The role of T-type calcium channels in human pancreatic β cell maturity

Sammanfattning: This PhD work has optimized a series of in vivo, ex vivo and in vitro approaches to recapitulate, intervene and appraise in vivo maturation of naïve β cells within human induced pluripotent stem cell-derived islets (hiPSC-islets). These approaches have also been applied to characterize hyperglycemia-induced dedifferentiation of mature β cells within native human islets grafted into the anterior chamber of the eye (ACE) of immunodeficient mice. It has verified the critical role of exaggerated T-type Ca2+ channels in these adverse events. The immunodeficient mouse ACE serves as a remarkable transplantation site for human islets including hiPSC-islets and native human islets. They are placed apart from each other on the iris and undergo satisfactory survival, engraftment and vascularization. This allows in vivo recapitulation and microscopy of hiPSC-islet insulin-expressing cell maturation and mature human islet β cell dedifferentiation. This also enables intact retrieval of intracameral human islet grafts for ex vivo measurements of cytosolic free Ca2+ concentration ([Ca2+]i) and patch clamp analysis as well as in vitro confocal microscopy/immunofluorescence labeling. In addition, intravitreally-infused drugs enter the ACE and act on intracameral human islets with special, beneficial pharmacokinetic, pharmacodynamic and toxicological properties. The feasibility and merits of these approaches bring about the following main results. Intracameral hiPSC-islets display heterogeneity in their survival, engraftment, vascularization, growth and fates during post-transplantation. They gradually mature at least in their insulin-secretory capacity and glucoseactivated [Ca2+]i dynamics. Intriguingly, naïve hiPSC-islet insulin-expressing cells mistakenly retain excessive T-type Ca2+ channels and in vivo inhibition of these channels significantly promotes development of glucose-dependent [Ca2+]i dynamics in intracameral hiPSC-islets. Furthermore, hyperglycemia induces mature β cell dedifferentiation in intracameral native human islets by activating Ttype Ca2+ channel-calcineurin signaling resulting in β cell HSF1 dislocation, VAMP- 2 reduction and exocytosis deterioration. This pathway is inactivated by inhibition of β cell T-type Ca2+ channels, calcineurin or both. These findings demonstrate that upregulated β cell T-type Ca2+ channels impede naïve human β cell maturation and promote mature human β cell dedifferentiation. This points out that T-type Ca2+ channel inhibition has a great potential counteracting these two detrimental events for generation of clinically transplantable hiPSC-islets and for effective treatment for diabetes.