Introduction: Intrahepatic islet transplantation in patients with type 1 diabetes is limited by donor availability and lack of engraftment. To overcome these limitations, new sources of β cells and alternative sites are needed. Organ decellularization is an emerging strategy in organ regeneration. Based on our experience with decellularized rat lung as scaffold for the generation of Vascularized Endocrine Pancreas (lung scaffold repopulated by neonatal pig islet and BOEC cells), we used the same platform to engineer an iPSC-based version named iVEP (iPSC-derived Vascularized Endocrine Pancreas).

Methods: iPSC-derived β (iβ) and endothelial (iEC) cells were characterized in flow cytometry. Rat lung was decellularized by vascular perfusion with 1% SDS and 0.1% Triton and seeded with commercially available iβ and iEC. The recellularized scaffold matured in vitro for 7 days in a customized perfusion bioreactor specifically designed to allow cell/compartment integration. The iβ death was estimated during ex vivo organ maturation compared to 7 days of iβ in vitro culture evaluating miR-375 expression by droplet digital PCR. At day 7, fluorangiography assay was performed to evaluate the vascular compartment structure and function while for the endocrine compartment, insulin production was measured by dynamic glucose perifusion and insulin quantification (ELISA/IF). Matured iVEPs were then transplanted subcutaneously into NSG diabetic mice followed for 30 days, compared to the deviceless (DL) implantation site and explanted iVEPs were assessed for IF evaluation.

Results: iEC/iβ maintained for 7 days in vitro their phenotype expressing endothelial (>95% CD31+/CD105+/CD73+/CD90- cells) and β-cell (>60% PDX1+/insulin+ cells) markers respectively. Matured iVEPs showed regenerated vascular network (CD31+) able to sustain the direct distribution of a perfusate with iβ cells (insulin+) integrated. In mature iVEP, iβ preserved insulin expression. miR-375, a marker of β cell death, was expressed in iβ but not in iECs. iVEP was able to significantly reduce β cell death: the amount of lost iβ was ≤18% during organ maturation, while >70% during in vitro culture (p<0.05). In iVEPs, lung ECM was able to sustain iβ engraftment and insulin secretion during maturation process (AUC first phase iVEPs: 3.9465±1.23 Vs 1.44±0.83 p<0.05). Preliminary preclinical results demonstrated the ability of iVEP to engraft and restore normoglycemia in diabetic recipient mice compared to the iβ implanted in the DL site.

Conclusion: We conclude that ex vivo bio-fabrication of iVEPs enables iβ engraftment, maturation and vascularization before transplantation. To our knowledge, we assembled the first human entirely iPSC-derived Vascularized Endocrine Pancreas able to provide both in vitro and in vivo insulin production and regenerated vascular network.