Insulin-producing Beta Cells Research Articles

Achieving efficient clonal beta cells transfection using nanostraw/nanopore-assisted electroporation.

The prospect of being able to efficiently inject large plasmids in insulin-producing beta cells is very attractive for diabetes research. However, conventional transfection methods suffer from high cytotoxicity or low transfection efficiency, which negatively affect their outcome. In contrast, nanostraw electroporation is a gentle method that can provide a high transfection efficiency while maintaining high cell viability. While nanostraw electroporation has gone through some method optimization in the past, such as tuning the pulse frequency, amplitude, and duration, the effect of other parameters has not been thoroughly investigated. Here, we demonstrate efficient transfection of clonal beta cells and investigate the effect of voltage at a fixed inter-electrode distance, cell density, and cargo solution conductivity on transfection efficiency. We used GFP-encoding DNA plasmids stained with an intercalating dye to enable immediate analysis and assessment of the electrophoretic transport of cargo. Moreover, we ran simulations to assess how cargo buffer conductivity impacts the transfection efficiency by affecting the voltage drop on the nanostraws and cell membrane during electroporation. Both experiments and simulations show that MilliQ water as the cargo buffer yields the best transfection efficiency. We also show that the cell density should be adjusted to maximize the number of cells interfacing the nanostraws and avoid cell stacking. Finally, we compared the transfection efficiency when using nanostraws and nanopores. Whereas the amount of GFP plasmids injected using nanostraws is larger than for nanopores, the outcome in terms of GFP fluorescence 48 h after transfection was worse than for nanopores. Moreover, when using nanostraws, fewer cells were found on the substrate 48 h after transfection compared to when using nanopores. This suggests that injecting substantial amounts of plasmids in cells can affect their proliferation and/or viability, and that nanopore electroporation, as a simpler method, is an interesting alternative to nanostraws in achieving efficient and gentle clonal beta cell transfection.

Enhancing human islet xenotransplant survival and function in diabetic immunocompetent mice through LRH-1/NR5A2 pharmacological activation.

The intricate etiology of type 1 diabetes mellitus (T1D), characterized by harmful interactions between the immune system and insulin-producing beta cells, has hindered the development of effective therapies including human islet transplantation, which requires strong immunosuppressants that impair beta cell survival and function. As such alternative immunomodulating therapies are required for successful transplantation. The discovery that pharmacological activation of the nuclear receptor LRH-1/NR5A2 can reverse hyperglycemia in mouse models of T1D by altering, and not suppressing the autoimmune attack, prompted us to investigate whether LRH-1/NR5A2 activation could improve human islet function/survival after xenotransplantation in immunocompetent mice. Human islets were transplanted under the kidney capsule of streptozotocin (STZ)-induced diabetic mice, and treatment with BL001 (LRH-1/NR5A2 agonist) or vehicle was administered one week post-transplant. Our study, encompassing 3 independent experiments with 3 different islet donors, revealed that mice treated for 8 weeks with BL001 exhibited lower blood glucose levels correlating with improved mouse survival rates as compared to vehicle-treated controls. Human C-peptide was detectable in BL001-treated mice at both 4 and 8 weeks indicating functional islet beta cells. Accordingly, in mice treated with BL001 for 8 weeks, the beta cell mass was preserved, while a significant decrease in alpha cells was observed compared to mice treated with BL001 for only 4 weeks. In contrast, vehicle-treated mice exhibited a reduction in insulin-expressing cells at 8 weeks compared to those at 4 weeks. These results suggest that BL001 significantly enhances the survival, engraftment, and functionality of human islets in a STZ-induced diabetic mouse model.

Renewable Human Cell Model for Type 1 Diabetes Research: EndoC-βH5/HUVEC Coculture Spheroids.

