Widespread use of pancreatic islet transplantation for treatment of type 1 diabetes (T1D) is currently limited by requirements for long-term immunosuppression, limited donor supply, and poor long-term engraftment and function. Upon isolation from their native microenvironment, islets undergo rapid apoptosis, which is further exacerbated by poor oxygen and nutrient supply following infusion into the portal vein. Identifying alternative strategies to restore critical microenvironmental cues, while maximizing islet health and function, is needed to advance this cellular therapy. We hypothesized that biophysical properties provided through type I oligomeric collagen macroencapsulation are important considerations when designing strategies to improve islet survival, phenotype, and function. Mouse islets were encapsulated at various Oligomer concentrations (0.5 -3.0 mg/ml) or suspended in media and cultured for 14 days, after which viability, protein expression, and function were assessed. Oligomer-encapsulated islets showed a density-dependent improvement in in vitro viability, cytoarchitecture, and insulin secretion, with 3 mg/ml yielding values comparable to freshly isolated islets. For transplantation into streptozotocin-induced diabetic mice, 500 islets were mixed in Oligomer and injected subcutaneously, where rapid in situ macroencapsulation occurred, or injected with saline. Mice treated with Oligomer-encapsulated islets exhibited rapid (within 24 h) diabetes reversal and maintenance of normoglycemia for 14 (immunocompromised), 90 (syngeneic), and 40 days (allogeneic). Histological analysis showed Oligomer-islet engraftment with maintenance of islet cytoarchitecture, revascularization, and no foreign body response. Oligomer-islet macroencapsulation may provide a useful strategy for prolonging the health and function of cultured islets and has potential as a subcutaneous injectable islet transplantation strategy for treatment of T1D.

Replacement of islets/β cells that provide long-lasting glucose-sensing and insulin-releasing functions has the potential to restore extended glycemic control in individuals with type 1 diabetes. Unfortunately, persistent challenges preclude such therapies from widespread clinical use including cumbersome administration via portal vein infusion, significant loss of functional islet mass upon administration, limited functional longevity, and requirement for systemic immunosuppression. Previously, fibril-forming type I collagen (oligomer) was shown to support subcutaneous injection and in-situ encapsulation of syngeneic islets within diabetic mice, with rapid (<24 hours) reversal of hyperglycemia and maintenance of euglycemia for beyond 90 days. Here, we further evaluated this macroencapsulation strategy, defining effects of islet source (allogeneic and xenogeneic) and dose (500 and 800), injection microenvironment (subcutaneous and intraperitoneal), and macrocapsule format (injectable and preformed implantable) on islet functional longevity and recipient immune response. We found that xenogeneic rat islets functioned similarly to or better than allogeneic mouse islets, with only modest improvements in longevity noted with dose. Additionally, subcutaneous injection led to more consistent encapsulation outcomes, along with improved islet health and longevity, when compared to intraperitoneal administration, while no significant differences were observed between subcutaneous injectable and preformed implantable formats. Collectively, these results document the benefits of incorporating natural collagen for islet/β cell replacement therapies.

Objective β-cell microRNA-21 (miR-21) is increased by islet inflammatory stress but it decreases glucose-stimulated insulin secretion (GSIS). Thus, we sought to define the effects of miR-21 on β-cell function using in vitro and in vivo systems. Methods We developed a tetracycline-on system of pre-miR-21 induction in clonal β-cells and human islets, along with transgenic zebrafish and mouse models of β-cell-specific pre-miR-21 overexpression. Results β-cell miR-21 induction markedly reduced GSIS and led to reductions in transcription factors associated with β-cell identity and increased markers of dedifferentiation, which led us to hypothesize that miR-21 induces β-cell dysfunction by loss of cell identity. In silico analysis identified transforming growth factor-beta 2 ( Tgfb2 ) and Smad family member 2 (Smad2) mRNAs as predicted miR-21 targets associated with the maintenance of β-cell identity. Tgfb2 and Smad2 were confirmed as direct miR-21 targets through RT-PCR, immunoblot, pulldown, and luciferase assays. In vivo zebrafish and mouse models exhibited glucose intolerance, decreased peak GSIS, decreased expression of β-cell identity markers, increased insulin and glucagon co-staining cells, and reduced Tgfb2 and Smad2 expression. Conclusions These findings implicate miR-21-mediated reduction of mRNAs specifying β-cell identity as a contributor to β-cell dysfunction by the loss of cellular differentiation.

We previously showed that β cell microRNA 21 (miR-21) is increased by islet inflammatory stress and diabetes, that miR-21 induces β cell dysfunction by targeting mRNAs maintaining β cell identity, and that β cell miR-21 induction in vivo leads to hyperglycemia. However, upstream regulators of β cell miR-21 are poorly defined. We hypothesized that increases in the transcriptional regulator Hypoxia Inducible Factor 1 Subunit Alpha (Hif1a) during diabetogenic islet stress activate β cell miR-21 transcription, thereby contributing to loss of β cell identity occurring under these conditions. To test this, we examined Hif1a and miR-21 levels in β cell lines and mouse islets. Hif1a was increased in islets from mice after multiple low dose streptozotocin (STZ) or 4 wks of 60% high fat diet (HFD). INS1 cells and flow-sorted β cells showed increases in both miR-21 and Hif1a mRNA after 24-hr IL1β treatment. Hif1a overexpression in INS1 cells increased miR-21 levels, insulin and glucagon co-expression, and aldehyde dehydrogenase 1a3 staining, all suggesting loss of β cell identity. By contrast, siRNA depletion of Hif1a abrogated cytokine-induced reductions in mRNAs regulating β cell identity. Chromatin immunoprecipitation studies verified increased HIF1a occupancy at the miR-21 promoter after IL1β treatment. To test if these HIF1a-mediated increases in β cell miR-21 exacerbate β cell dysfunction under diabetogenic conditions in vivo, transgenic mice harboring a β cell tamoxifen inducible miR-21 transgene (Tg(β-miR-21, Ins1(CreERT2)Thor) were treated with STZ or HFD. Compared to controls, STZ and HFD-induced hyperglycemia was exacerbated in Tg(β-miR-21) mice following tamoxifen administration. By contrast, after STZ, conditional β cell miR-21 knock out (Ins1(Cre)Thor) mice showed improved glycemia. In conclusion, these findings implicate Hif1a-miR-21 signaling as a contributor to β cell dysfunction under conditions of inflammation and diabetes. Disclosure S. Ibrahim: None. C. Stephens: None. R.E. Moore: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. E.K. Sims: None.