Beta Cell Replacement Therapy Research Articles

Advances in Type 1 Diabetes Mellitus Management in Children.

Recent advancements in the management of type 1 diabetes mellitus (T1DM) have significantly improved outcomes and quality of life for patients, particularly children. Technological innovations, such as continuous glucose monitoring (CGM) systems and insulin pump therapy, including hybrid closed-loop systems, have enhanced glycemic control by providing real-time data and automated insulin delivery. Ultrarapid-acting insulins and adjunctive pharmacotherapies, like sodium-glucose transport protein 2 (SGLT2) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists, offer improved postprandial glucose management and reduced insulin requirements. Immunotherapy and beta-cell replacement therapies, including stem cell research and encapsulation devices, aim to preserve or restore endogenous insulin production. Digital health platforms and telemedicine have expanded access to education and support, fostering better self-management. Future directions in precision medicine, artificial intelligence, and microbiome research hold promise for personalized and potentially curative treatments. Collectively, these advances are transforming T1DM management, reducing disease burden, and enhancing the prospects for children with T1DM.

The role of genetic risk factors, diet, and gut microbiota in type 1 diabetes mellitus, pancreas and pancreatic islet transplantation.

Despite advances in insulin delivery and glucose monitoring technology, prevention of the progression of secondary complications in patients with type 1 diabetes (T1DM) remains a challenge. Beta cell replacement therapy in the form of islet or pancreas transplantation can restore long-term normoglycaemia with sustained periods of insulin independence among T1DM patients. However, the same genetic, behavioural, or gut microbiota-related factors that promoted autoimmunity and primary islet destruction may also affect the function of transplanted islets and the ultimate results of transplant procedures. In such cases, identifying genetic risk factors and modifying behavioural factors and those related to gut microbiota may be beneficial for the outcomes of transplant procedures. Herein, we review related literature to the identified current gap in knowledge to be addressed in future clinical trials.

Efficient Vascular and Neural Engraftment of Stem Cell-Derived Islets.

Pluripotent stem cell-derived islets (SC-islets) now emerge as a new source for beta-cell replacement therapy. While the function of human islet transplants is hampered by excessive cell death post-transplantation, contributing factors include inflammatory reactions, insufficient revascularization and islet amyloid formation, there is a gap in knowledge on the engraftment process of the SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at three months post-transplantation, and observed that the cell apoptosis rates were lower, but the vascular density was similar in SC-islets to that of human islets. While the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. The oxygenation of the SC-islet grafts was twice as high as in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, also the reinnervation of the SC-islets was four- to five-fold higher than the human islets. Both SC-islets and the human islets contained amyloid at one and three months post-transplantation. We conclude that the vascular and neural engraftment of SC-islets is superior to human islets, but that grafts of both origins develop amyloid with potential long-term consequences.

Molecular Retention Limitations for Prevascularized Subcutaneous Sites for Islet Transplantation.

Beta cell replacement therapies utilizing the subcutaneous space have inherent advantages to other sites: the potential for increased accessibility, noninvasive monitoring, and graft extraction. Site prevascularization has been developed to enhance islet survivability in the subcutaneous zone while minimizing potential foreign body immune responses. Molecular communication between the host and prevascularized implant site remains ill-defined. Poly(ethylene oxide)s (PEOs) of various hydrated radii (i.e., ∼11-62 Å) were injected into prevascularized subcutaneous sites in C57BL/6 mice, and the clearance and organ biodistribution were characterized. Prevascularization formed a barrier that confined the molecules compared with the unmodified site. Molecular clearance from the prevascularized site was inversely proportional to the molecular weight. The upper limit in molecular size for entering the vasculature to be cleared was determined to be 35 kDa MW PEO. These findings provide insight into the impact of vascularization on molecular retention at the injection site and the effect of molecular size on the mobility of hydrophilic molecules from the prevascularized site to the host. This information is necessary for optimizing the transplantation site for increasing the beta cell graft survival.

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.

Adipose-Derived Stromal Cells Preserve Pancreatic Islet Function in a Transplantable 3D Bioprinted Scaffold.

