Allogeneic Islet Research Articles

310.6: Analysis of islet isolation from DCD for allogeneic human islet transplantation to type1 diabetes – single center experience

310.6: Analysis of islet isolation from DCD for allogeneic human islet transplantation to type1 diabetes – single center experience

235.7: Analysis of collagenase dose on allogeneic islet isolation outcomes and glucose-stimulated insulin release

235.7: Analysis of collagenase dose on allogeneic islet isolation outcomes and glucose-stimulated insulin release

400.2: Long-term insulin independence following allogenic islet transplant and childbirth

400.2: Long-term insulin independence following allogenic islet transplant and childbirth

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.

Long-Term Survival and Induction of Operational Tolerance to Murine Islet Allografts by Co-Transplanting Cyclosporine A Microparticles and CTLA4-Ig.

One strategy to prevent islet rejection is to create a favorable immune-protective local environment at the transplant site. Herein, we utilize localized cyclosporine A (CsA) delivery to islet grafts via poly(lactic-co-glycolic acid) (PLGA) microparticles to attenuate allograft rejection. CsA-eluting PLGA microparticles were prepared using a single emulsion (oil-in-water) solvent evaporation technique. CsA microparticles alone significantly delayed islet allograft rejection compared to islets alone (p < 0.05). Over 50% (6/11) of recipients receiving CsA microparticles and short-term cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4-Ig) therapy displayed prolonged allograft survival for 214 days, compared to 25% (2/8) receiving CTLA4-Ig alone. CsA microparticles alone and CsA microparticles + CTLA4-Ig islet allografts exhibited reduced T-cell (CD4+ and CD8+ cells, p < 0.001) and macrophage (CD68+ cells, p < 0.001) infiltration compared to islets alone. We observed the reduced mRNA expression of proinflammatory cytokines (IL-6, IL-10, INF-γ, and TNF-α; p < 0.05) and chemokines (CCL2, CCL5, CCL22, and CXCL10; p < 0.05) in CsA microparticles + CTLA4-Ig allografts compared to islets alone. Long-term islet allografts contained insulin+ and intra-graft FoxP3+ T regulatory cells. The rapid rejection of third-party skin grafts (C3H) in islet allograft recipients suggests that CsA microparticles + CTLA4-Ig therapy induced operational tolerance. This study demonstrates that localized CsA drug delivery plus short-course systemic immunosuppression promotes an immune protective transplant niche for allogeneic islets.

Intraperitoneal injection of cell-engineered pancreas in rats with experimental type i diabetes (preliminary results)

Creation of a bioartificial pancreas, including a cell-engineered construct (CEC) formed from pancreatic islets (islets of Langerhans) and a biocompatible matrix mimicking the native microenvironment of pancreatic tissue, is one of the approaches to the treatment of type 1 diabetes mellitus (T1D).Objective: to conduct preliminary in vivo studies of the functional efficacy of intraperitoneal injection of a cell-engineered pancreatic endocrine construct and a suspension of rat pancreatic islets in an experimental T1D model.Materials and methods. Tissue-specific scaffold was obtained by decellularization of human pancreatic fragments. The viability and functional activity of rat islets isolated with collagenase were determined. Experimental T1D was modeled by intraperitoneal injection of low-dose streptozotocin and incomplete Freund’s adjuvant into rats. The rats were intraperitoneally injected twice with pancreatic CEC (n = 2) or islet suspension (n = 1). Glucose levels in the blood and urine of the rats were assessed. Histological examination of organs (pancreas and kidneys) of the experimental animals was carried out.Results. After the first injection, blood glucose levels gradually decreased in all animals by more than 47% of the initial values; by follow-up day 24, the glucose level rose to the initial hyperglycemic values. After repeated administration, a 63.4% decrease in glycemic level was observed in the rats with pancreatic CEC and a 47.5% decrease in the one with islet suspension. At week 5 of the experiment, blood glucose levels gradually increased in all animals. At the same time, the glycemic index of the rat with injected pancreatic CEC was 62% lower than the glycemic index of the rat with injected islets.Conclusion. Allogeneic pancreatic islets in pancreatic CEC increase the duration of stable glycemic level in T1D rats.