In vitro drug screening for type 1 diabetes therapies has largely been conducted on human organ donor islets for proof of efficacy. While native islets are the ultimate target of these drugs (either in situ or for transplantation), significant benefit can be difficult to ascertain due to the highly heterogeneous nature of individual donors and the overall scarcity of human islets for research. We present an in vitro coculture model based on immortalized insulin-producing beta-cell lines with human endothelial cells in 3D spheroids that aims to recapitulate the islet morphology in an effort towards developing a standardized cell model for in vitro diabetes research. Human insulin-producing immortalized EndoC-βH5 cells are cocultured with human endothelial cells in varying ratios to evaluate 3D cell culture models for type 1 diabetes research. Insulin secretion, metabolic activity, live cell fluorescence staining, and gene expression assays were used to compare the viability and functionality of spheroids composed of 100% beta-cells, 1 : 1 beta-cell/endothelial, and 1 : 3 beta-cell/endothelial. Monoculture and βH5/HUVEC cocultures formed compact spheroids within 7 days, with average diameter ~140 μm. This pilot study indicated that stimulated insulin release from 0 to 20 mM glucose increased from ~8-fold for monoculture and 1 : 1 coculture spheroids to over 20-fold for 1 : 3 EndoC-βH5/HUVEC spheroids. Metabolic activity was also ~12% higher in the 1 : 3 EndoC-βH5/HUVEC group compared to other groups. Stimulating monoculture beta-cell spheroids with 20 mM glucose +1 μg/mL glycine-modified INGAP-P increased the insulin stimulation index ~2-fold compared to glucose alone. Considering their availability and consistent phenotype, EndoC-βH5-based spheroids present a useful 3D cell model for in vitro testing and drug screening applications.

Teplizumab: an FDA- approved drug, marks milestone in treatment of type 1 diabetes

Diabetes type-1 is a chronic disease characterized by immune-mediated destruction of insulin-producing beta cells in the pancreas. It progresses through distinct stages at varying rates, with stage 1 being β-cell autoimmunity with normal blood sugar, stage 2 being β-cell autoimmunity with dysglycemia, and stage 3 being symptomatic disease onset.

Integrating multi-omics data to analyze the potential pathogenic mechanism of CTSH gene involved in type 1 diabetes in the exocrine pancreas.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing pancreatic islet beta cells. Despite significant advancements, the precise pathogenesis of the disease remains unknown. This work integrated data from expression quantitative trait locus (eQTL) studies with Genome wide association study (GWAS) summary data of T1D and single-cell transcriptome data to investigate the potential pathogenic mechanisms of the CTSH gene involved in T1D in exocrine pancreas. Using the summary data-based Mendelian randomization (SMR) approach, we obtained four potential causative genes associated with T1D: BTN3A2, PGAP3, SMARCE1 and CTSH. To further investigate these genes'roles in T1D development, we validated them using a scRNA-seq dataset from pancreatic tissues of both T1D patients and healthy controls. The analysis showed a significantly high expression of the CTSH gene in T1D acinar cells, whereas the other three genes showed no significant changes in the scRNA-seq data. Moreover, single-cell WGCNA analysis revealed the strongest positive correlation between the module containing CTSH and T1D. In addition, we found cellular ligand-receptor interactions between the acinar cells and different cell types, especially ductal cells. Finally, based on functional enrichment analysis, we hypothesized that the CTSH gene in the exocrine pancreas enhances the antiviral response, leading to the overexpression of pro-inflammatory cytokines and the development of an inflammatory microenvironment. This process promotes β cells injury and ultimately the development of T1D. Our findings offer insights into the underlying pathogenic mechanisms of T1D.

Glyburide-treated human monocyte-derived dendritic cells loaded with insulin represent tolerogenic features with anti-inflammatory responses and modulate autologous T cell responses in vitro