Intra-portal islet transplantation is currently the only clinically approved beta cell replacement therapy, but its outcome is hindered by limited cell survival due to a multifactorial reaction against the allogeneic tissue in liver. Adipose-derived stromal cells (ASCs) can potentially improve the islet micro-environment by their immunomodulatory action. The challenge is to combine both islets and ASCs in a relatively easy and consistent long-term manner in a deliverable scaffold. Manufacturing the 3D bioprinted double-layered scaffolds with primary islets and ASCs using a mix of alginate/nanofibrillated cellulose (NFC) bioink is reported. The diffusion properties of the bioink and the supportive effect of human ASCs on islet viability, glucose sensing, insulin secretion, and reducing the secretion of pro-inflammatory cytokines are demonstrated. Diabetic mice transplanted with islet-ASC scaffolds reach normoglycemia seven days post-transplantation with no significant difference between this group and the group received islets under the kidney capsules. In addition, animals transplanted with islet-ASC scaffolds stay normoglycemic and show elevated levels of C-peptide compared to mice transplanted with islet-only scaffolds. The data present a functional 3D bioprinted scaffold for islets and ASCs transplanted to the extrahepatic site and suggest a possible role of ASCs on improving the islet micro-environment.

338.6: A conceptual supportive cell based implant for beta cell replacement therapy

338.6: A conceptual supportive cell based implant for beta cell replacement therapy

336.3: Identification of novel cell surface markers for the characterization of human beta cell replacement therapies

336.3: Identification of novel cell surface markers for the characterization of human beta cell replacement therapies

European Society for Organ Transplantation (ESOT) Consensus Statement on the Role of Pancreas Machine Perfusion to Increase the Donor Pool for Beta Cell Replacement Therapy.

The advent of Machine Perfusion (MP) as a superior form of preservation and assessment for cold storage of both high-risk kidney's and the liver presents opportunities in the field of beta-cell replacement. It is yet unknown whether such techniques, when applied to the pancreas, can increase the pool of suitable donor organs as well as ameliorating the effects of ischemia incurred during the retrieval process. Recent experimental models of pancreatic MP appear promising. Applications of MP to the pancreas, needs refinement regarding perfusion protocols and organ viability assessment criteria. To address the "Role of pancreas machine perfusion to increase the donor pool for beta cell replacement," the European Society for Organ Transplantation (ESOT) assembled a dedicated working group comprising of experts to review literature pertaining to the role of MP as a method of improving donor pancreas quality as well as quantity available for transplant, and to develop guidelines founded on evidence-based reviews in experimental and clinical settings. These were subsequently refined during the Consensus Conference when this took place in Prague.

289-LB: Intestinal Stem Cells as a Source of Insulin-Positive Beta-Like Cells for Treating Diabetes

Shortage of functional pancreatic beta-cells is the basic cause of type 1 and some late-stage type 2 diabetes, making generation of new beta-cells from stem cells a promising strategy for treating diabetes. Intestinal stem cells (ISCs) are adult stem cells found in the base of the crypts of the adult intestine, which is responsible for the continuous life-long renewal of intestinal epithelia cells and thus could also be used a potential source for autologous transplantation. Unlike the most extensively studied bone marrow derived stem cells, ISCs share the advantage of having a common endodermic origin with beta-cells, making their differentiation into beta-cells easier. However, purifying ISCs has been a challenge. Here, we reported a novel method that successfully purifies ISCs from wildtype mice by exploiting their specific binding to a lectin. The only other cell type in the intestine that also binds to this lectin was found to be Paneth cells, which could be excluded through a negative selection for CD24. The purified Lectin+CD24-ISCs, confirmed by gene profiling, were labeled with a fluorescent tag and shown to differentiate into insulin-producing beta-like cells when injected into day 11.5 mouse embryonic pancreatic explants and grown for a week. Further analysis of gene profile alterations and signaling pathways allowed us to develop a time-sensitive protocol with recombinant growth factors to optimize the differentiation of ISCs into beta-like cells in vitro, without the need for an embryonic environment. Our results not only demonstrate a new method for purifying ISCs, but also their potential to differentiate into insulin-producing beta-like cells. This study provides a promising strategy for beta-cell replacement therapy in diabetes. Disclosure Y. Xiao: None. Y. Xiao: None. Y. Jiang: None. Funding University of Pittsburgh; Children?s Hospital of Pittsburgh; Cochrane-Weber Endowed Fund in Diabetes Research

Replenishable prevascularized cell encapsulation devices increase graft survival and function in the subcutaneous space.