1808-PUB: Effective and Durable Stem-Cell-Enabled Therapy of Autoimmune T1DM in Rodents and Dogs Provides the Scientific Basis for the First-in-Human Trial Using I.P. Administered “Neo-Islets,” 3-D Organoids of Allogeneic Islet, and Mesenchymal Stromal Cells—The Omentum Becomes the New Endocrine Pancreas without the Need for Antirejection Drugs

Two initial reports from Clinical Trials with cell-based interventions for the urgently needed therapy of T1DM show: (a) Open, s.c.-placed encapsulation devices containing insulin-producing progenitor cells that require anti-rejection drugs have failed to adequately improve glycemic control. (b) The intraportal administration of stem cell-derived insulin-producing cells, requiring anti-rejection drugs, has shown promising results in 2 or 3 subjects. However, the inability to retrieve these cells and the need for anti-rejection drugs remain a concern. Our approach harnesses the immune-modulating and -isolating, anti-inflammatory, angiogenic, anti-apoptotic and other trophic actions of Mesenchymal Stromal Cells (MSCs). First, we demonstrated that allogeneic Neo-islets (NIs), islet-sized organoids composed of equal numbers of MSCs and culture expanded Islet Cells do, when given i.p., omentally engraft, redifferentiate, re-express all islet hormones and permanently restore euglycemia in auto-immune diabetic NOD mice, i.e., without encapsulation devices or antirejection drugs. The omentum becomes the new endocrine pancreas. Second, i.p. therapy of spontaneously diabetic pet dogs with allogeneic canine NIs significantly improved glycemia and insulin needs (&amp;gt; 3 years), and this without immune response (abstract this meeting). Third, human NIs permanently correct streptozotocin-induced diabetes in NOD/SCID mice. Removal of the omentally-engrafted NIs promptly restores the diabetic state. No long-term complications of the NI therapy have been observed. Based on this body of evidence, a successful Pre-IND meeting with the FDA was held and final Proof-of-Concept studies that are expected to lead IND approval are ongoing. Disclosure A.Gooch: Board Member; SymbioCellTech, LLC, Employee; SymbioCellTech, LLC, Stock/Shareholder; SymbioCellTech, LLC. C.Westenfelder: Board Member; SymbioCellTech, LLC., Employee; SymbioCellTech, LLC., Research Support; SymbioCellTech, LLC., Stock/Shareholder; SymbioCellTech, LLC.

91-OR: AdoShell Islet, a New Biomaterial for Allogenic Islet Encapsulation Shows Six Months Insulin Secretion in Diabetic Immunocompetent Rats

Islet transplantation is an effective cure for T1D but the requirement for immunosuppression makes its application limited. AdoShell® is an implantable and fully retrievable scaffold for islet transplantation without immunosuppression. It is based on a permselective hydrogel film that allows the diffusion of insulin and prevents immune cell invasion. AdoShell® is a soft biomaterial with 95% water, synthetized from non-degradable polymers cross-linked by bio-orthogonal click chemistry. Encapsulated human islets displayed a gluco-responsive insulin secretion comparable to naked islets in perifusion experiments after 7 days and 1 month (N=3 donors 7 days / N=2 donors 1 month). Wistar rat allogenic islets encapsulated in AdoShell® were implanted in immunocompetent STZ-induced diabetic rats. Insulin levels in vivo were on average 121pM/day over 41 days (n=22 rats), a significant increase versus negative control (45pM/day, n=9 rats) (M.W. t-test, p&amp;lt;0.0001). AdoShell® Islets led to physiological weight gain, reduction of fasting hyperglycemia, and sustained C-peptide and insulin secretion for over 6 months in diabetic rats. It was well tolerated and did not trigger inflammatory reaction nor fibrosis after the 6-month implantation period in rat peritoneal cavity. Neither biodegradation, nor immune cell penetration were observed. Disclosure A.Geissler: Employee; Adocia, Stock/Shareholder; Adocia. O.Jouannot: Employee; ADOCIA, Stock/Shareholder; ADOCIA. A.Gaffuri: Employee; ADOCIA, Stock/Shareholder; ADOCIA. C.Gautier: Employee; Centre européen d'étude du diabète, Biomeostasis, Charles Rivers Laboratories, Institut de recherche contre les cancers de l'appareil digestif, GenOway, NAMSA. Y.P.Courbebaisse: None. K.Bouzakri: Consultant; Adocia, Stock/Shareholder; ILONOV. R.Eloy: Stock/Shareholder; Adocia. O.Soula: Employee; Adocia, Stock/Shareholder; Adocia.

