Human endocrine cell differentiation and islet morphogenesis play critical roles in determining islet cell mass and function, but the events and timeline of these processes are incompletely defined. To better understand early human islet cell development and maturation, we collected 123 pediatric pancreata and mapped morphological and spatiotemporal changes from birth through the first ten years of life. Using quantitative analyses and a combination of complementary tissue imaging approaches, including confocal microscopy and whole-slide multiplex imaging, we developed an integrated model for endocrine cell formation and islet architecture, including endocrine cell type heterogeneity and abundance, endocrine cell proliferation, and islet vascularization and innervation. We also assessed insulin and glucagon secretory profiles in isolated islet preparations from pediatric donors aged 2 months to 10 years and found a temporal difference in the maturation of insulin secretion compared to glucagon secretion. This comprehensive summary of postnatal and pediatric pancreatic islet development provides a framework for future studies and for integrating emerging genetic and genomic data related to islet biology and diabetes risk.

Introduction and Objective: Pancreatic islet function is closely related to cell-cell communication and paracrine signaling, suggesting that the spatial arrangement of endocrine cells could play a critical role in islet physiology. However, quantitative analyses linking islet spatial organization to functional outcomes are limited. This study systematically characterized the distribution and spatial arrangement of endocrine cells and their relationship to islet function. Methods: We analyzed the phenotyping data of isolated human islets from 578 organ donors without diabetes obtained through Integrated Islet Distribution Program (IIDP) and Human Pancreas Analysis Program (HPAP), representing different age, sex, BMI, and race/ethnicity. Immunofluorescence images of islet histological sections were processed with machine learning-based pipelines to annotate cell types and extract spatial organization features from 54,974 islets (diameter ≥ 50 μm). Statistical modeling and graph-based deep learning identified associations between cell organization and secretory function. Results: Endocrine cell organization was non-random, with significant enrichment of homotypic (i.e., α-α, β-β, and δ-δ cell) contacts (p<10-100). The enrichment level varied substantially between donors (p<10-100). Deep learning models demonstrated that the spatial arrangement of α, β, and δ cells provided predictive power for hormone secretion beyond endocrine cell composition alone. Despite being the smallest of the three endocrine cell types, δ cells showed the strongest associations with stimulated insulin secretion. Both higher δ cell number (p<0.001) and larger per-δ-cell cytoplasmic area (p<0.01) correlated with reduced insulin secretory responses. Conclusion: The spatial organization of endocrine cells in human islets exhibits substantial inter-donor heterogeneity yet follows non-random patterns. Neighborhood relationships among endocrine cells, particularly δ cells, are key determinants of human islet function in vitro. Disclosure F. Feng: None. T. Bate: None. A.L. Hopkirk: None. D.C. Saunders: None. S. Mei: None. A. Coldren: None. J.R. Taylor: None. H. Durai: None. C. Davis: None. C. Reihsmann: None. A. Eskaros: None. R. Jenkins: None. A.C. Powers: None. J.C. Niland: None. C. Evans-Molina: Advisory Panel; Ended; Sanofi. Speaker's Bureau; Ended; Sanofi. Other - Shared grant and project; Current; Neurodon. Advisory Panel; Current; Diogenyx. M. Brissova: None.

Phenotyping and genotyping initiatives within the Integrated Islet Distribution Program (IIDP), the largest source of human islets for research in the U.S., provide standardized assessment of islet preparations distributed to researchers and enable the integration of multiple data types. Data from islets of the first 299 organ donors without diabetes analyzed using this pipeline highlights substantial heterogeneity in islet cell composition associated with hormone secretory traits, sex, reported race and ethnicity, genetically predicted ancestry, and genetic risk for type 2 diabetes (T2D). While α and β cell composition influenced insulin and glucagon secretory traits, the abundance of δ cells showed the strongest association with insulin secretion and was also associated with the genetic risk score (GRS) for T2D. These findings have important implications for understanding mechanisms underlying diabetes heterogeneity and islet dysfunction and may provide insight into strategies for personalized medicine and β cell replacement therapy.

