Tacrolimus and Belatacept.

Calcineurin inhibitors (CNIs) are critical in preventing rejection posttransplantation but pose an increased risk of post-transplant diabetes (PTD). Recent studies show that late conversion from CNIs to belatacept, a costimulation blocker, improves HbA1c in kidney transplant recipients with PTD or de novo diabetes. This study investigates whether the observed effects on PTD stem solely from CNI withdrawal or if belatacept influences PTD independently. The study assessed the impact of tacrolimus and belatacept on insulin secretion in MIN6 cells (a beta cell line) and rat islets. Tacrolimus and belatacept were administered to the cells and islets, followed by assessments of cell viability and insulin secretion. Tacrolimus impaired insulin secretion without affecting cell viability, while belatacept showed no detrimental effects on either parameter. These findings support clinical observations of improved HbA1c upon switching from tacrolimus to belatacept. Belatacept holds promise in islet or pancreas transplantation, particularly in patients with unstable diabetes. Successful cases of islet transplantation treated with belatacept without severe hypoglycemia highlight its potential in managing PTD. Further research is needed to fully understand the metabolic changes accompanying the transition from CNIs to belatacept. Preserving insulin secretion emerges as a promising avenue for investigation in this context.

Advancing diabetes treatment: the role of mesenchymal stem cells in islet transplantation.

Diabetes mellitus, a prevalent global health challenge, significantly impacts societal and economic well-being. Islet transplantation is increasingly recognized as a viable treatment for type 1 diabetes that aims to restore endogenous insulin production and mitigate complications associated with exogenous insulin dependence. We review the role of mesenchymal stem cells (MSCs) in enhancing the efficacy of islet transplantation. MSCs, characterized by their immunomodulatory properties and differentiation potential, are increasingly seen as valuable in enhancing islet graft survival, reducing immune-mediated rejection, and supporting angiogenesis and tissue repair. The utilization of MSC-derived extracellular vesicles further exemplifies innovative approaches to improve transplantation outcomes. However, challenges such as MSC heterogeneity and the optimization of therapeutic applications persist. Advanced methodologies, including artificial intelligence (AI) and single-cell RNA sequencing (scRNA-seq), are highlighted as potential technologies for addressing these challenges, potentially steering MSC therapy toward more effective, personalized treatment modalities for diabetes. This review revealed that MSCs are important for advancing diabetes treatment strategies, particularly through islet transplantation. This highlights the importance of MSCs in the field of regenerative medicine, acknowledging both their potential and the challenges that must be navigated to fully realize their therapeutic promise.

The Importance of Intra-Islet Communication in the Function and Plasticity of the Islets of Langerhans during Health and Diabetes.

Islets of Langerhans are anatomically dispersed within the pancreas and exhibit regulatory coordination between islets in response to nutritional and inflammatory stimuli. However, within individual islets, there is also multi-faceted coordination of function between individual beta-cells, and between beta-cells and other endocrine and vascular cell types. This is mediated partly through circulatory feedback of the major secreted hormones, insulin and glucagon, but also by autocrine and paracrine actions within the islet by a range of other secreted products, including somatostatin, urocortin 3, serotonin, glucagon-like peptide-1, acetylcholine, and ghrelin. Their availability can be modulated within the islet by pericyte-mediated regulation of microvascular blood flow. Within the islet, both endocrine progenitor cells and the ability of endocrine cells to trans-differentiate between phenotypes can alter endocrine cell mass to adapt to changed metabolic circumstances, regulated by the within-islet trophic environment. Optimal islet function is precariously balanced due to the high metabolic rate required by beta-cells to synthesize and secrete insulin, and they are susceptible to oxidative and endoplasmic reticular stress in the face of high metabolic demand. Resulting changes in paracrine dynamics within the islets can contribute to the emergence of Types 1, 2 and gestational diabetes.

Illuminating the complete ß-cell mass of the human pancreas- signifying a new view on the islets of Langerhans.

Pancreatic islets of Langerhans play a pivotal role in regulating blood glucose homeostasis, but critical information regarding their mass, distribution and composition is lacking within a whole organ context. Here, we apply a 3D imaging pipeline to generate a complete account of the insulin-producing islets throughout the human pancreas at a microscopic resolution and within a maintained spatial 3D context. These data show that human islets are far more heterogenous than previously accounted for with regards to their size distribution and cellular make up. By deep tissue 3D imaging, this in-depth study demonstrates that 50% of the human insulin-expressing islets are virtually devoid of glucagon-producing α-cells, an observation with significant implications for both experimental and clinical research.