89-OR: Immunoprivileged Hydrogels Extend Functionality of Islets in Immunocompetent Animals

Background: Despite the promise of current islet cell transplantation, the need for systemic immune suppression significantly limits its utility for patients with diabetes. Until safe and immune-evasive cells are realized, physical immuno-protection offers an alternate route to allogeneic islet implants. Our proprietary synthetic hydrogels provide shape-agnostic, robust and retrievable Islet environments with anti-fouling and non-fibrotic properties. Methods: Rat and human donor islets were encapsulated and implanted in the peritoneal space of streptozotocin (STZ)-induced diabetic immunocompetent mice and pigs to assess islet functionality over time through measurement of blood glucose, C-peptide, and HbA1c. Next generation shape-agnostic hydrogels were also tested for in-vitro and in-vivo function. Results: Our hydrogels enabled rapid and persistent blood glucose control for 140+ days in immunocompetent mice with marginal loads of 1000 rat IEQ/mouse. Human donor islet function in mice was extended to day 45 (~8x longer than free human islets), with C-peptide detected at day 50, and corresponding HbA1c decreases. Our hydrogels showed minimal fibrosis when explanted after 45 days and showed robustness and integrity at day 340 post-implant in immunocompetent mice. Next-gen shape-agnostic hydrogels successfully excluded immune components including IgG and maintained good islet function in vitro. Our data also demonstrate function of human donor islets in immunocompetent pigs, based on C-peptide and cell viability data. Conclusion: Our proprietary synthetic and retrievable hydrogels show strong immune protection in xenogenic transplants of donor islets into immune-competent animals. We are currently scaling to therapeutic doses in large animals, in pursuit of a curative therapy based on an optimal balance of Islet protection and functionality, without immunosuppression. Disclosure H.Stover: Employee; Allarta Life Science Inc.

137-LB: Modified Personal Glycemic State Model Predicts the Need for Islet Transplantation in Human

Continuous glucose monitoring (CGM) has become a valuable tool for assessing glycemic control. The personal glycemic state (PGS)1 is a mathematical model that considers 5 CGM data elements (mean blood glucose, percent time in range, glucose variability percentage, and frequency of moderate and severe hypoglycemia) equally to generate a score representing the degree of metabolic control. Our adapted personal glycemic state for islet transplant (PGSFIT) targets normoglycemia in islet transplantation by limiting the percent time in range to 70-140mg/dl instead of commonly used 70-180mg/dl, and uses weighted components without limiting the influence of any individual component on the total score. This allowed us to account for T1D patients keeping blood glucose high to avoid hypoglycemia or low to avoid long term complications despite increased hypoglycemia. Using PGS and PGSFIT to analyze glycemic control in 16 subjects with T1D post allogenic islet transplantations showed concordance in classifying the glycemic control as acceptable (PGS/PGSFIT &amp;lt;15) or unacceptable (≥15) in 88% of the cases when variability was low (standard deviation of PGS components ≤ 0.1), and both PGSFIT and PGS models had excellent correlation with fasting c-peptide results. In contrast, when variability was high (standard deviation of PGS components &amp;gt; 0.1), 71% of patients deemed clinically suitable for repeat transplant had PGSFIT score ≥15 but only 14% had PGS score ≥15. In patients who had not yet received their final islet infusion, high parameter variability and PGSFIT ≥15 was associated with lower fasting C-peptide compared to patients with high parameter variability and PGSFIT ≤15 who received the maximum therapeutic dose. PGSFIT significantly improved after repeat transplant in patients with high PGSFIT and high component variability. These results suggest PGSFIT may also be used to identify subjects suitable for first islet transplant. Disclosure C. Orr: None. J. Hacker-stratton: None. M. El-shahawy: None. E. Forouhar: None. K. Omori: None. M. Qi: None. F. R. Kandeel: None. Funding National Institutes of Health (U24DK098085)