Human endocrine cell differentiation and islet morphogenesis play critical roles in determining islet cell mass and function, but the events and timeline of these processes are incompletely defined. To better understand early human islet cell development and maturation, we collected 115 pediatric pancreata and mapped morphological and spatiotemporal changes from birth through the first ten years of life. Using quantitative analyses and a combination of complementary tissue imaging approaches, including confocal microscopy and whole-slide imaging, we developed an integrated model for endocrine cell formation and islet architecture, including endocrine cell type heterogeneity and abundance, endocrine cell proliferation, and islet vascularization and innervation. We also assessed insulin and glucagon secretory profiles in isolated islet preparations from pediatric donors aged 2 months to 10 years and found a temporal difference in the maturation of insulin secretion compared to glucagon secretion. This comprehensive summary of postnatal and pediatric pancreatic islet development provides a framework for future studies and integration of emerging genetic and genomic data related to islet biology and diabetes risk.

In type 1 diabetes (T1D), α cell function is dysregulated with loss of glucagon secretion in response to hypoglycemia that precedes a secretory impairment of major α cell stimulus, epinephrine, produced by adrenal chromaffin cells. Recurrent hypoglycemic episodes, which correlate with progressive β cell loss in T1D, lead to an impairment of sympathoadrenal responses and the risk of a life-threatening hypoglycemia unawareness syndrome. To discover new molecules that activate signaling pathways in human α cells and promote glucagon secretion during hypoglycemia, we used our primary human pseudoislet system for screening of 1027 FDA-approved compounds from ion channel and GPCR compound library (APExBIO). The compound screen was optimized for pseudoislet formation in 96-well format including state-of-the-art liquid-handling platforms for compound addition and analysis of glucagon secretion from a single pseudoislet in the presence of carefully selected internal standards. Using this approach, we identified several ‘hit’ compounds targeting both ion channels and GPCRs that increased glucagon secretion above positive controls including 1.7 mM glucose + 20 mM arginine. Notably, nearly 30 compounds targeting GPCRs including opioid, oxytocin, histamine H1, dopamine, 5-HT, adrenergic, and muscarinic receptors elicited robust glucagon responses across multiple human islet donors (N=3). Additionally, we leveraged our single cell and bulk RNA-seq datasets to evaluate target specificity and facilitate the compound selection process based on target expression changes in T1D α cells. Overall, the primary human pseudoislet system is ideal for the screen because it maintains 3-D islet cell arrangement, islet microenvironment, and allows for generation of α cell-enriched, T1D-like pseudoislets as a model system for further compound validation. This could lead to repurposing of FDA-approved drugs for treatment of hypoglycemia and rapid translation for clinical testing. Disclosure T.S.R. Bate: None. C. Reihsmann: None. R. Aramandla: None. C. Davis: None. S. Mei: None. A. Bradley: None. J. Bauer: None. S. Parker: Research Support; Pfizer Inc. D.C. Saunders: None. A.C. Powers: None. M. Brissova: None.

Introduction & Objective: Islet amyloid composed of islet amyloid polypeptide (IAPP) is often present in the pancreas in type 2 diabetes. Cell culture and transgenic rodent models have been used to study islet amyloid toxicity, but less is known about the effects of endogenously-produced IAPP on human islet function and health. Methods: To evaluate acute and chronic effects of amyloid formation, we established a pseudoislet platform with adenoviral delivery of constructs expressing human IAPP (huIAPP) or mouse IAPP (moIAPP), which is not amyloidogenic. After 7 days in culture +/- exposure to the fatty acid palmitate, pseudoislets were evaluated for secretory function, islet structure and amyloid formation by ThioS staining. Pseudoislets were also transplanted into the anterior chamber of the eye of immunodeficient mice for assessment of secretory function for up to 3 months. Results: After 7 days in culture, transduced pseudoislets had 6.3 fold greater cellular IAPP content than untransduced pseudoislets. HuIAPP expression resulted in greater amyloid formation (0.49 ± 0.12 vs 0.17 ± 0.07 % islet area), but no changes in insulin or glucagon secretion or cellular toxicity, compared with moIAPP pseudoislets. High resolution microscopy showed ThioS+ fibrils in the ER of huIAPP-expressing beta cells. No extracellular amyloid was seen. Exposure to 0.5mM palmitate for 3 days further increased amyloid deposition and impaired insulin secretion by huIAPP pseudoislets, but not moIAPP. HuIAPP pseudoislets transplanted into immunodeficient mice had normal insulin secretion after 4 and 8 weeks, but impaired secretion after 12 weeks, compared to moIAPP pseudoislets. Conclusion: In vitro overexpression of IAPP in human pseudoislets induces intracellular amyloid formation, and in combination with lipotoxic stress, impairs insulin secretion. In vivo, human IAPP overexpression impairs insulin secretion after three months. This model will be valuable to elucidate mechanisms of islet dysfunction in type 2 diabetes. Disclosure J.J. Wright: None. P.B. Jackson: None. R. Aramandla: None. C. Davis: None. M. Brissova: None. A.C. Powers: None. Funding National Institutes of Health (K08DK133691); Veterans Administration (IK2BX005910)

