CCL21 and beta-cell antigen releasing hydrogels as tolerance-inducing therapy in Type I diabetes.

Type-I Diabetes (T1D) is caused by defective immunotolerance mechanisms enabling autoreactive T cells to escape regulation in lymphoid organs and destroy insulin-producing ß-cells in the pancreas, leading to insulin dependence. Strategies to promote ß-cell tolerance could arrest T1D. We previously showed that secretion of secondary lymphoid chemokine CCL21 by CCL21 transgenic ß-cells induced tolerance and protected non-obese diabetic (NOD) mice from T1D. T1D protection was associated with formation of lymph node-like stromal networks containing tolerogenic fibroblastic reticular cells (FRCs). Here, we developed a polyethylene glycol (PEG) hydrogel platform with hydrolytically degradable PEG-diester dithiol crosslinkers to provide controlled and sustained delivery of CCL21 and ß-cell antigens for at least 28 days in vitro and recapitulate properties associated with the tolerogenic environment of CCL21 transgenic ß-cells in our previous studies. CCL21 and MHC-II restricted antigens were tethered to gels via simple click-chemistry while MHC-I restricted antigens were loaded in PEG-based polymeric nanovesicles and incorporated in the gel networks. CCL21 and antigen release kinetics depended on the PEG gel tethering strategy and the linkers. Importantly, in vitro functionality, chemotaxis, and activation of antigen-specific T cells were preserved. Implantation of CCL21 and ß-cell antigen gels under the kidney capsule of pre-diabetic NOD mice led to enrichment of adoptively transferred antigen-specific T cells, formation of gp38 + FRC-like stromal cell networks, and increased regulation of specific T cells with reduced accumulation within pancreatic islets. Thus, our platform for sustained release of ß-cell antigens and CCL21 immunomodulatory molecule could enable the development of antigen-specific tolerance therapies for T1D.

Parallel Evaluation of Polyethylene Glycol Conformal Coating and Alginate Microencapsulation as Immunoisolation Strategies for Pancreatic Islet Transplantation.

Pancreatic islet transplantation improves metabolic control and prevents complications in patients with brittle type 1 diabetes (T1D). However, chronic immunosuppression is required to prevent allograft rejection and recurrence of autoimmunity. Islet encapsulation may eliminate the need for immunosuppression. Here, we analyzed in parallel two microencapsulation platforms that provided long-term diabetes reversal in preclinical T1D models, alginate single and double capsules versus polyethylene glycol conformal coating, to identify benefits and weaknesses that could inform the design of future clinical trials with microencapsulated islets. We performed in vitro and in vivo functionality assays with human islets and analyzed the explanted grafts by immunofluorescence. We quantified the size of islets and capsules, measured capsule permeability, and used these data for in silico simulations of islet functionality in COMSOL Multiphysics. We demonstrated that insulin response to glucose stimulation is dependent on capsule size, and the presence of permselective materials augments delays in insulin secretion. Non-coated and conformally coated islets could be transplanted into the fat pad of diabetic mice, resulting in comparable functionality and metabolic control. Mac-2+ cells were found in conformally coated grafts, indicating possible host reactivity. Due to their larger volume, alginate capsules were transplanted in the peritoneal cavity. Despite achieving diabetes reversal, changes in islet composition were found in retrieved capsules, and recipient mice experienced hypoglycemia indicative of hyperinsulinemia induced by glucose retention in large capsules as the in silico model predicted. We concluded that minimal capsule size is critical for physiological insulin secretion, and anti-inflammatory modulation may be beneficial for small conformal capsules.

Performance of islets of Langerhans conformally coated via an emulsion cross-linking method in diabetic rodents and nonhuman primates.

Polyethylene glycol (PEG)-based conformal coating (CC) encapsulation of transplanted islets is a promising ß cell replacement therapy for the treatment of type 1 diabetes without chronic immunosuppression because it minimizes capsule thickness, graft volume, and insulin secretion delay. However, we show here that our original CC method, the direct method, requiring exposure of islets to low pH levels and inclusion of viscosity enhancers during coating, severely affected the viability, scalability, and biocompatibility of CC islets in nonhuman primate preclinical models of type 1 diabetes. We therefore developed and validated in vitro and in vivo, in several small- and large-animal models of type 1 diabetes, an augmented CC method-emulsion method-that achieves hydrogel CCs around islets at physiological pH for improved cytocompatibility, with PEG hydrogels for increased biocompatibility and with fivefold increase in encapsulation throughput for enhanced scalability.

Conformal Coating of Stem Cell-Derived Islets for ß Cell Replacement in Type 1 Diabetes.

The scarcity of donors and need for immunosuppression limit pancreatic islet transplantation to a few patients with labile type 1 diabetes. Transplantation of encapsulated stem cell-derived islets (SC islets) might extend the applicability of islet transplantation to a larger cohort of patients. Transplantation of conformal-coated islets into a confined well-vascularized site allows long-term diabetes reversal in fully MHC-mismatched diabetic mice without immunosuppression. Here, we demonstrated that human SC islets reaggregated from cryopreserved cells display glucose-stimulated insulin secretion in vitro. Importantly, we showed that conformally coated SC islets displayed comparable in vitro function with unencapsulated SC islets, with conformal coating permitting physiological insulin secretion. Transplantation of SC islets into the gonadal fat pad of diabetic NOD-scid mice revealed that both unencapsulated and conformal-coated SC islets could reverse diabetes and maintain human-level euglycemia for more than 80 days. Overall, these results provide support for further evaluation of safety and efficacy of conformal-coated SC islets in larger species.