Tolerogenic dendritic cells (TolDCs) are attractive therapeutic options for autoimmune disorders because they suppress autologous T-cell responses. Dendritic cells (DCs) are equipped with pattern recognition receptors (PRR), including nucleotide-binding and oligomerization domain-like receptors (NLRs) such as NLRP3. Abnormal NLRP3 activation has been reported to be correlated with the occurrence of autoimmune disorders. Accordingly, we hypothesized that glyburide treatment of DCs by blocking the ATP-sensitive K+ (kATP) channels generates TolDCs by inhibiting NLRP3. Insulin was even loaded on a group of glyburide-treated mature DCs (mDCs) to investigate the antigen (Ag) loading effects on glyburide-treated mDCs' phenotypical and functional features. Consequently, T lymphocytes' mediated responses ensuing co-culture of them with control mDCs, insulin loaded and unloaded glyburide treated mDCs were evaluated to determine generated TolDCs' capacity in inhibition of T cell responses that are inducer of destruction in insulin-producing pancreatic beta cells in Type 1 Diabetes Mellitus (T1DM). Our findings indicated that glyburide generates desirable TolDCs with decreased surface expression of maturation and Ag presentation related markers and diminished level of inflammatory but increased level of anti-inflammatory cytokines, which even insulin loading demonstrated more anti-inflammatory functions. In addition, co-cultured T cells showed regulatory or T helper 2 phenotype instead of T helper 1 features. Our findings suggested that insulin-loaded and unloaded glyburide-treated DCs are promising therapeutic approaches for autoimmune patients, specifically DCs loaded with insulin for T1DM patients. However, further research is required before this technique can be applied in clinical practice.

Imaging in Type 1 Diabetes, Current Perspectives and Directions.

Type 1 diabetes (T1D) is characterized by the autoimmune-mediated attack of insulin-producing beta cells in the pancreas, leading to reliance on exogenous insulin to control a patient's blood glucose levels. As progress is being made in understanding the pathophysiology of the disease and how to better develop therapies to treat it, there is an increasing need for monitoring technologies to quantify beta cell mass and function throughout T1D progression and beta cell replacement therapy. Molecular imaging techniques offer a possible solution through both radiologic and non-radiologic means including positron emission tomography, magnetic resonance imaging, electron paramagnetic resonance imaging, and spatial omics. This commentary piece outlines the role of molecular imaging in T1D research and highlights the need for further applications of such methodologies in T1D.

Novel time-resolved reporter mouse reveals spatial and transcriptional heterogeneity during alpha cell differentiation.

Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. We established the mouse model, 'Gcg-Timer', in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090.

OR18-05 Interleukin 21 Drives The Development Of Autoimmune Diabetes Mellitus During Immune Checkpoint Inhibitor Cancer Therapy

Abstract Disclosure: J.G. Ortega: None. L.N. Scott: None. S. Wang: None. S. Zhang: None. H. Chen: None. N. Huang: None. M. Lechner: None. Autoimmune diabetes mellitus is a rare but life-threatening side effect of immune checkpoint inhibitor (ICI) cancer therapy. Checkpoint inhibitors increase immune activation by blocking natural regulatory molecules on T cells, including programmed death protein (PD1) and cytotoxic T lymphocyte antigen (CTLA4). As a result of increased immune activation, up to 60% of patients may develop autoimmunity in healthy tissues. Endocrine tissues are among the most often affected by these immune related adverse events (IRAEs). ICI-associated diabetes mellitus (ICI-T1DM) results from autoimmune destruction of insulin-producing pancreatic beta-cells. Patients with ICI-T1DM frequently present in diabetic ketoacidosis and require life-long insulin therapy. Unfortunately, the cause of many IRAEs remains poorly understood. We recently developed a mouse model of IRAEs in which anti-PD1 + anti-CTLA4 antibody treatment of autoimmunity-prone non-obese diabetic mice leads to multi-organ autoimmune infiltrates, including acceleration of insulinitis and DM. ICI-treated mice had increased CD4+ T follicular (Tfh; PD1+ICOS+CXCR5+) and T peripheral helper (Tph; PD1+ICOS+CXCR5-) cells compared to isotype-treated controls. Such Tfh and Tph cells secrete interleukin (IL)-21 and can promote cytotoxic CD8+ T cell function in autoimmunity. Indeed, CD8+ T cells expressing Granzyme B, interferon gamma (IFNγ), and chemokine receptor CXCR6 ("CD8+ autoimmune mediators") were expanded in ICI-treated mice. Furthermore, in vitro, IL21 induced the autoimmune mediator phenotype in CD8+ T cells, with upregulation of IFNγ, cytotoxic and trafficking molecules. Using mice with genetic deletion of T cell receptor alpha (NOD.TCRa-/-), we showed that CD4+ and CD8+ T cells were required for the development of IRAEs in ICI-treated mice. In addition, adoptive transfer of CD4+ and CD8+ T cells into immunodeficient NOD.SCID mice confirmed that these populations were sufficient to cause DM during ICI treatment. Thus, we predicted that IL21 from Tfh and Tph cells contributes to the development of pancreas IRAEs by activating CD8+ autoimmune mediator T cells. Indeed, in our model, mice with genetic blockade of IL21 signaling (IL21R-/-) were protected from developing diabetes. After 4 weeks of ICI treatment, only one of 11 (9.1%) IL21R-/- mice developed DM with onset at day 28, compared to 80% of ICI-treated wild-type (WT) mice, with a median DM-free time of 10 days from start of ICI therapy (p<0.0001, Log Rank test); no isotype-treated WT or IL21R-/- mice developed diabetes. Histologic analyses of pancreatic tissue from ICI-treated mice also showed a significantly lower insulinitis index in IL21R-/- compared to WT mice (mean 0.19±0.06 vs. 0.7±0.14, p<0.01). In summary, we identify IL21+ CD4+ Tfh/Tph cells as drivers of pancreas IRAEs and a potential therapeutic target to prevent ICI-T1DM in cancer patients. Presentation: Saturday, June 17, 2023