Pancreatic islet endocrine cell phenotype and function are tightly regulated by transcription factor (TF) regulatory networks. In a mouse model of pancreas organogenesis, the key islet-enriched TF NKX2.2 governs both β and α cell specification. Further, loss of NKX2.2 in adult mouse β cells results in glucose intolerance, reduced insulin content, and polyhormonal β cells, demonstrating its importance in the maintenance of β cell phenotype. To understand the undefined role of NKX2.2 in adult human islets, we utilized adenoviral delivery of CRISPR and shRNA constructs in our primary human pseudoislet system to knockout (gNKX2-2) or knockdown (shNKX2-2) NKX2-2 in islet cells, respectively (n=4-6 donors without diabetes). Immunofluorescence analyses of gNKX2-2 pseudoislets demonstrated loss of NKX2.2 signal across β (81 ± 2.0%; p<0.001), α (58 ± 5.5%; p=0.001), and δ cells (83 ± 2.2%; p<0.001) compared to scrambled controls (gSCR). Surprisingly, functional assessment by dynamic perifusion revealed increased insulin secretion in gNKX2-2 versus gSCR pseudoislets measured as area under the curve in response to 16.7 mM glucose (6.64 ± 1.53 vs. 3.55 ± 1.53 ng/100 islet equivalents (IEQs); p=0.007) and cAMP-evoked potentiation with 16.7 mM glucose + 100 μM isobutylmethylxanthine (IBMX) (5.43 ± 0.50 vs. 3.15 ± 0.59 ng/100 IEQs p=0.003) without changes in total insulin content (gNKX2-2 vs. gSCR: 3.64 ± 0.75 vs. 4.78 ± 0.87 ng/IEQ, p=0.45). Furthermore, these findings were recapitulated in shNKX2-2 versus shSCR human pseudoislets (16.7 mM glucose: 10.17 ± 2.46 vs. 6.50 ± 2.51 ng/100 IEQs, p=0.02; 16.7 mM glucose + IBMX: 8.43 ± 1.31 vs. 4.21 ± 1.33 ng/100 IEQs, p=0.0002; insulin content: 6.18 ± 1.05 vs. 7.53 ± 1.26 ng/IEQ, p=0.36), where we observed a reduction in NKX2.2hi β (65 ± 10%; p=0.008), α (52 ± 5.5%; p=0.003), and δ cells (83 ± 4.6%; p=0.003). Collectively, our data indicate a critical role of NKX2-2 in the maintenance of primary adult human β cell function and suggest differential roles in islet function across species. Disclosure Y.D.Pettway: None. S.Parker: Research Support; Pfizer Inc. A.C.Powers: None. M.Brissova: None. J.Walker: None. C.Dai: None. R.Aramandla: None. A.L.Hopkirk: None. C.Reihsmann: None. C.Davis: None. R.Jenkins: None. L.Sussel: None.

