Maike Sander - US grants

DESCRIPTION (provided by applicant): Diabetes mellitus results from loss or dysfunction of the insulin-producing beta-cells in the pancreas. Despite refined insulin injection regimens, diabetic patients suffer from long-term complications, such as blindness and kidney failure. An ultimate cure for diabetes could be achieved through the generation of replacement insulin-producing cells. To develop these replacement cells, we need to identify the molecular pathways that initiate beta-cell formation and insulin-production. Using genetically engineered mouse models, this proposal examines the role of NKX6 class transcription factors in beta-cell differentiation and function. The specific hypothesis is that different NKX6 transcription factors partially compensate for each other's function in pancreatic cell differentiation. This hypothesis is based on the observation that beta-cell numbers are diminished in Nkx6.1 mutant mice, while Nkx6.1/Nkx6.2 double mutant mice show a reduction in both insulin producing beta- and glucagon-producing alpha-cells. Experiments are proposed to dissect the role of NKX6 factors in pancreatic endocrine development using compound mouse mutants for Nkx6 genes. Aim 1 is to define the role of NKX6 factors in the beta-cell differentiation pathway by attempting to restore beta-cell development in Nkx6.1 mutant mice with different transgenes. Aim 2 examines in mice if pancreatic progenitors are reverted into alternate cellular fates in the absence of NKX6 activity. Aim 3 focuses on the role of NKX6 factors in adult beta-cell function. Using selective inactivation of Nkx6 genes in beta-cells, it will be studied if NKX6 factors control aspects of beta-cell function, such as insulin synthesis or insulin secretion.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to identify and characterize putative stem/progenitor cells in the adult pancreas. The identification of such cells would facilitate the development of a cell replacement therapy for patients with diabetes. Such therapy, though highly effective, is currently limited by the shortage of transplantable islets from cadaver tissue. Adult progenitors would be a particularly attractive source for the differentiation of replacement insulin-producing beta-cells, as they could possibly be isolated from the patient's own pancreas, thereby avoiding the immune response associated with the transplantation of foreign tissue. It is, however, still unclear whether a stem or progenitor cell population resides in the pancreas beyond the embryonic period. In preliminary studies, we have identified the transcription factor SOX9 as a marker for progenitor cells in the embryonic pancreas and found that Sox9 is essential for their expansion and maintenance. Strikingly, its expression persists in the adult pancreas exclusively in a subset of ductal cells;a cell type that is regarded as a potential reservoir of pancreatic stem/progenitor cells. Given the crucial role of SOX9 in maintaining undifferentiated, pluripotent progenitors of the embryonic pancreas, we hypothesize that this factor also marks and maintains a stem cell compartment in the adult pancreas. Experiments are proposed to test whether SOX9 plays a role in pancreas regeneration and whether the cells marked by SOX9 in adult pancreas can function as pluripotent pancreas progenitor cells. Using inducible gene ablation, Aim 1 is to define the role of Sox9 in pancreatic cell differentiation and maintenance throughout development and adulthood. Aim 2 examines if Sox9 is required for pancreas regeneration after partial pancreatectomy or STZ treatment. Aim 3 will define whether Sox9-expressing cells have characteristics and properties of multipotential stem/progenitor cells. This will be tested by transcriptional profiling of isolated cells and by tracking their fate in cell transplantation experiments.