237-OR: Gastrin Improves Single Islet Transplant Outcomes

Background: Multiple allogenic islet transplants (ITs) are usually required to supply adequate islet mass (~10,000 IEQ/kg) to induce insulin independence. Improving outcomes of a SINGLE islet transplant are needed. Gastrin is a gut hormone that is involved in normal fetal pancreas development. Evidence by our group and others have shown gastrin can support islet cell expansion/function and have anti-inflammatory and pro-angiogenesis effects. We compared outcomes of IT alone across 3 clinical trials using: Edmonton-based (ITA), T-cell depleting (TCD), and TCD immunosuppression with an investigational gastrin analogue (GAS). Methods: Between 2004-2022, a total of 31 evaluable T1D patients without a prior history of transplant received IT-alone under the ITA (n=17), TCD (n=7), and GAS (n=7) clinical trials. Participants in ITA and TCD were eligible to receive up to 3 ITs each. GAS participants received only a single IT and two 30-day courses of gastrin (SQ, BID) at the time of IT and again 6 months later. Immunosuppression included basiliximab or daclizumab + etanercept (ITA) and T-cell depletion (anti-thymoglobulin) + etanercept + anakinra (TCD and GAS). Results: Progressive improvements in engraftment were observed in the ITA, TCD, and GAS trials. Average insulin requirements were reduced by 18, 26, and 33 u/day. GAS subjects showed the most rapid reduction of 30.9 u/day (94%) in the first 2 weeks post-IT, compared to 17.5 u/day (67%) in TCD. Rates of subjects achieving insulin independence with a SINGLE islet transplant were 0, 15, and 57%. Subjects required an average of 3 ITs in ITA, 2 in TCD, and only one IT in GAS to achieve insulin independence. Average numbers of islets required to achieve insulin independence were 13,280, 8,433 and 4,837 IEQ/kg. Conclusion: Gastrin use in the peri-transplant period leads to rapid engraftment, larger magnitude of insulin reduction, and permitted achievement of insulin independence in more than half of subjects after a single islet transplant of less than half the normally targeted islet dose. Disclosure J.Hacker-stratton: None. C.Orr: None. K.Omori: None. M.Qi: None. E.Forouhar: None. M.El-shahawy: None. F.R.Kandeel: None. Funding National Institutes of Health (U24DK098085)

Association between primary graft function and 5-year outcomes of islet allogeneic transplantation in type 1 diabetes: a retrospective, multicentre, observational cohort study in 1210 patients from the Collaborative Islet Transplant Registry