The Integrated Islet Distribution Program (IIDP) is currently the largest source of human islets for research in the U.S. The IIDP Human Islet Phenotyping Program (HIPP) provides standardized islet assessment and makes these data available to researchers. Since 2016, HIPP has phenotyped 337 islet samples from nondiabetic organ donors (40%F / 60%M, 45±12 years, BMI 29.0±5.4; 52% European, 26% Latino, 8% African, 3% Asian, and 11% other ancestry) . To define the islet structure-function relationships, we integrated physiological profiling of hormone secretion with islet cell composition measured by immunohistochemistry­. We found that % β cells varied significantly (range 34 - 92%; CV 21%) and had a strong negative correlation with % α (range 3 - 59%; CV 33%; r -0.95; p<1.78E-163) and % δ cells (range 1 - 19%; CV 44%; r -0.32, p=2.69E-09) . This variation did not impact basal insulin secretion at 5.6 mM glucose, but insulin secretory responses stimulated by high 16.7 mM glucose, cAMP-evoked potentiation, and KCl-mediated depolarization, were all negatively correlated with % α and δ cells. By contrast, glucagon response to basal 5.6 mM glucose had the strongest negative correlation with % β cells (r -0.31, p=4.25E-09) , but not % δ cells (r 0.03, p=0.63) . Furthermore, the β cell composition did not correlate with age or BMI, but was higher in males vs. females (59.1±12.2% vs. 55.7±11.0%, p=0.0107) and in islets from donors of Asian ancestry (71.2±14.7%, p<0.01) . Finally, we noted that insulin response to KCl-mediated depolarization was significantly lower in islets from donors of African vs. European or Asian ancestry (8.3±4.0 vs. 16.5±8.8 and 13.0±7.0ng/IEQ; p<0.01) . Thus, these data highlight the importance of endocrine cell composition in islet hormone secretion possibly influencing β-α cell paracrine interactions and setpoint of glucagon secretion in response to low glucose. In addition, they suggest that β cell composition and insulin secretion are influenced by sex and ancestry. Disclosure M. Brissova: None. G. Poffenberger: None. D. C. Saunders: None. A. C. Powers: None. J. C. Niland: None. C. Evans-molina: Advisory Panel; Avotres Inc., DiogenX, Isla Technologies, MaiCell Therapeutics, Provention Bio, Inc., Other Relationship; Astellas Pharma Inc., Dompé, Lilly, Research Support; BMS, Nimbus Therapeutics. H. H. Durai: None. S. Mei: None. A. Coldren: None. C. Davis: None. C. Reihsmann: None. A. L. Hopkirk: None. D. Gibson: None. R. Aramandla: None.

ATP release from β cell secretory vesicles during insulin exocytosis is postulated to modulate islet hormone secretion via purinergic signaling in an autocrine or paracrine manner. Ecto-enzymes, including a family of ectonucleotidases (NTPDase) , control the concentration of extracellular nucleotides. One member of this family, ectonucleotidase triphosphate diphosphohydrolase 3 (NTPDase3) , is specifically expressed in human β cells during postnatal maturation, and its enzymatic activity is present in isolated adult human islets. The role of NTPDase3 in islet hormone secretion is not understood. We hypothesized that NTPDase3 regulates islet hormone secretion by modulating local extracellular nucleotide levels that signal through purinergic receptors expressed by islet cells. To define the cellular distribution of purinergic ecto-enzymes and nucleotide receptors in human islets, we used single-cell RNA sequencing (scRNA-seq) data. In addition to β cell NTPDase3 expression, ecto-enzymes ENPP1, and ENPP2 were abundant in α and ε cells while ecto-5'-NT and ADA were expressed in endothelial and stellate cells. These ecto-enzymes can further hydrolyze β cell-derived ATP to ADP, AMP, and adenosine which all bind purinergic receptors. We found that the ATP receptor, P2RY1, was enriched in β cells, and an adenosine receptor, ADORA2A, was expressed in α cells by both bulk and scRNA-seq. To study the function of this purinergic niche, we further assessed extracellular ATP levels which increased concomitantly (4.2E-12 vs. 4.3E-13 moles/100 IEQ/hour; n=9; p=0.03) with insulin secretion (21 vs. 60 ng/100 IEQ/hour; n=9; p=0.02) upon stimulation with 16.7 mM glucose + IBMX. To directly test NTPDase3 loss of function, we genetically manipulated human pseudoislets using an shRNA knockdown approach, resulting in a 1.7-fold increase of insulin secretion (AUC=3 vs. 5 ng/100 IEQs; n=3) . Overall, these studies provide an insight into how extracellular nucleotides and purinergic signaling mechanisms fine-tune human islet hormone secretion. Disclosure T. M. Richardson: None. D. C. Saunders: None. K. C. Coate: None. S. C. Robson: Advisory Panel; eGenesis, Consultant; Puretech, Synlogic, Research Support; Tizona Inc, Stock/Shareholder; EPurines, Purinomia. A. C. Powers: None. M. Brissova: None. C. Dai: None. R. Aramandla: None. H. H. Durai: None. C. Reihsmann: None. C. Davis: None. S. Mei: None. D. Gibson: None. S. Shrestha: None.