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop strategies for deriving glucose-responsive insulin-producing (-cells from human embryonic stem (hES) cells or patient-derived induced pluripotent stem (iPS) cells. With this objective our proposal will advance one of the focus areas of the NIH-NIDDK Beta Cell Biology Consortium, which is to use cues from pancreatic development to directly differentiate (-cells from stem/progenitor cells for use in cell-replacement therapies for diabetes. To achieve our objectives we have assembled a consortium of five investigators, which includes experts in (-cell and stem cell biology as well as genomics. By genome-wide mapping of key histone modifications in a variety of primary embryonic and adult human cells and tissues, we will define epigenetic signatures that define pancreatic progenitors and their endocrine descendants. This knowledge will be used to guide efforts for improving preexisting in vitro differentiation protocols of hES cells into pancreatic progenitors and eventually glucose-responsive insulin-producing p-cells. Key to our proposal is a novel cellular microarray technology that allows for combinatorial screening of extracellular matrix components, factors and/or molecular pathways for their ability to support efficient generation of each intermediary precursor along the step-wise differentiation path from hES cell to mature (-cell. The epigenetic signatures will be used as endpoints to assess how closely the in vitro-generated, hES cell-derived cells resemble their in vivo pancreatic counterparts. hES cell-derived (-cells will eventually be tested for their ability to correct elevated blood glucose levels upon transplantation into diabetic mouse models.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our lab has recently identified Sox9 as a factor that exclusively marks the stem/progenitor cell compartment in the embryonic pancreas. In this proposal, I will test how Sox9 alters the properties of a cell to ensure that the cell maintains progenitor cell characteristics. By comparing normal progenitors to Sox9-deficient progenitors, I have already determined which genes are activated by Sox9 in the cell. I found that many of these genes are important for allowing cells to make specific connections with other cells, thus ensuring communication between cells. Using time-specific gene inactivation in mice, I here propose to test how inactivation of Sox9 in multipotential pancreas progenitors affects the ability of progenitors to give rise to beta-cells. I will then test whether Sox9 controls beta-cell formation by allowing cells to make appropriate contacts with their neighbors.

DESCRIPTION (provided by applicant): Maintenance of glucose homeostasis is central to our health and its failure results in diabetes mellitus. Despite current treatment regimens of several insulin injections per day, blood glucose levels still fluctuate significantly in diabetic patients, making diabetes the sixth leading cause of death in the United States. Alternative approaches to insulin injections include attempts to develop a cell therapy for diabetes by producing insulin- secreting ?-cells from human embryonic stem cells (hESCs) cells or by reprogramming other cell types into ?- cells. However, despite some success to differentiate hESCs into insulin-expressing cells, these cells express multiple hormones and are not capable of reversing diabetes. The main bottleneck for generating true functional ?-cells from other cell sources is the paucity of knowledge of how ?-cells are specified. We observed that conditional, stable activation of the transcription factor Nkx6.1 in endocrine progenitors introduces a ?-cell fate bias and disfavors differentiation into non-?-cell islet cell types. Moreover, we found that Nkx6.1 is required to maintain expression of ?-cell-specific programs of gene expression in adult mice. Research under this proposal will (a) define the molecular cues that specify ?-cells and repress alternative endocrine cell fates, (b) directly test whether the Nkx6.1 can reprogram non-?-cell islet cells into ?-cells in vivo and (c) genetically dissect the gene regulatory network controlled by Nkx6.1 in the regulation of adult ?-cell fate maintenance, proliferation and function. Knowledge gained from these studies will help devise strategies to resolve mixed endocrine lineage patterns in hESC-derived endocrine cells and to reprogram other cell types into fully functional ?-cells.