Spontaneous Akt2 deficiency in a colony of NOD mice exhibiting early diabetes.

Diabetes constitutes a major public health problem, with dramatic consequences for patients. Both genetic and environmental factors were shown to contribute to the different forms of the disease. The monogenic forms, found both in humans and in animal models, specially help to decipher the role of key genes in the physiopathology of the disease. Here, we describe the phenotype of early diabetes in a colony of NOD mice, with spontaneous invalidation of Akt2, that we called HYP. The HYP mice were characterised by a strong and chronic hyperglycaemia, beginning around the age of one month, especially in male mice. The phenotype was not the consequence of the acceleration of the autoimmune response, inherent to the NOD background. Interestingly, in HYP mice, we observed hyperinsulinemia before hyperglycaemia occurred. We did not find any difference in the pancreas' architecture of the NOD and HYP mice (islets' size and staining for insulin and glucagon) but we detected a lower insulin content in the pancreas of HYP mice compared to NOD mice. These results give new insights about the role played by Akt2 in glucose homeostasis and argue for the ß cell failure being the primary event in the course of diabetes.

Identification and removal of unexpected proliferative off-target cells emerging after iPSC-derived pancreatic islet cell implantation.

Differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSCs) has been thoroughly investigated for application in cell therapy against diabetes. In the context of induced pancreatic endocrine cell implantation, previous studies have reported graft enlargement resulting from off-target pancreatic lineage cells. However, there is currently no documented evidence of proliferative off-target cells beyond the pancreatic lineage in existing studies. Here, we show that the implantation of seven-stage induced PSC-derived pancreatic islet cells (s7-iPICs) leads to the emergence of unexpected off-target cells with proliferative capacity via in vivo maturation. These cells display characteristics of both mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs), termed proliferative MSC- and SMC-like cells (PMSCs). The frequency of PMSC emergence was found to be high when 108 s7-iPICs were used. Given that clinical applications involve the use of a greater number of induced cells than 108, it is challenging to ensure the safety of clinical applications unless PMSCs are adequately addressed. Accordingly, we developed a detection system and removal methods for PMSCs. To detect PMSCs without implantation, we implemented a 4-wk-extended culture system and demonstrated that putative PMSCs could be reduced by compound treatment, particularly with the taxane docetaxel. When docetaxel-treated s7-iPICs were implanted, the PMSCs were no longer observed. This study provides useful insights into the identification and resolution of safety issues, which are particularly important in the field of cell-based medicine using PSCs.

Unraveling the significance of PPP1R1A gene in pancreatic ß-cell function: A study in INS-1 cells and human pancreatic islets.

BACKGROUND AND AIMS: The protein phosphatase 1 regulatory inhibitor subunit 1A (PPP1R1A) has been linked with insulin secretion and diabetes mellitus. Yet, its full significance in pancreatic ß-cell function remains unclear. This study aims to elucidate the role of the PPP1R1A gene in ß-cell biology using human pancreatic islets and rat INS-1 (832/13) cells. RESULTS: Disruption of Ppp1r1a in INS-1 cells was associated with reduced insulin secretion and impaired glucose uptake; however, cell viability, ROS, apoptosis or proliferation were intact. A significant downregulation of crucial ß-cell function genes such as Ins1, Ins2, Pcsk1, Cpe, Pdx1, Mafa, Isl1, Glut2, Snap25, Vamp2, Syt5, Cacna1a, Cacna1d and Cacnb3, was observed upon Ppp1r1a disruption. Furthermore, silencing Pdx1 in INS-1 cells altered PPP1R1A expression, indicating that PPP1R1A is a target gene for PDX1. Treatment with rosiglitazone increased Ppp1r1a expression, while metformin and insulin showed no effect. RNA-seq analysis of human islets revealed high PPP1R1A expression, with α-cells showing the highest levels compared to other endocrine cells. Muscle tissues exhibited greater PPP1R1A expression than pancreatic islets, liver, or adipose tissues. Co-expression analysis revealed significant correlations between PPP1R1A and genes associated with insulin biosynthesis, exocytosis machinery, and intracellular calcium transport. Overexpression of PPP1R1A in human islets augmented insulin secretion and upregulated protein expression of Insulin, MAFA, PDX1, and GLUT1, while silencing of PPP1R1A reduced Insulin, MAFA, and GLUT1 protein levels. CONCLUSION: This study provides valuable insights into the role of PPP1R1A in regulating ß-cell function and glucose homeostasis. PPP1R1A presents a promising opportunity for future therapeutic interventions.

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology.