Allogeneic islet transplantation is a validated therapy in type 1 diabetes; however, there is decline of transplanted islet graft function over time and the mechanisms underlying this decline are unclear. We evaluated the distinct association between primary graft function (PGF) and 5-year islet transplantation outcomes. In this retrospective, multicentre, observational cohort study, we enrolled all patients from the Collaborative Islet Transplant Registry who received islet transplantation alone (ITA recipients) or islet-after-kidney transplantation (IAK recipients) between Jan 19, 1999, and July 17, 2020, with a calculable PGF (exposure of interest), measured 28 days after last islet infusion with a validated composite index of islet graft function (BETA-2 score). The primary outcome was cumulative incidence of unsuccessful islet transplantation, defined as an HbA1c of 7·0% (53 mmol/mol) or higher, or severe hypoglycaemia (ie, requiring third-party intervention to correct), or a fasting C-peptide concentration of less than 0·2 ng/mL. Secondary outcomes were graft exhaustion (fasting C-peptide <0·3 ng/mL); inadequate glucose control (HbA1c ≥7·0% [53 mmol/mol] or severe hypoglycaemia); and requirement for exogenous insulin therapy (≥14 consecutive days). Associations between PGF and islet transplantation outcomes were explored with a competing risk analysis adjusted for all covariates suspected or known to affect outcomes. A predictive model based on PGF was built and internally validated by using bootstraps resampling method. In 39 centres worldwide, we enrolled 1210 patients with a calculable PGF (of those without missing data, mean age 47 years [SD 10], 712 [59·5%] were female, and 865 (97·9%) were White), who received a median of 10·8 thousand islet-equivalents per kg of bodyweight (IQR 7·4-13·5). 986 (82·4%) were ITA recipients and 211 (17·6%) were IAK recipients. Of 1210 patients, 452 (37·4%) received a single islet infusion and 758 (62·6%) received multiple islet infusions. Mean PGF was 14·3 (SD 8·8). The 5-year cumulative incidence of unsuccessful islet transplantation was 70·7% (95% CI 67·2-73·9), and was inversely and linearly related to PGF, with an adjusted subhazard ratio (sHR) of 0·77 (95% CI 0·72-0·82) per 5-unit increase of BETA-2 score (p<0·0001). Secondary endpoints were similarly related to PGF. The model-adjusted median C-statistic values of PGF for predicting 5-year cumulative incidences of unsuccessful islet transplantation, graft exhaustion, inadequate glucose control, and exogenous insulin therapy were 0·70 (range 0·69-0·71), 0·76 (0·74-0·77), 0·65 (0·64-0·66), and 0·72 (0·71-0·73), respectively. This global multicentre study reports a linear and independent association between PGF and 5-year clinical outcomes of islet transplantation. The main study limitations are its retrospective design and the absence of analysis of complications. Public Health Service Research, National Institutes of Health, Juvenile Diabetes Research Foundation International, Agence National de la Recherche, Fondation de l'Avenir, and Fonds de Dotation Line Renaud-Loulou Gasté.

Research Highlights.

Research Highlights.

Human hypoimmune primary pancreatic islets avoid rejection and autoimmunity and alleviate diabetes in allogeneic humanized mice.

Transplantation of allogeneic pancreatic donor islets has successfully been performed in selected patients with difficult-to-control insulin-dependent diabetes and impaired awareness of hypoglycemia (IAH). However, the required systemic immunosuppression associated with this procedure prevents this cell replacement therapy from more widespread adoption in larger patient populations. We report the editing of primary human islet cells to the hypoimmune HLA class I- and class II-negative and CD47-overexpressing phenotype and their reaggregation into human HIP pseudoislets (p-islets). Human HIP p-islets were shown to survive, engraft, and ameliorate diabetes in immunocompetent, allogeneic, diabetic humanized mice. HIP p-islet cells were further shown to avoid autoimmune killing in autologous, diabetic humanized autoimmune mice. The survival and endocrine function of HIP p-islet cells were not impaired by contamination of unedited or partially edited cells within the p-islets. HIP p-islet cells were eliminated quickly and reliably in this model using a CD47-targeting antibody, thus providing a safety strategy in case HIP cells exert toxicity in a future clinical setting. Transplantation of human HIP p-islets for which no immunosuppression is required has the potential to lead to wider adoption of this therapy and help more diabetes patients with IAH and history of severe hypoglycemic events to achieve insulin independence.

No Time to Die-How Islets Meet Their Demise in Transplantation.

Islet transplantation represents an effective treatment for patients with type 1 diabetes mellitus (T1DM) and severe hypoglycaemia unawareness, capable of circumventing impaired counterregulatory pathways that no longer provide protection against low blood glucose levels. The additional beneficial effect of normalizing metabolic glycaemic control is the minimisation of further complications related to T1DM and insulin administration. However, patients require allogeneic islets from up to three donors, and the long-term insulin independence is inferior to that achieved with solid organ (whole pancreas) transplantation. This is likely due to the fragility of islets caused by the isolation process, innate immune responses following portal infusion, auto- and allo-immune-mediated destruction and β-cell exhaustion following transplantation. This review covers the specific challenges related to islet vulnerability and dysfunction that affect long-term cell survival following transplantation.