SAT102 Natural History Of Type 1 Diabetes In Humanized Mouse Model

Abstract Disclosure: A. Aldasouqi: None. S. Rafiqi: None. J.C. Jaume: None. S. Imam: None. Type 1 diabetes (T1D) is an autoimmune disease caused by an imbalance in T-regulatory and T-effector cells characterized by the destruction of insulin-producing beta cells by T-effector cells, leading to insulin deficiency and complexities like diabetic nephropathy, retinopathy, and neuropathy. Researchers at The University of Toledo have developed a humanized mice model which spontaneously develops T1D at 3-5 weeks of age and mimics human T1D. This study aims to monitor blood glucose levels before the onset of T1D (preweaning stage mice) and track the dynamics of immune cells using flow cytometry throughout the progression of the disease. Our results show that T-regulatory cells constitute 4.773% ± 0.81% cells at preweaning which drops to 2.60± 0.35 (%) in males and 1.78 ± 0.58 (%) in females at the 10th week while CD8+ T cells producing interferon-gamma (cytotoxic lymphocytes, CTLs) constitute 3.32 ± 0.60 (%) at preweaning, which increased to 27.625± 1.43 (%) in males and 21.13± 2.87 (%) in females as the disease progresses. This reduction in T-regulatory cells and enrichment of CD8s and CTLs leads to the destruction of beta cells as seen in humans as well. Dysregulation of immune responses can be correlated with the progression of diabetes in terms of blood glucose levels which increased from 147mg/dl (preweaning), peaked to 373mg/dl in 5th week, and was observed in partial remission stage around the 8th week as observed in human T1D ‘honeymoon period’. This study will help us to understand the immune dynamics in the progression of T1D as it helps us to present a better picture of the status of immune cells in a human-like immune system and provides benchmark data of the interplay of the immune system. Presentation: Saturday, June 17, 2023

Disease-modifying therapies and features linked to treatment response in type 1 diabetes prevention: a systematic review

