Abstract
Ca(V)3 channels are ontogenetically downregulated with the maturation of certain electrically excitable cells, including pancreatic beta cells. Abnormally exaggerated Ca(V)3 channels drive the dedifferentiation of mature beta cells. This led us to question whether excessive Ca(V)3 channels, retained mistakenly in engineered human-induced pluripotent stem cell-derived islet (hiPSC-islet) cells, act as an obstacle to hiPSC-islet maturation. We addressed this question by using the anterior chamber of the eye (ACE) of immunodeficient mice as a site for recapitulation of in vivo hiPSC-islet maturation in combination with intravitreal drug infusion, intravital microimaging, measurements of cytoplasmic-free Ca2+ concentration ([Ca2+](i)) and patch clamp analysis. We observed that the ACE is well suited for recapitulation, observation and intervention of hiPSC-islet maturation. Intriguingly, intraocular hiPSC-islet grafts, retrieved intact following intravitreal infusion of the Ca(V)3 channel blocker NNC55-0396, exhibited decreased basal [Ca2+](i) levels and increased glucose-stimulated [Ca2+](i) responses. Insulin-expressing cells of these islet grafts indeed expressed the NNC55-0396 target Ca(V)3 channels. Intraocular hiPSC-islets underwent satisfactory engraftment, vascularization and light scattering without being influenced by the intravitreally infused NNC55-0396. These data demonstrate that inhibiting Ca(V)3 channels facilitates the maturation of glucose-activated Ca2+ signaling in hiPSC-islets, supporting the notion that excessive Ca(V)3 channels as a developmental error impede the maturation of engineered hiPSC-islet insulin-expressing cells.