The overall objective of our Enrichment Program is to create linkages, collaborations and interactions among the DRC members at our five institutions and to foster the next generation of diabetes research leaders. The Program is responsible for and implements DRC communications, academic enrichment, and planning and organization for DRC activities. The Enrichment Program also provides materials for the Website and utilizes the listserv, integrating with the Administrative Core and the Executive Committee. The Program is run by a Seminar, Meeting, and Retreat Committee (SMRC) that is responsible for organizing our Annual DRC Day retreats and organizing the P&F Symposium. The SMRC is also responsible for enhancing the integration of our five institutions with other centers, institutes, and programs. The committee plans and organizes seminars, research symposia and lectures, communicates with the Administrative Core's Information Manager to coordinate announcements to the greater diabetes community via the listserv, and provides information for the website about events and activities. They also advance, coordinate and enhance the training programs and integrate them with the DRC activities, fostering the next generation of diabetes researchers.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to define the mechanisms that underlie the formation of endocrine cells in the pancreas and to apply this knowledge to instruct human embryonic stem cells (hESCs) to produce functional insulin-secreting beta cells. During the past funding period, work under this grant has determined that ductal progenitors in the pancreas are the major source of endocrine cells during embryonic development. Mechanistic studies in the PI's laboratory have further shown that the transcription factor Sox9 is necessary to bestow competence upon ductal progenitors to initiate endocrine gene expression programs. Moreover, we have demonstrated that Sox9 expression is under control of the Fgf and Notch signaling pathways, suggesting that progenitors need to be exposed to Fgf and Notch signals for endocrine cell differentiation to be initiated. In preliminary studies presented to support a continuation of these studies, we show that Fgf and Notch signaling are aberrantly regulated in current differentiation protocols of hESCs towards pancreatic endocrine beta cells. We hypothesize that the aberrant Fgf and Notch signaling environment accounts for the malfunction of beta- like cells produced in vitro. In this continuing renewal application, the PI proposes a combination of mouse genetic and hESC-based approaches to (a.) further define the mechanisms by which Fgf and Notch signaling orchestrate endocrine cell development and (b.) apply this knowledge to generate functional endocrine cells from hESCs in vitro. To better understand the specific signaling environment necessary for endocrine cell differentiation, Aim 1 will investigate how the Fgf and Notch signaling pathways coordinately control the specification and differentiation of endocrine cells. To aid these experiments, the PI's laboratory has developed unique genetic mouse models. In Aim 2, we will employ a novel live imaging technology established in the PI's laboratory to monitor the initiation of endocrine cell differentiation at single cell resolution in real time. Based on evidence in othe tissues and organs, experiments under this aim will explore a possible connection between cell division, Notch activity, and the initiation of cell differentiation. In Aim 3, we will apply paradgms learned from our mouse genetic experiments to direct hESCs towards the beta cell lineage. Experiments under this Aim will directly test how changes in Fgf and Notch signaling affect the maturity of endocrine cells produced from hESCs in vitro. Preliminary evidence from the PI's laboratory suggests that endocrine cell maturity can be improved by providing a Fgf and Notch signaling environment that more closely resembles the environment during normal development. Together, these experiments will aid the identification of culture conditions that support the differentiation of functional beta cells from hESCs in vitro.

? DESCRIPTION (provided by applicant): An ideal system for identifying disease mechanisms of diabetes and screening for new therapeutics would be a renewable source of beta cells and the ability to study patient-specific cells. Such a system could help identify beta cell-intrinsic mechanisms of cell death in type I diabetes and help establish genotype-phenotype correlations. 2D cell culture systems have been the mainstay of attempts to culture human cadaveric islets or to differentiate human pluripotent stem cells (hPSCs) toward the pancreatic beta cell fate. However, human islets cannot be maintained for prolonged periods of time with these systems, nor can functional beta cells be produced from hPSCs. Since current 2D culture conditions do not take into account critical cell-cell and cell- matrix interactions for beta cell development and function, there is a need for new 3D culture models of human islets that more accurately mimic the in vivo environment. Our multidisciplinary team of a stem cell/islet biologist a vascular biologist and two bioengineers proposes to develop a novel in vitro platform to create a human islet micro-organ perfused with human microvessels in a microfluidic device with all components derived from a single human induced pluripotent stem cell (hiPSC) source. First, we will optimize conditions and cell ratios by creating a 3D in vitro human islet micro-organ in static cultures outside the device that is comprised of islet endocrine cells, stromal cells, pancreas-specific extracellular matrix, and human endothelial cells (Aim 1). Next, we will assemble these 3D human islet micro-organs in a microfluidic device, so that nutrients are delivered and waste products are removed through a perfused capillary bed. This 3D islet micro- organ will closely mimic the dynamic metabolic changes typical for the in vivo beta cell environment (Aim 2). While a hiPSC-derived islet micro-organ is the ultimate goal, we will pursue a parallel approach with each Aim, using human cadaveric islets as a cell source, as experiments with primary human islets will provide important insight into the microenvironment necessary for maintaining mature beta cells ex vivo. Our model, which fully mimics in vivo physiology and is amenable to high throughput screening, will provide a platform for identifying regulators of beta cell maturation, replication, failure, and survival and will help reveal the causes of human diabetes. Our microfluidic platform has the flexibility to combine islet micro-organs with additional micro-organs (e.g. liver) in a continuous vascular network to simulate the complex inter-organ interactions relevant to human beta cell physiology. Thus, our platform will enable studies into the role of inter-organ cross talk in the pathogenesis of diabetes.

