ObjectivesReadily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new... Show moreObjectivesReadily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-βH5 cells, the latest in the EndoC-βH cell family.MethodsEndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed transcriptome, immunological and extensive functional assays.ResultsReady to use EndoC-βH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-βH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-βH5 cells elicit specific A2-alloreactive CD8 T cell activation.ConclusionsEndoC-βH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models. Show less
Noordstra, I.; Berg, C.M. van den; Boot, F.W.J.; Katrukha, E.A.; Yu, K.L.; Tas, R.P.; ... ; Akhmanova, A. 2022
Insulin secretion in pancreatic beta-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these... Show moreInsulin secretion in pancreatic beta-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5 beta (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5 beta or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchangewere stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release. Show less
