Macroencapsulation of human stem cell-derived beta cells ameliorates rejection in CAR T cell humanized mice

Problem: Type 1 diabetes is characterized by the destruction of insulin-producing beta cells in pancreatic islets, requiring life-long exogenous insulin therapy. Cell therapies using allogeneic stem cell-derived beta cells (sBC) are a promising alternative therapy with the potential to eliminate long-term complications associated with imperfect insulin administration. However, allogeneic therapies require chronic systemic immunosuppression that presents acute risks of infections and malignancies. Moreover, stem cell-based therapies carry the potential for teratoma formation in immunosuppressed patients. To overcome these issues, we have developed a hydrogel injection molding-based macroencapsulation method to reduce the need for immunosuppression with allogeneic cell-based therapies. Macroencapsulation also enhances graft safety by enabling graft containment and retrieval in the case of adverse events. This method is a high-throughput biomanufacturing platform capable of manufacturing encapsulated stem cell-based products at scale and generates spiral alginate geometries with high surface area-to-volume ratios for optimal oxygen transport.
Objective: Here, we evaluate spiral alginate encapsulated sBC cluster viability and function in vitro and in vivo using longitudinal in vivo imaging, and the efficacy of macroencapsulation to prevent T cell-mediated rejection in a humanized mouse model.
Methodology: The Mel1 INSGFP cell line was used to generate sBC clusters expressing human HLA-A-2 with GFP expression driven by the insulin promoter and constitutive luciferase expression. In vitro characterization of alginate spiral-encapsulated sBC clusters via Alamar Blue metabolic activity, live/dead confocal microscopy, and glucose-stimulated insulin secretion assay demonstrated encapsulated sBCs performed comparably to unencapsulated controls after 48 hr of culture. In vitro co-culture of encapsulated sBC with effector T cells (Teff) expressing a chimeric antigen receptor (CAR) specific for HLA-A-2 (A2 CAR Teff) demonstrated a significant reduction of granzyme B positive T cell proliferation relative to unencapsulated controls. A total of 600 alginate spiral-encapsulated luciferase-expressing sBC clusters transplanted into the epididymal fat pads (EFP) of non-obese diabetic severe combined immunodeficiency gamma chain mutant mice (NSG) demonstrated comparable survival to unencapsulated controls via in vivo bioluminescence imaging out to 54 days post-transplant, with no change in signal compared to day 1. Longitudinal human C-peptide measurements were comparable between encapsulated and control sBCs, at 37 and 36 pg/mol on day 33, respectively. Additionally, select grafts were whole mount imaged for lectin-positive vasculature integration with encapsulation devices, showing close proximity of vasculature to GFP positive sBCs. On day 54, A2 CAR Teff cells were administered to sBC-engrafted NSG mice. Within 3 weeks, a greater reduction in luciferase signal was observed in the unencapsulated sBC group relative to encapsulated sBC.
Conclusion: Overall, we demonstrate that hydrogel injection molding-based macroencapsulation results in human sBC viability and function comparable to unencapsulated cells both in vitro and in vivo in immune compromised mice, and that macroencapsulation can protect encapsulated sBC from T cell-mediated destruction. Further histological studies are evaluating differences in T cell infiltration and spatial distribution within grafts and confirming sBC insulin positivity post-T cell challenge.