Introduction: When first generated, AG MC were intended to offer selective permeable and biocompatible physical shields for the immune-protection of pancreatic islet grafts in diabetic recipients. In house development of unique methods for AG ultra-purification had allowed us to initiate pilot human clinical trials of microencapsulated islet allograft in T1D. We aimed to achieve significant advances in the MC membrane’s engineering, to make these coatings suitable for molecules regulated delivery and stem cell differentiation in T1D.

Materials and methods: A) Towards T1D therapy by microencapsulated human induced pluripotent stem cells (hiPSC). Thawed hiPSC upon culture on Vitronectin XF™ and NutriStem underwent a differentiation protocol into beta cell-like spheroids according to Millman. To activate spheroid formation, cells were incubated at a density of 2x106 cells/ml in NutriStem XFTM supplemented with 10 μM ROCK inhibitor under agitation. We then enveloped the spheroids within highly biocompatible and selective permeable and porous AG-MC, prior to intraperitoneal graft in NOD/SCID mice. B) Prevention of T1D. Rat G3C hybridoma cells were enveloped in specially engineered AG-MC. The new MC prototype was designed to alter the original membrane’s m.w. cutoff, by meticulously re-calculating AG-polyaminoacidic stoichiometric molar ratios, to allow egress of IgM, secreted by the G3C, targeting Tc GITR+. GITR+ triggering would expand the recipient’s Treg population and promote acquired immune-tolerance. G3C containing AG-MC were grafted intraperitoneally in naïve NOD mice (70% of females develop T1D) intraperitoneally.

Results: A) Immunofluorescence for pluripotency markers confirmed the presence of Oct4, Sox2, Nanog, c-Myc and Ki67 on all cells of each cluster and cytofluorimetry confirmed that over 90% of cells were triple positive for Sox2, Oct4 and Nanog. qPCR for the various differentiation factors (PDX-1, NKX6.1, SOX9, PTF1a, FOXA2, SOX17, CXCR4, c-KIT, Glut and GK), and those expressed by Beta-cells (MafA, MAfB, NKX2.2 and NKX6.1) showed best visibility at day 27 of maturation, although not a full differentiation. 30 days after TX, we observed that the cell aggregates had increased in size with well-preserved viability. DTZ staining showed the presence of a significant amount of insulin, unlike the encapsulated control cells in vitro. B) None of the recipient NOD’s grafted with G3C AG-MC developed T1D over 110 days of follow-up, unlike control mice receiving empty AG-MC. Quantitative morphometry of the retrieved pancreases documented interruption of the disease process in mice receiving G3C containing AG-MC grafts unlike controls.

Conclusions: Clinical grade AG-MC other than constituting effective immune-barriers, serve for 3D bioartificial matrix for in vivo maturation of hiPSC. Pro AG-MC plasticity, looser AG-MC represent an excellent biomolecules delivery system that by release of G3C-derived IgM, targeting GITR+ Tc, may enable discontinuation of the disease process leading to overt T1D in NOD mice.