Beta cell replacement therapy (BCRT) for patients with type 1 diabetes (T1D) improves blood glucose regulation by replenishing the endogenous beta cells destroyed by autoimmune attack. Several limitations, including immune isolation, prevent this therapy from reaching its full potential. Cell encapsulation devices used for BCRT provide a protective physical barrier for insulin-producing beta cells, thereby protecting transplanted cells from immune attack. However, poor device engraftment posttransplantation leads to nutrient deprivation and hypoxia, causing metabolic strain on transplanted beta cells. Prevascularization of encapsulation devices at the transplantation site can help establish a host vascular network around the implant, increasing solute transport to the encapsulated cells. Here, we present a replenishable prevascularized implantation methodology (RPVIM) that allows for the vascular integration of replenishable encapsulation devices in the subcutaneous space. Empty encapsulation devices were vascularized for 14 days, after which insulin-producing cells were inserted without disrupting the surrounding vasculature. The RPVIM devices were compared with nonprevascularized devices (Standard Implantation Methodology [SIM]) and previously established prevascularized devices (Standard Prevascularization Implantation Methodology [SPVIM]). Results show that over 75% of RPVIM devices containing stem cell-derived insulin-producing beta cell clusters showed a signal after 28 days of implantation in subcutaneous space. Notably, not only was the percent of RPVIM devices showing signal significantly greater than SIM and SPVIM devices, but the intraperitoneal glucose tolerance tests and histological analyses showed that encapsulated stem-cell derived insulin-producing beta cell clusters retained their function in the RPVIM devices, which is crucial for the successful management of T1D.

Nanothin Conformal Coating with Poly(N-vinylpyrrolidone) and Tannic Acid (PVPON/TA) Preserves Murine and Human Pancreatic Islets Function.

Beta cell replacement therapies can restore glycemic control to select individuals living with type 1 diabetes. However, the obligation of lifelong immunosuppression restricts cell therapies from replacing exogenous insulin administration. Encapsulation strategies can reduce the inherent adaptive immune response; however, few are successfully translated into clinical testing. Herein, we evaluated if the conformal coating of islets with poly(N-vinylpyrrolidone) (PVPON) and tannic acid (TA) (PVPON/TA) could preserve murine and human islet function while conferring islet allograft protection. In vitro function was evaluated using static glucose-stimulated insulin secretion, oxygen consumption rates, and islet membrane integrity. In vivo function was evaluated by transplanting human islets into diabetic immunodeficient B6.129S7-Rag1tm1Mom/J (Rag-/-) mice. The immunoprotective capacity of the PVPON/TA-coating was assessed by transplanting BALB/c islets into diabetic C57BL/6 mice. Graft function was evaluated by non-fasting blood glucose measurements and glucose tolerance testing. Both coated and non-coated murine and human islets exhibited indistinguishable in vitro potency. PVPON/TA-coated and control human islets were able to restore euglycemia post-transplant. The PVPON/TA-coating as monotherapy and adjuvant to systemic immunosuppression reduced intragraft inflammation and delayed murine allograft rejection. This study demonstrates that PVPON/TA-coated islets may be clinically relevant as they retain their in vitro and in vivo function while modulating post-transplant immune responses.

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

Operational Tolerance of Murine Islet Allografts Co-Transplanted With Cyclosporine A Eluting Microparticles

Beta cell replacement therapy is a proven strategy to restore glycemic control, reduce hypoglycemic awareness and stabilize glycated hemoglobin (HbA1c) for a subset of patients with type 1 diabetes. However, the absence of an effective strategy to prevent islet allograft rejection and recurrent autoimmunity restricts patient inclusion and durable insulin independence. Herein, we explore the utility of islet graft localized cyclosporine A (CsA) delivery via co-transplanted drug-eluting poly(lactic-co-glycolic acid) (PLGA) microparticles to attenuate allograft rejection.

From islet of Langerhans transplantation to the bioartificial pancreas

Type 1 diabetes is a disease resulting from autoimmune destruction of the insulin-producing beta cells in the pancreas. When type 1 diabetes develops into severe secondary complications, in particular end-stage nephropathy, or life-threatening severe hypoglycemia, the best therapeutic approach is pancreas transplantation, or more recently transplantation of the pancreatic islets of Langerhans. Islet transplantation is a cell therapy procedure, that is minimally invasive and has a low morbidity, but does not display the same rate of functional success as the more invasive pancreas transplantation because of suboptimal engraftment and survival. Another issue is that pancreas or islet transplantation (collectively known as beta cell replacement therapy) is limited by the shortage of organ donors and by the need for lifelong immunosuppression to prevent immune rejection and recurrence of autoimmunity.A bioartificial pancreas is a construct made of functional, insulin-producing tissue, embedded in an anti-inflammatory, immunomodulatory microenvironment and encapsulated in a perm-selective membrane allowing glucose sensing and insulin release, but isolating from attacks by cells of the immune system. A successful bioartificial pancreas would address the issues of engraftment, survival and rejection. Inclusion of unlimited sources of insulin-producing cells, such as xenogeneic porcine islets or stem cell-derived beta cells would further solve the problem of organ shortage.This article reviews the current status of clinical islet transplantation, the strategies aiming at developing a bioartificial pancreas, the clinical trials conducted in the field and the perspectives for further progress.