Bioengineered omental transplant site promotes pancreatic islet allografts survival in non-human primates

The transplanting islets to the liver approach suffers from an immediate posttransplant loss of islets of more than 50%, progressive graft dysfunction over time, and precludes recovery of grafts should there be serious complications such as the development of teratomas with grafts that are stem cell-derived islets (SC-islets). The omentum features an attractive extrahepatic alternative site for clinical islet transplantation. We explore an approach in which allogeneic islets are transplanted onto the omentum, which is bioengineered with a plasma-thrombin biodegradable matrix in three diabetic non-human primates (NHPs). Within 1week posttransplant, each transplanted NHP achieves normoglycemia and insulin independence and remains stable until termination of the experiment. Success was achieved in each case with islets recovered from a single NHP donor. Histology demonstrates robust revascularization and reinnervation of the graft. This preclinical study can inform the development of strategies for β cell replacement including the use of SC-islets or other types of novel cells in clinical settings.

Engineering pancreatic islets with a novel form of thrombomodulin protein to overcome early graft loss triggered by instant blood-mediated inflammatory reaction

The instant blood-mediated inflammatory reaction (IBMIR) is initiated by innate immune responses that cause substantial islet loss after intraportal transplantation. Thrombomodulin (TM) is a multifaceted innate immune modulator. In this study, we report the generation of a chimeric form of thrombomodulin with streptavidin (SA-TM) for transient display on the surface of islets modified with biotin to mitigate IBMIR. SA-TM protein expressed in insect cells showed the expected structural and functional features. SA-TM converted protein C into activated protein C, blocked phagocytosis of xenogeneic cells by mouse macrophages and inhibited neutrophil activation. SA-TM was effectively displayed on the surface of biotinylated islets without a negative effect on their viability or function. Islets engineered with SA-TM showed improved engraftment and established euglycemia in 83% of diabetic recipients when compared with 29% of recipients transplanted with SA-engineered islets as control in a syngeneic minimal mass intraportal transplantation model. Enhanced engraftment and function of SA-TM–engineered islets were associated with the inhibition of intragraft proinflammatory innate cellular and soluble mediators of IBMIR, such as macrophages, neutrophils, high-mobility group box 1, tissue factor, macrophage chemoattractant protein-1, interleukin-1β, interleukin-6, tumor necrosis factor-α, interferon-γ. Transient display of SA-TM protein on the islet surface to modulate innate immune responses causing islet graft destruction has clinical potential for autologous and allogeneic islet transplantation.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

Engineering the lymph node environment promotes antigen-specific efficacy in type 1 diabetes and islet transplantation

Antigen-specific tolerance is a key goal of experimental immunotherapies for autoimmune disease and allograft rejection. This outcome could selectively inhibit detrimental inflammatory immune responses without compromising functional protective immunity. A major challenge facing antigen-specific immunotherapies is ineffective control over immune signal targeting and integration, limiting efficacy and causing systemic non-specific suppression. Here we use intra-lymph node injection of diffusion-limited degradable microparticles that encapsulate self-antigens with the immunomodulatory small molecule, rapamycin. We show this strategy potently inhibits disease during pre-clinical type 1 diabetes and allogenic islet transplantation. Antigen and rapamycin are required for maximal efficacy, and tolerance is accompanied by expansion of antigen-specific regulatory T cells in treated and untreated lymph nodes. The antigen-specific tolerance in type 1 diabetes is systemic but avoids non-specific immune suppression. Further, microparticle treatment results in the development of tolerogenic structural microdomains in lymph nodes. Finally, these local structural and functional changes in lymph nodes promote memory markers among antigen-specific regulatory T cells, and tolerance that is durable. This work supports intra-lymph node injection of tolerogenic microparticles as a powerful platform to promote antigen-dependent efficacy in type 1 diabetes and allogenic islet transplantation.

Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice.

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.