Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Synaptotagmin I is a synaptic vesicle-associated protein essential for synchronous neurotransmission. We investigated its impact on the intracellular Ca 2؉ -dependence of large dense-core vesicle (LDCV) exocytosis by combining Ca 2؉ -uncaging and membrane capacitance measurements in adrenal slices from mouse synaptotagmin I null mutants. Synaptotagmin I-deficient chromaffin cells displayed prolonged exocytic delays and slow, yet Ca 2؉ -dependent fusion rates, resulting in strongly reduced LDCV release in response to short depolarizations. Vesicle recruitment, the shape of individual amperometric events, and endocytosis appeared unaffected. These findings demonstrate that synaptotagmin I is required for rapid, highly Ca 2؉ -sensitive LDCV exocytosis and indicate that it regulates the equilibrium between a slowly releasable and a readily releasable state of the fusion machinery. Alternatively, synaptotagmin I could function as calcium sensor for the readily releasable pool, leading to the destabilization of the pool in its absence.T he release of neurotransmitters from nerve terminals and hormones from neuroendocrine cells occurs through exocytosis of secretory vesicles in response to increases in the intracellular Ca 2ϩ concentration [Ca 2ϩ ] i (1). The supralinear Ca 2ϩ dependence of neurosecretion suggests that the binding of at least 3-5 Ca 2ϩ ions to Ca 2ϩ -sensing entities on the fusion machinery is required to trigger the rapid fusion of secretory vesicle with the plasma membrane (2-6). At present, the exact mechanism of Ca 2ϩ -dependent exocytosis and the molecular identity of the involved Ca 2ϩ sensor(s) remain matters of debate. Numerous studies indicate that the synaptic vesicle protein synaptotagmin I, a brain-enriched member of the synaptotagmin family, plays a key role in Ca 2ϩ -dependent neurosecretion. Synaptotagmin I has been described to interact with several synaptic proteins including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin (7) and SNAP-25 (8), the assembled SNARE complex (9-11), and the clathrin assembly protein complex AP-2 (12). Through the first of its two C 2 domains (C 2 A), synaptotagmin I binds Ca 2ϩ and rapidly interacts with phospolipid membranes in a Ca 2ϩ -dependent manner (9, 13). Functional evidence has been presented that implicate synaptotagmin I in vesicle docking (14), fusion (15)(16)(17)(18)(19)(20), and recycling (21). Most strikingly, gene mutation studies in mice (20,22) demonstrated that synaptotagmin I is specifically required for rapid synchronous neurotransmission but not for asynchronous or Ca 2ϩ -independent release (i.e., spontaneous release and release triggered by hypertonic solutions or ␣-latrotoxin). This finding led to the hypothesis that synaptotagmin I is the major Ca sensor, whereas the kinetics of exocytosis were simultaneously determined by using high time-resolution membrane capacitance (C m ) measurements. Using these methods, we demonstrate that synaptotagmin I-deficient chromaffin cells d...

Ca2؉ elevations in Chinese hamster ovary cells stably expressing OX 1 receptors were measured using fluorescent Ca 2؉ indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca 2؉ elevations with an EC 50 around 1 nM. When the extracellular [Ca 2؉ ] was reduced to a submicromolar concentration, the EC 50 was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mM external Ca 2؉ was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.The recently described hypothalamic peptides called orexins (1) or hypocretins (2) mediate their effects through G proteincoupled receptors called OX 1 and OX 2 receptors (1). The peptides and their receptors are widespread in the hypothalamus, cortex, and brainstem (2-5). The orexin/hypocretin peptides are encoded by a single mRNA giving rise to a 33-residue orexin-A peptide containing disulfide bridges and a linear 28-residue orexin-B (1). Orexin-A has a 10 -100-fold higher affinity and potency for OX 1 receptor as compared with orexin-B, whereas no preference is displayed by the OX 2 receptor (1). The orexins cause robust increases in intracellular Ca 2ϩ both in neurons cultured from rat medial and lateral hypothalamus (6) and spinal cord (7), and when studied using recombinant receptors (1). This has led to the suggestion that the receptors are coupled to the G q family G proteins. Interestingly, the response in neurons is partially dependent on extracellular Ca 2ϩ , which may suggest that the receptors are connected to a Ca 2ϩ influx pathway in neurons (6). Several different pathways for receptor-stimulated Ca 2ϩ entry have been suggested based on functional studies with other G protein-coupled receptors. Suggested pathways include store-operated Ca 2ϩ channels, second messenger-operated channels, as well as Ca 2ϩ -activated Ca 2ϩ channels (reviewed in Refs. 8 and 9). The aim of this study was to examine in detail the Ca 2ϩ mobilizing actions of orexins on recombinant OX 1 receptors expressed in CHO 1 -K1 cells. The results reveal the presence of a novel amplification mechanism at the level of phospholipase C that is dependent on activation of Ca 2ϩ influx pathway upstream of phospholipase C. EXPERIMENTAL PROCEDURESCell Cultures-To prepare the CHO-hOX 1 -C1 cells used in this study CHO-K1 cells were transfected with a bicistronic vector containing the coding sequence of human OX 1 receptor as described previously for chemokine receptors (10). Neomycin resistant clones were then isolated by limited dilution. They were grown in nutrient mixture (Ham's F-12) medium (Life Technologies, Inc., Paisley, United Kingdom) supplemented with 100 units/ml penicillin G (Sigma), 80 units/ml streptomycin (Sigma), 400 g/ml Geneticin (G418; Life Technologies, Inc.) and 10% (v/v) fetal calf serum (Life Te...