Pankreasinseltransplantation – State of the Art

In specific patient populations with unstable diabetes, biological beta cell replacement therapy is a valid approach to attain the desirable metabolic control. The main indications for allogeneic islet transplantation are frequent hypoglycemia and severe metabolic lability despite intensive insulin therapy, or simultaneous or sequential islet–kidney transplantation in uremic patients with type 1 diabetes. Clear outcome definitions of function and failure have been instrumental for a broader positive recognition of islet transplantation among diabetologists/endocrinologists. International collaboration has resulted in continuous improvements in cell processing techniques, patient management, and development of specific immunotherapy protocols, thus, significantly improving outcomes after islet transplantation. Excellent metabolic control, reliable prevention of hypoglycemia, and positive effects on diabetes-related complications and quality of life have been demonstrated. Islet transplantation is a safe, minimally invasive procedure that can potentially cure type 1 diabetes. Yet, limited availability of donor organs and the need for systemic immunosuppression make islet transplantation an option for only highly selected patients.

O007 Pancreas and islet transplantation in the United Kingdom during the COVID-19 era

IntroductionWHO declared a pandemic of COVID-19 in March 2020. This study analyses the impact of COVID-19 on beta-cell replacement therapy in the UK.MethodsPancreas and islet donation and transplant activity in the period March 2020/2021 was compared with the same period the previous year.Results2,180 patients had a functioning graft during March 2020/2021. 5.8%(n=126) tested positive for COVID-19 and two died (1%). In this period there was a 43% reduction in solid organ donors n=1,615, compared with the previous year, n=2,840. Of the 625 solid organ donors with a pancreas offered, 32% had the pancreas retrieved compared with 51% the previous period. 97 whole pancreas and islet transplants were performed in the UK down 54% from the prior period. Of the 84 pancreas transplant recipients; four tested positive for COVID-19 but none died, and two grafts failed within the first week from vascular thrombosis (neither were COVID-19 positive). Of the 13 SIK and islet alone transplant recipients, two tested positive for COVID-19 but neither died. Of these SIK transplants, one is known to have failed within a month and this is equivalent to that seen in the previous time period. To our knowledge, no patient receiving beta cell replacement therapy died of COVID during the first year of the pandemic despite immunosuppression.ConclusionIn the UK, pancreas, and islet transplantation have continued during the pandemic at a lower rate. Outcomes following transplantation within the COVID era are, so far, similar to those in the period prior.Take-home messageOutcomes following transplantation within the COVID era are, so far, similar to those in the period prior.

β-cell replacement therapy for type 1 diabetes: closer and closer.

β-cell replacement therapy for type 1 diabetes: closer and closer.

Transgenic pigs expressing near infrared fluorescent protein-A novel tool for noninvasive imaging of islet xenotransplants.

Islet xenotransplantation is a promising concept for beta-cell replacement therapy. Reporter genes for noninvasive monitoring of islet engraftment, graft mass changes, long-term survival, and graft failure support the optimization of transplantation strategies. Near-infrared fluorescent protein (iRFP) is ideal for fluorescence imaging (FI) in tissue, but also for multispectral optoacoustic tomography (MSOT) with an even higher imaging depth. Therefore, we generated reporter pigs ubiquitously expressing iRFP. CAG-iRPF720 transgenic reporter pigs were generated by somatic cell nuclear transfer from FACS-selected stable transfected donor cells. Neonatal pig islets (NPIs) were transplanted into streptozotocin-diabetic immunodeficient NOD-scid IL2Rgnull (NSG) mice. FI and MSOT were performed to visualize different numbers of NPIs and to evaluate associations between signal intensity and glycemia. MSOT was also tested in a large animal model. CAG-iRFP transgenic NPIs were functionally equivalent with wild-type NPIs. Four weeks after transplantation under the kidney capsule, FI revealed a twofold higher signal for 4000-NPI compared to 1000-NPI grafts. Tenweeks after transplantation, the fluorescence intensity of the 4000-NPI graft was inversely correlated with glycemia. After intramuscular transplantation into diabetic NSG mice, MSOT revealed clear dose-dependent signals for grafts of 750, 1500, and 3000 NPIs. Dose-dependent MSOT signals were also revealed in a pig model, with stronger signals after subcutaneous (depth ∼6mm) than after submuscular (depth ∼15mm) placement of the NPIs. Islets from CAG-iRFP transgenic pigs are fully functional and accessible to long-term monitoring by state-of-the-art imaging modalities. The novel reporter pigs will support the development and preclinical testing of novel matrices and engraftment strategies for porcine xeno-islets.