? DESCRIPTION (provided by applicant): The high mortality rate of pancreatic ductal adenocarcinoma (PDA) is mainly due to a lack of highly sensitive and specific tools to detect the disease at an early stage and, as such, tumors are typically diagnosed after metastasis. There is an urgent need to further improve our understanding of molecular events leading to PDA development and to identify strategies for diagnosing and treating PDA in its preinvasive state before metastasis occurs. Intraductal papillary mucinous neoplasias (IPMNs) are macroscopically identifiable, non- invasive, preneoplastic cystic lesions that are precursors to PDA. IPMNs as a group can be categorized into different histological subtypes that are associated with different survival outcomes. While knowledge of the IPMN subtype in patients could be of prognostic value, preoperative diagnostic subtyping of IPMNs is difficult. Identifying biomarkers for IPMN subtypes, such as unique gene mutations that can be detected in aspirates from pancreatic cysts or serum, would help distinguish cysts with low and high malignant potential. To further our understanding of IPMN biology and identify markers predictive of malignant potential, we have developed the first mouse model of IPMN that fully recapitulates the clinicopathological features of human IPMN formation and progression. Based on observations in human patients showing that dysregulation of the PI3K/PTEN signaling pathway is associated with IPMN-PDA, we employed a genetic strategy to inducibly ablate the duct- enriched tumor suppressor Pten specifically in pancreatic ductal cells in mice. These mice spontaneously form IPMN lesions of multiple histological subtypes. Interestingly, in a subset of mice that progress to invasive PDA, we detected spontaneous activating mutations of Kras in the associated IPMN lesions. These activating Kras mutations were specifically found in pancreatobiliary IPMNs (PB-IPMN), suggesting that combined loss of Pten and Kras activation drives initiation and/or progression of this IPMN subtype. To test this hypothesis, we will genetically determine if activation of Kras and loss of Pten synergize in mouse pancreatic ductal cells to drive the formation and/or progression of PB-IPMNs (Aim 1). Further, we propose to employ this IPMN mouse model to identify novel mutations involved in IPMN initiation and progression by performing exome sequencing on tissue samples from our mouse model (Aim 2). Finally, we will employ a human tissue microarray of pancreatic lesions to examine whether the same mutations are present in human IPMNs and associate with specific IPMN subtypes (Aim 2). Results from our proposed studies will not only identify candidate diagnostic biomarkers for pre-operatively assessing malignant potential of IPMNs, but may also reveal novel signaling pathways to target for drug discovery.