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the pancreas endocrine and exocrine physiology and interplay. Much has been learned from the study of isolated pancreatic islets, but this prevents examining their function and interactions in the context of the whole tissue. Pancreas slices provide a unique opportunity to explore the physiology of normal, inflamed, and structurally damaged islets within their native environment, in turn allowing the study of interactions between endocrine and exocrine compartments to better investigate the complex dynamics of pancreatic tissue. Thus, the adoption of the living pancreas slice platform represents a significant advancement in the field. This protocol describes how to generate living tissue slices from deceased organ donors by tissue embedding in agarose and vibratome slicing as well as their utilization to assess functional readouts such as dynamic secretion and live cell imaging.

Innovations in bio-engineering and cell-based approaches to address immunological challenges in islet transplantation.

Human allogeneic pancreatic islet transplantation is a life-changing treatment for patients with severe Type 1 Diabetes (T1D) who suffer from hypoglycemia unawareness and high risk of severe hypoglycemia. However, intensive immunosuppression is required to prevent immune rejection of the graft, that may in turn lead to undesirable side effects such as toxicity to the islet cells, kidney toxicity, occurrence of opportunistic infections, and malignancies. The shortage of cadaveric human islet donors further limits islet transplantation as a treatment option for widespread adoption. Alternatively, porcine islets have been considered as another source of insulin-secreting cells for transplantation in T1D patients, though xeno-transplants raise concerns over the risk of endogenous retrovirus transmission and immunological incompatibility. As a result, technological advancements have been made to protect transplanted islets from immune rejection and inflammation, ideally in the absence of chronic immunosuppression, to improve the outcomes and accessibility of allogeneic islet cell replacement therapies. These include the use of microencapsulation or macroencapsulation devices designed to provide an immunoprotective environment using a cell-impermeable layer, preventing immune cell attack of the transplanted cells. Other up and coming advancements are based on the use of stem cells as the starting source material for generating islet cells 'on-demand'. These starting stem cell sources include human induced pluripotent stem cells (hiPSCs) that have been genetically engineered to avoid the host immune response, curated HLA-selected donor hiPSCs that can be matched with recipients within a given population, and multipotent stem cells with natural immune privilege properties. These strategies are developed to provide an immune-evasive cell resource for allogeneic cell therapy. This review will summarize the immunological challenges facing islet transplantation and highlight recent bio-engineering and cell-based approaches aimed at avoiding immune rejection, to improve the accessibility of islet cell therapy and enhance treatment outcomes. Better understanding of the different approaches and their limitations can guide future research endeavors towards developing more comprehensive and targeted strategies for creating a more tolerogenic microenvironment, and improve the effectiveness and sustainability of islet transplantation to benefit more patients.

Investigating the Impact of IL6 on Insulin Secretion: Evidence from INS-1 Cells, Human Pancreatic Islets, and Serum Analysis.

Interleukin-6 (IL6) is a pleiotropic cytokine implicated in metabolic disorders and inflammation, yet its precise influence on insulin secretion and glucose metabolism remains uncertain. This study examined IL6 expression in pancreatic islets from individuals with/without diabetes, alongside a series of functional experiments, including siRNA silencing; IL6 treatment; and assessments of glucose uptake, cell viability, apoptosis, and expression of key ß-cell genes, which were conducted in both INS-1 cells and human islets to elucidate the effect of IL6 on insulin secretion. Serum levels of IL6 from Emirati patients with type 2 diabetes (T2D) were measured, and the effect of antidiabetic drugs on IL6 levels was studied. The results revealed that IL6 mRNA expression was higher in islets from diabetic and older donors compared to healthy or young donors. IL6 expression correlated negatively with PDX1, MAFB, and NEUROD1 and positively with SOX4, HES1, and FOXA1. Silencing IL6 in INS-1 cells reduced insulin secretion and glucose uptake independently of apoptosis or oxidative stress. Reduced expression of IL6 was associated with the downregulation of Ins, Pdx1, Neurod1, and Glut2 in INS-1 cells. In contrast, IL6 treatment enhanced insulin secretion in INS-1 cells and human islets and upregulated insulin expression. Serum IL6 levels were elevated in patients with T2D and associated with higher glucose, HbA1c, and triglycerides, regardless of glucose-lowering medications. This study provides a new understanding of the role of IL6 in ß-cell function and the pathophysiology of T2D. Our data highlight differences in the response to IL6 between INS-1 cells and human islets, suggesting the presence of species-specific variations across different experimental models. Further research is warranted to unravel the precise mechanisms underlying the observed effects of IL-6 on insulin secretion.