Directed self-assembly of a xenogeneic vascularized endocrine pancreas for type 1 diabetes

Type 1 diabetes (T1D) currently requires glucose monitoring and exogenous insulin injections, which fail to perfectly replicate the dynamic glucose-insulin homeostasis of a healthy pancreas. β-cell replacement through islet or pancreas transplantation offers an alternative, but organ donor scarcity and immune rejection remain significant hurdles. The ideal solution requires an unlimited source of insulin-producing cells and biocompatible scaffolds that promote cell interaction and integration. Previous research demonstrated the potential of decellularized lung as a scaffold for β-cell replacement. This study builds upon that foundation by integrating neonatal porcine islets (NPIs) – a readily available and scalable source – and human blood outgrowth endothelial cells (BOECs) to engineer a xenogeneic vascularized endocrine pancreas (VEP). The rationale is that the decellularized lung matrix provides a supportive environment, while the BOECs establish a functional vascular network within the scaffold, enhancing islet survival, maturation, and integration.

Extensive research explores alternative islet sources, including stem cell-derived products and pig islets. Pigs are attractive due to their similar physiology to humans, high fecundity, and susceptibility to genetic engineering to mitigate immune rejection and reduce the risk of zoonosis. However, NPIs have a lower insulin content than adult pig islets and require maturation. Previous attempts to foster endocrine maturation *in vitro* using native or synthetic extracellular matrix (ECM) components have shown limited success in achieving immediate, orchestrated mature function. The microenvironment's role in cell behavior, differentiation, and function is well-established. ECM components, geometry, and stiffness modulate intracellular signaling, influencing cell maturation. Prior work demonstrated that a decellularized lung scaffold significantly improved β-cell survival, function, and *in vivo* performance. This supports the hypothesis that using a decellularized organ ECM can positively impact endocrine cell survival and maturation.

This study involved several key steps: **1. BOEC Characterization:** BOECs were isolated from healthy donors and characterized for their endothelial phenotype using immunofluorescence (CD31, Von Willebrand factor, VE-cadherin, VEGFR2) and flow cytometry (CD31, VE-cadherin, KDR). Their proliferation and tube formation capacity were assessed in both standard BOEC culture media (BCM) and a modified VEP media. **2. VEP Assembly:** NPIs (both iRFP transgenic and wild-type) were isolated from neonatal piglets and, after *in vitro* maturation, were seeded along with BOECs into decellularized rat lung left lobes. BOECs were initially seeded via the pulmonary artery and vein, followed by seeding of NPIs and additional BOECs via the trachea. The constructs were then cultured for 7 days in a two-phase media protocol (angiogenic medium followed by stabilization medium). **3. Functional Assessment *in vitro*:** The engineered VEPs were assessed for cell integration, vascular network functionality (using microsphere perfusion), and endocrine maturation (insulin, glucagon, somatostatin mRNA and protein expression). Dynamic insulin secretion tests were conducted to evaluate the glucose-responsive insulin release. β-cell death was monitored using miR-375, a miRNA highly expressed in pancreatic β-cells, whose release is a potential indicator of β-cell death. **4. *In vivo* Transplantation:** Mature VEPs were transplanted into immunodeficient NSG mice with severe diabetes. Their performance was compared to control groups: NPIs transplanted subcutaneously (device-less), under the kidney capsule, or into the liver. Glucose levels were monitored for 9, 14, or 18 weeks. Explanted VEPs were analyzed for iRFP signal, vascular density, and immune cell infiltration. **Additional in vitro and in vivo experiments:** Further experiments were performed to investigate the role of BOECs in NPIs maturation and engraftment using VEPs generated without BOECs. The vascular density of explanted VEPs was analyzed using AngioTool software. Immunofluorescence analysis was used to assess both human and murine endothelium, as well as nerves within the VEPs. Transmission electron microscopy (TEM) was used to examine the ultrastructure of explanted VEPs. Detailed descriptions of all procedures are included in the Methods section of the original paper.

The key findings of this study include: * **Successful VEP Biofabrication:** The researchers successfully engineered a functional xenogeneic VEP using a decellularized rat lung scaffold, neonatal porcine islets, and human BOECs. * **Enhanced Islet Maturation:** NPIs within the VEP showed significantly enhanced maturation *in vitro*, as evidenced by increased insulin, glucagon, and somatostatin expression at both mRNA and protein levels, and improved physiological insulin secretion kinetics compared to NPIs cultured in standard conditions. The presence of a vascularized ECM, created by the BOECs, was crucial for this enhanced maturation. * **Functional Vascular Network:** The VEP's vascular network, formed by the BOECs, was shown to be functional, enabling efficient perfusion of the islet cells. * **Low β-cell Death:** MiR-375 quantification showed a very low rate of β-cell death (0-5%) during the 7-day VEP maturation process, indicating the biocompatibility of the scaffold. * **Superior *in vivo* Performance:** VEP transplantation resulted in significantly better glycemic control in diabetic mice compared to control groups with NPIs transplanted to other sites (device-less subcutaneous space, kidney capsule, or liver). VEP-implanted mice achieved normoglycemia rapidly and maintained it for up to 18 weeks. The explantation of the VEPs resulted in a rapid return to hyperglycemia in the recipient mice, confirming the graft's functional contribution to normoglycemia. * **Role of BOECs:** Experiments using VEPs without BOECs showed a significant delay in achieving normoglycemia, confirming the crucial role of BOECs in both islet maturation and engraftment. * **Vascular and Nerve Integration:** Explanted VEPs showed progressive integration of the vascular structure from host to the recipient and the presence of nerve fibers in close proximity to the endocrine cells.

The results demonstrate a significant advancement in bioengineered endocrine pancreas technology for T1D treatment. The use of a decellularized lung scaffold, combined with the self-assembly of NPIs and autologous BOECs, creates a biocompatible, vascularized environment that promotes islet maturation and function, resulting in superior *in vivo* performance compared to traditional transplantation methods. The functional vascular network within the VEP ensures adequate nutrient and oxygen delivery, crucial for islet survival and function. The low rate of β-cell death during the *in vitro* maturation process highlights the biocompatibility of the scaffold. Furthermore, the study's findings underscore the crucial role of BOECs not only in creating the vascular network but also in promoting islet maturation and engraftment. The long-term maintenance of normoglycemia in the preclinical model strongly supports the translational potential of this technology. The immediate function of the VEP post-implantation in the subcutaneous site, contrasting with the failure of device-less NPI transplantation, highlights the advantages of this approach.

This study successfully demonstrated the feasibility of creating a functional vascularized endocrine pancreas using a novel biofabrication approach. The results show that the VEP platform can successfully direct the self-assembly of immature NPIs and BOECs into a functional organ that achieves rapid and sustained normoglycemia in a preclinical model. This technology offers a promising strategy for developing a clinically applicable treatment for T1D. Further research should focus on scaling up the production process for clinical grade VEPs, optimizing the scaffold composition, and exploring the application of this approach in combination with gene-editing techniques to produce immune-tolerant xenogeneic islets.

The study was conducted in immunodeficient mice, which may not fully reflect the immune response in human recipients. The long-term effects of the VEP in immunocompetent animals or humans require further investigation. The current VEP fabrication process is based on a rat lung scaffold, and further research is needed to optimize the process for human clinical application, particularly the scalability and reproducibility of the procedure, and to identify potential long-term complications. It is also important to carefully assess the long term safety and efficacy of this treatment in the context of potential rejection or disease transmission.