BackgroundType 1 diabetes (T1D) results from immune-mediated destruction of insulin-producing beta cells. Prevention efforts have focused on immune modulation and supporting beta cell health before or around diagnosis; however, heterogeneity in disease progression and therapy response has limited translation to clinical practice, highlighting the need for precision medicine approaches to T1D disease modification.MethodsTo understand the state of knowledge in this area, we performed a systematic review of randomized-controlled trials with ge50 participants cataloged in PubMed or Embase from the past 25 years testing T1D disease-modifying therapies and/or identifying features linked to treatment response, analyzing bias using a Cochrane-risk-of-bias instrument.ResultsWe identify and summarize 75 manuscripts, 15 describing 11 prevention trials for individuals with increased risk for T1D, and 60 describing treatments aimed at preventing beta cell loss at disease onset. Seventeen interventions, mostly immunotherapies, show benefit compared to placebo (only two prior to T1D onset). Fifty-seven studies employ precision analyses to assess features linked to treatment response. Age, beta cell function measures, and immune phenotypes are most frequently tested. However, analyses are typically not prespecified, with inconsistent methods of reporting, and tend to report positive findings.ConclusionsWhile the quality of prevention and intervention trials is overall high, the low quality of precision analyses makes it difficult to draw meaningful conclusions that inform clinical practice. To facilitate precision medicine approaches to T1D prevention, considerations for future precision studies include the incorporation of uniform outcome measures, reproducible biomarkers, and prespecified, fully powered precision analyses into future trial design.

Automated high-throughput, high-content 3D imaging of intact pancreatic islets.

Diabetes poses a global health crisis affecting individuals across age groups and backgrounds, with a prevalence estimate of 700 million people worldwide by 2045. Current therapeutic strategies primarily rely on insulin therapy or hypoglycemic agents, which fail to address the root cause of the disease - the loss of pancreatic insulin-producing beta-cells. Therefore, bioassays that recapitulate intact islets are needed to enable drug discovery for beta-cell replenishment, protection from beta-cell loss, and islet-cell interactions. Standard cancer insulinoma beta-cell lines MIN6 and INS-1 have been used to interrogate beta-cell metabolic pathways and function but are not suitable for studying proliferative effects. Screening using primary human/rodent intact islets offers a higher level of physiological relevance to enhance diabetes drug discovery and development. However, the 3-dimensionality of intact islets have presented challenges in developing robust, high-throughput assays to detect beta-cell proliferative effects. Established methods rely on either dissociated islet cells plated in 2D monolayer cultures for imaging or reconstituted pseudo-islets formed in round bottom plates to achieve homogeneity. These approaches have significant limitations due to the islet cell dispersion process. To address these limitations, we have developed a robust, intact ex vivo pancreatic islet bioassay in 384-well format that is capable of detecting diabetes-relevant endpoints including beta-cell proliferation, chemoprotection, and islet spatial morphometrics.

Estimating residual undifferentiated cells in human chemically induced pluripotent stem cell derived islets using lncRNA as biomarkers

Human pluripotent stem cells (hPSCs) can generate insulin-producing beta cells for diabetes treatment, but residual undifferentiated cells may cause tumors. We developed a highly sensitive assay to detect these cells in islet cells derived from human chemically induced pluripotent stem cells (hCiPSCs), which are transgene-free and safer. We used RNA-seq data to find protein-coding and non-coding RNAs that were only expressed in hCiPSCs, not in islet cells. We confirmed these biomarkers by RT-qPCR and ddPCR. We chose long non-coding RNA (lncRNA) markers, which performed better than protein-coding RNA markers. We found that LNCPRESS2, LINC00678 and LOC105370482 could detect 1, 1 and 3 hCiPSCs in 106 islet cells by ddPCR, respectively. We tested our method on several hCiPSC lines, which could quantify 0.0001% undifferentiated cell in 106 islet cells by targeting hCiPSCs-specific lncRNA transcripts, ensuring the safety and quality of hCiPSC-derived islet cells for clinical use.

Incidence of Type 1 Diabetes Mellitus in Hamadan West Region of Iran2019 - 2020

Objective: Type 1 diabetes mellitus (T1DM) results from the autoimmune destruction of insulin-producing beta cells of the pancreas. The objective of the present study was to describe the one year (2019-2020) incidence of T1DM in Hamadan west region of Iran.
 Materials and Methods: All 25 new patients with T1DM resident of Hamadan and its suburbs registered in Besat Hospital, Hamadan, Iran, from March 2019 to 2020 were prospectively reviewed. Information including age, sex, place of residence (urban or rural), season of diagnosis and season of birth were taken from parents through face-to-face interviews.
 Results: During 1 year survey, a total of 57 cases of newly diagnosed T1DM were identified in our center. Among these, 25 patients were residents of Hamadan city and suburbs. Based on this15 per 100 000 per year new cases T1DM occurred in the resident population of Hamadan areas, higher in boys than in girls (18 vs. 11 per 100 000).The mean age of diagnosis was 8.03 yr. The peak incidence was observed at the age group of 10- 14 years. 92% were urban .Most cases were diagnosed in the autumn (44%). 52% had a history of neonatal jaundice and 16% had stressful life events in the family before the onset of diabetes.
 Conclusion: Our study showed a relatively high incidence of T1DM according category of the WHO, being greater in boys and in the age group of 10 to 14 years. The incidence is lower than in some Middle Eastern countries.

