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There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs via inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules via beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A*02:01, underwent transplantation of 1,000 human HLA-A*02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below in vivo detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin+/PD-L1+ sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3+ and CD45+ immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first in vivo studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.

Revascularization of transplanted pancreatic islets is critical for survival and treatment of type 1 diabetes. Questions concerning how islets influence local microvascular networks and how networks form connections with islets remain understudied and motivate the need for new models that mimic the complexity of real tissue. Recently, our laboratory established the rat mesentery culture model as a tool to investigate cell dynamics involved in microvascular growth. An advantage is the ability to observe blood vessels, lymphatics, and immune cells. The objective of this study was to establish the rat mesentery tissue culture model as a useful tool to investigate islet tissue integration. DiI-labeled islets were seeded onto adult rat mesentery tissues and cultured for up to 3 days. Live lectin labeling enabled time-lapse observation of vessel growth. During culture, DiI-positive islets remained intact. Radial lectin-positive capillary sprouts with DiI labeling were observed to form from islets and connect to host networks. Lectin-positive vessels from host networks were also seen growing toward islets. PECAM and NG2 labeling confirmed that vessels sprouting from islets contained endothelial cells and pericytes. Our results introduce the rat mesentery culture model as a platform for investigating dynamics associated with the initial revascularization of transplanted islets.