In vivo maturation and resilience of human pluripotent stem cell derived islet clusters

• Applicants: Professor Anthony Gavalas, Ph.D.; Professor Dr. Stephan Speier
• Subject Area: Endocrinology, Diabetology, Metabolism
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 540762991

Functional performance and post-transplantation graft safety represent two main hurdles for the clinical application of human pluripotent stem (hPS) cell-derived islet-like clusters (SC-islets). This is most likely due to the persisting immature state of in vitro derived SC-islets prior to transplantation. SC-islets have been shown to undergo further differentiation and maturation after transplantation, but do not reach the required functional and safety status. We hypothesize that detailed analysis of the temporal and mechanistic aspects of in vivo maturation will uncover essential, so far unknown, new cues to further promote in vitro differentiation and maturation of SC-islets, thus improving the outcome of transplantations. The aim of this project is to identify the mechanisms implicated in the in vivo maturation of SC-islets and leverage them to accelerate their maturation in vitro. To this aim, we will employ our anterior chamber of the eye (ACE) platform, which takes advantage of the ACE as a natural body window and applies laser-scanning microscopy to in vivo image the transplanted SC-islets at high resolution. Using the hPS cell reporter lines that we have developed, we will longitudinally investigate the kinetics of in vivo maturation and function of SC-islets after their transplantation into the ACE of immune deficient mouse. Additionally, the safety of SC-islets will be evaluated by closely monitoring the SC-islet mass and, in particular, the non-granulated cell mass which would result from the expansion of residual progenitor cells in the SC-islets. These in vivo data will be complemented by functional physiological characterization assays of the SC-islets before transplantation in vitro and after graft retrieval ex vivo. Single-cell transcriptomics of the grafted SC-islet grafts, retrieved at key transition time points of the in vivo maturation, will identify candidate molecules and signalling pathways implicated in the maturation process. Using our reporter lines and a high content SC-islet imaging procedure that we have established, we will apply these findings in a combinatorial manner to identify conditions promoting SC-islet differentiation and maturation in vitro and develop advanced procedures for the generation of mature SC-islets. The survival and function of these advanced SC-islets will then be assessed in metabolically challenging systemic conditions that SC-islets would encounter following transplantation in a diabetic patient. These comprehensive in vivo, in vitro and ex vivo datasets will provide unprecedented mechanistic insights into endocrine cell in vivo maturation. The findings will be leveraged to promote in vitro differentiation and maturation of SC-islets toward the development of a safe and effective cell replacement therapy for diabetic patients.

• DFG Programme: Research Grants