Beyond Syringes and Pills: Advances in Drug Delivery Systems for Diabetes

Diabetes is a chronic disease that affects millions of people worldwide, and its prevalence is increasing. The two primary subtypes, type 1 and 2, have different causes and mechanisms, but both result in abnormal glucose metabolism. The standard of care for diabetes includes insulin therapy, oral anti-diabetic medications, diet, exercise, weight loss, and frequent self-monitoring of blood glucose levels. However, these treatments have limitations that can lead to poor patient compliance and suboptimal outcomes. Alternative insulin delivery systems such as inhalers, patches, and oral sprays offer potential benefits such as increased convenience, reduced pain, and improved adherence. Non-insulin injectables, long-acting basal insulins, and GLP-1 agonists have also shown promise in improving glycemic control and reducing the risk of complications. Nanoparticle-based systems like SLNs are a novel approach that offers several advantages for diabetic management. They allow for targeted drug delivery, controlled release, and improved biocompatibility, enhancing drug efficacy and reducing side effects. SLNs have shown potential in animal models for reducing extracellular matrix degradation, inhibiting carbohydrate digestive enzymes, and enhancing the regeneration of insulin-producing beta cells. More studies are needed to validate their safety and efficacy in humans, but the potential benefits of SLNs make them a promising option for diabetes management.

In vitro oxygen imaging of acellular and cell-loaded beta cell replacement devices

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices’ full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices’ in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO2) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin–lattice relaxation rate (R1) of oxygen-sensitive spin probes such as trityl OX071 and pO2 to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the “Oxygen Measurement Core” was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members’ laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO2 measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO2 calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO2 measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO2 imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D.

Short-term function and immune-protection of microencapsulated adult porcine islets with alginate incorporating CXCL12 in healthy and diabetic non-human primates without systemic immune suppression: A pilot study.

Replacement of insulin-producing pancreatic beta-cells by islet transplantation offers a functional cure for type-1 diabetes (T1D). We recently demonstrated that a clinical grade alginate micro-encapsulant incorporating the immune-repellent chemokine and pro-survival factor CXCL12 could protect and sustain the integrity and function of autologous islets in healthy non-human primates (NHPs) without systemic immune suppression. In this pilot study, we examined the impact of the CXCL12 micro encapsulant on the function and inflammatory and immune responses of xenogeneic islets transplanted into the omental tissue bilayer sac (OB; n = 4) and diabetic (n = 1) NHPs. Changes in the expression of cytokines after implantation were limited to 2-6-fold changes in blood, most of which did not persist over the first 4 weeks after implantation. Flow cytometry of PBMCs following transplantation showed minimal changes in IFNγ or TNFα expression on xenoantigen-specific CD4+ or CD8+ T cells compared to unstimulated cells, and these occurred mainly in the first 4 weeks. Microbeads were readily retrievable for assessment at day 90 and day 180 and at retrieval were without microscopic signs of degradation or foreign body responses (FBR). In vitro and immunohistochemistry studies of explanted microbeads indicated the presence of functional xenogeneic islets at day 30 post transplantation in all biopsied NHPs. These results from a small pilot study revealed that CXCL12-microencapsulated xenogeneic islets abrogate inflammatory and adaptive immune responses to the xenograft. This work paves the way toward future larger scale studies of the transplantation of alginate microbeads with CXCL12 and porcine or human stem cell-derived beta cells or allogeneic islets into diabetic NHPs without systemic immunosuppression.