306.1: Clinical Metrics Predict Defective Physiologic Responses to Hypoglycemia in Long-standing Type 1 Diabetes and May Facilitate Early Consideration of Referral for Beta-Cell Replacement Therapy

Repeated hypoglycemia exposure leads to impaired awareness of hypoglycemia (IAH) and the development of defective counterregulatory responses. To date, only islet transplantation has demonstrated normalization of endogenous glucose production (EGP) during insulin-induced hypoglycemia, in addition to improving hypoglycemia awareness. This study aims to validate clinical metrics of IAH (Clarke score), hypoglycemia severity (HYPO score), glycemic lability (lability index, LI), and CGM measures of hypoglycemia exposure and glucose variability as predictors of defective glucose counterregulation, defined by absent EGP, and hypoglycemia unawareness, defined by absent autonomic symptom (AS) recognition, measured in response to insulin-induced hypoglycemia. 42 subjects with mean±SD age 43±13 years and T1D duration 29±12 years including 32 with IAH (Clarke unaware;19 pre-islet [Cohort 0], 13 pre-CGM [Cohort 1]) and 10 with intact hypoglycemia awareness (aware), and 12 non-diabetic control subjects, matched for age, sex and BMI (Cohort 3) underwent single-blinded randomized-paired hyperinsulinemic euglycemic and hypoglycemic clamp experiments using a stable glucose isotope tracer at the University of Pennsylvania between 2008–2021. Glycemic thresholds for EGP and AS responses were determined by the plasma glucose at which the response >95%CI of the euglycemic clamp control experiment. ROC and sensitivity analyses were performed to assess metric prediction of defective counterregulation. HYPO and LI were greater in Clarke unaware (Cohorts 0 and 1) vs aware cohort (Cohort 3; p<0.0001 and p=0.03, respectively). During hypoglycemic clamp experiments, final step EGP response was greatest in controls (1.4±0.4 mg/kg/min) and greater in aware (0.9±0.4 mg/kg/min) than unaware cohorts (0.5±0.4 mg/kg/min, p<0.01) which remained greater than in euglycemia (0.2±0.6 mg/kg/min, p=0.004). AS responses were comparable in aware and control groups (7.9±2.9 vs 8.0±6.7) however significantly less in the unaware group (2.4±3.0, p<0.001) which were no different than during euglycemia (1.0±2.2). Dichotomized Clarke score (</≥4) and Composite HYPO score/LI (</≥75th%) demonstrated good predictive value for determining glycemic threshold for EGP (Clarke: AUC=0.73, p=0.03; HYPO/LI: AUC=0.71, p=0.03) and AS (Clarke: AUC=0.71, p=0.04; HYPO/LI: AUC=0.71, p=0.03) responses. CGM time spent in hypoglycemia showed good ability to predict AS by 240 minutes but not EGP response. Parallel assessment of Clarke and hypoglycemia metrics increased the sensitivity and specificity to predict absent AS response at 240 minutes. Defective EGP response at 240 minutes was best predicted by a composite of Clarke and variability / lability metrics however at a lower specificity. These data suggest presence of IAH together with hypoglycemia exposure or increased glucose variability may identify individuals with defective counterregulation appropriate for early consideration of beta-cell replacement therapy. The project is supported by Public Health Services research grants R01 DK091331 (to M.R.R.), U01 DK070430 (University of Pennsylvania Center for Clinical Islet Transplantation), UL1 TR001878 (University of Pennsylvania Center for Human Phenomic Science), P30 DK19525 (University of Pennsylvania Diabetes Research Center), T32 DK00734 (University of Pennsylvania Training Grant in Diabetes, Endocrinology and Metabolic Diseases), and the Charles B. Humpton, Jr. Endowed Fellowship in Diabetes Research (to A.F.). The authors thank Dr. John Millar of the University of Pennsylvania Institute for Diabetes, Obesity and Metabolism Metabolic Tracer Resource for performance of the gas chromatography-mass spectrometry analysis of the 6,6-2H2-glucose tracer.