Endocrine Abstracts

Introduction: Stem cell-derived βcells are currently in phase 3 cell therapy trials in people with type 1 diabetes. Human induced pluripotent stem cells (iPSC) represent an inexhaustible source of functional islet cells. Generation of iPSC from a patient eliminates the problem of allogeneic rejection and allows grafts to be engineered prior to transplantation to stimulate vascularization, glucose sensing and insulin release, ultimately promoting graft survival. This unique model allows to create new organoids composed of different cells designed to form a niche. Beyond the supply of oxygen and nutrients, intra-islet endothelial cells (EC) and secreted components of the extracellular matrix form a niche necessary for the correct function of βcells and adaptation to their environment. We therefore generated “vascularized” islet organoids containing EC and endocrine cells derived from the same human iPSC, ensuring genetic concordance. The impact of EC on the maturation, self-organization and function of endocrine cells was assessed in vitro.

Methods: Our laboratory has implemented a 7-step protocol to generate functional islet-like aggregates from iPSC on a large scale. Following our coculture strategy, we generated functional iPSC-derived EC and islets from the same iPSC line to generate isogenic endothelial-endocrine organoids. The two cell types were characterized independently and then in coculture in terms of identity and function by immunostaining, RT-qPCR and dynamic insulin secretion in response to glucose.

Results: Our iPSC-derived EC and islets showed the expression of specific markers, both at the gene (VEGFR1/2, CD144 and PDX1, NKX6.1, MAFA, respectively) and protein level (CD31, von Willebrand factor, vimentinand insulin, glucagon, somatostatin, respectively). Oxidized-low density lipoprotein incorporation assaysconfirmed the functional maturity of iPSC-EC. We incorporated highly differentiated iPSC-EC at the endof the 7-step endocrine differentiation. Kinetic monitoring of the morphology of the organoids showedaggregates with endothelial characteristics (migration, assembly). Two distinct cell populations wereobserved in the vascularized condition, with larger and smaller clusters. No changes in endocrine geneexpression were observed at 1 and 2 weeks, but the expression of endothelial markers declined progressivelyto low levels after 2 weeks of coculture. Dynamic glucose-stimulated insulin secretion studiesshowed a similar secretory profile (stimulation index: vascularized 2.53 vs control 2.54) but reduced insulincontent in the vascularized condition compared with control.

Conclusion: We have succeeded in generating functional EC and islets from the same iPSC line and established coculture that durably preserves insulin secretion. To counteract the gradual disappearance of EC, we will establish a new culture method involving the progressive mixing of endocrine and endothelial media to improve EC survival. The human “vascularized” islet organoids will be tested in a preclinical in vivo model by transplantation into mice. Ultimately, these strategies will advance iPSC-islet replacement therapies in type 1 diabetes.

Keywords: iPSC, βcells, endothelial cells, vascularized organoids, large scale.