Distinct physiological characteristics and altered glucagon signaling in GHRH knockout mice: Implications for longevity.

Our previous research has demonstrated that mice lacking functional growth hormone-releasing hormone (GHRH) exhibit distinct physiological characteristics, including an extended lifespan, a preference for lipid utilization during rest, mild hypoglycemia, and heightened insulin sensitivity. They also show a further increase in lifespan when subjected to caloric restriction. These findings suggest a unique response to fasting, which motivated our current study on the response to glucagon, a key hormone released from the pancreas during fasting that regulates glucose levels, energy expenditure, and metabolism. Our study investigated the effects of an acute glucagon challenge on female GHRH knockout mice and revealed that they exhibit reduced glucose production, likely due to suppressed gluconeogenesis. However, these mice showed an increase in energy expenditure. We also observed alterations in pancreatic islet architecture, with smaller islets and a reduction of insulin-producing beta cells but no changes in glucagon-producing alpha cells. Additionally, the analysis of hepatic glucagon signaling showed a decrease in glucagon receptor expression and phosphorylated CREB. In conclusion, our findings suggest that the unique metabolic phenotype observed in these long-lived mice may be partly explained by changes in glucagon signaling. Further exploration of this pathway may lead to new insights into the regulation of longevity in mammals.

Cystine/glutamate antiporter System xc- deficiency impairs insulin secretion in mice

Aims/hypothesisGlutamate-induced cytotoxicity (excitotoxicity) has been detected in pancreatic beta cells. The cystine/glutamate antiporter System xc- exports glutamate to the extracellular space and is therefore implicated as driving excitotoxicity. As of yet, it has not been investigated whether System xc- contributes to pancreatic islet function.MethodsThis study describes the implications of deficiency of System xc- on glucose metabolism in both constitutive and myeloid cell-specific knockout mice using metabolic tests and diet-induced obesity. Pancreatic islets were isolated and analysed for beta cell function, glutathione levels and ER stress.ResultsConstitutive System xc- deficiency led to an approximately threefold decrease in glutathione levels in the pancreatic islets as well as cystine shortage characterised by upregulation of Chac1. This shortage further manifested as downregulation of beta cell identity genes and a tonic increase in endoplasmic reticulum stress markers, which resulted in diminished insulin secretion both in vitro and in vivo. Myeloid-specific deletion did not have a significant impact on metabolism or islet function.Conclusions/interpretationThese findings suggest that System xc- is required for glutathione maintenance and insulin production in beta cells and that the system is dispensable for islet macrophage function.Graphical

Advancements in biomaterials and biofabrication for enhancing islet transplantation

Type 1 diabetes (T1D) is characterized by the degeneration of insulin-producing beta cells within pancreatic islets, resulting in impaired endogenous insulin synthesis, which necessitates exogenous insulin therapy. Although intensive insulin therapy has been effective in many patients, a subset of individuals with unstable T1D encounter challenges in maintaining optimal glycemic control through insulin injections. Pancreatic islet transplantation has emerged as a promising therapeutic alternative for such patients, offering enhanced glucose regulation, reduced risk of complications, and liberation from exogenous insulin reliance. However, impediments such as immune rejection and the need for an optimal transplantation environment limit the success of islet transplantation. Revascularization, a crucial requirement for proper islet functionality, poses a challenge in transplantation settings. Biomaterial-based biofabrication approaches have attracted considerable attention to address these challenges. Biomaterials engineered to emulate the native extracellular matrix provide a supportive environment for islet viability and functionality. This review article presents the recent advancements in biomaterials and biofabrication technologies aimed at engineering cell delivery systems to enhance the efficacy of islet transplantation. Immune protection and vascularization strategies are discussed, key biomaterials employed in islet transplantation are highlighted, and various biofabrication techniques, including electrospinning, microfabrication, and bioprinting, are explored. Furthermore, the future directions and challenges in the field of cell delivery systems for islet transplantation are discussed. The integration of appropriate biomaterials and biofabrication methods has significant potential to promote successful islet transplantation by facilitating vascularization and bolstering the immune defense mechanisms.