? DESCRIPTION (provided by applicant): More than 27 million Americans suffer from Type 2 diabetes (T2D). GWAS have identified 128 lead SNPs associated with T2D and/or fasting hyperglycemia, but little is known about how these variants contribute to T2D pathogenesis. A major challenge in functionally characterizing variants found in GWAS is that each lead SNP directly associated with a trait is in LD with a collection of additional variants, and thus identifying the precise variant(s) underlying the association requires extensive computational and experimental analyses. Additionally, the majority of the associated SNPs are located within non-coding regions, where inferring functional consequences of sequence variants remains challenging. Finally, when associated SNPs are identified as candidate regulatory variants, functional testing is frequently hampered by a lack of appropriate experimental models. To address these challenges we have assembled a team of highly accomplished researchers in genomics (Frazer), epigenomics (Ren) and T2D biology (Sander). We propose to combine state-of-the-field computational methods, high throughput molecular assays, and disease modeling in human embryonic stem cells to comprehensively annotates T2D GWAS data and test variants for their gene regulatory function. In Aim 1 we will analyze 5,150 whole-genomes for variants in T2D and fasting hyperglycemia GWAS risk-associated loci. We estimate that these analyses will identify ~1,000,000 variants with a MAF > 1% in the intervals of interest. Additionally, we will identify rare variants that are enriched in T2D patients. We estimate that intersecting these data with existing epigenomic datasets will identify ~99,000 variants in putative regulatory elements in T2D-relevant tissues. In Aim 2 we will use three high throughput molecular assays to characterize these 99,000 candidate regulatory variants in T2D-relevant cell types. First, we will carry out massively parallel reporter assays (MPRA) to test the potentia of each SNP-harboring sequence element to act as a transcriptional enhancer, and if so, whether enhancer activity is affected by the candidate variant. Second, we will carry out a high throughput in vitro binding assay (SELEX) to determine whether the candidate variants affect DNA binding of relevant transcription factors. Third, we will predict target genes of the candidate variants using high throughput chromosome conformation capture (Hi-C) assays. In Aim 3, results from Aim 1 and Aim 2 will be integrated to prioritize 20 beta cell-relevant SNPs for functional validation. Key criteria include: (1) the variant resides in an active beta cell enhance, (2) disrupts transcription factor binding, and (3) targets T2D-relevant genes in Hi-C assays. We will validate these variants by (1) genetic engineering of an embryonic stem cell-derived cell model of human beta cells, testing how deletion of the cis-regulatory element or introduction of the risk variant affects target gene expression; (2) genetic engineering of mouse models, testing whether candidate enhancer/target gene pairs control glucose metabolism in vivo. The proposed study will provide key insights into the underpinnings of regulatory variants identified through GWAS in T2D etiology.

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is a complex disease that affects 1.25 million individuals in the United States and is characterized by autoimmune destruction of insulin-producing beta cells in the pancreatic islets. T1D GWAS studies have identified 114 index SNPs mapping to 58 loci that influence disease risk, but how these loci contribute to T1D pathogenesis is largely unknown. A major challenge in functionally characterizing T1D loci is identifying the precise causal variant(s) underlying disease risk at each locus. As the majority of T1D-risk variants are non-coding, an additional challenge is inferring the target genes of T1D-risk signals. Finally, functional validation of causal T1D-risk variants requires the use of appropriate experimental models for the key pathogenic cell types. To address these challenges we have assembled a team of highly accomplished investigators in human genome sciences (Gaulton and Frazer), epigenomics and high-throughput molecular assays (Ren), as well as T1D biology and diabetes models of human pluripotent stem cells (hPSC) (Sander). We propose to combine state-of-the-field computational methods, high-throughput molecular assays, and hPSC-based cell models to comprehensively identify T1D causal variants, annotate these variants across T1D- relevant cell types, and define their regulatory functions and target genes. In Aim 1 we will utilize T1D GWAS data, epigenomic annotation and high-throughput transcription factor binding assays to fine-map known T1D loci and identify and fine-map novel loci. These studies will annotate variants across cell types and thus be a broadly informative resource for the T1D community for hypothesis generation of variant function. In preliminary studies we discovered that ~50% of independent T1D-risk signals map to regulatory elements active in islets or pancreatic progenitors, suggesting a possible role of these variants in beta cell and progenitor function. In Aim 2 we will utilize a collection of 100 whole-genome-sequenced hPSCs, including 24 hPSC lines from T1D patients, to derive pancreatic progenitors and then generate Hi-C chromatin conformation, RNA-seq, ATAC-seq, H3K27ac ChIP-seq, and DNA methylation profiles. We will utilize these data combined with similar available datasets from human islets to annotate molecular QTLs at T1D-risk variants, as well as to evaluate potential contributions of rare variants to disease etiology in the 24 T1D patients. These analyses will provide insight into how T1D-risk variants alter local (<10kb) chromatin states and gene regulation in islets and their progenitors. In Aim 3 we will further uncover variant/target gene relationships by integrating the genetic data from Aim 1 and the local QTL data from Aim 2 with 3D chromatin contact maps to identify T1D-risk variants in enhancers that are distal (>10kb) QTLs for promoter sites. To validate predicted T1D enhancer-target gene(s) interactions and potentially discover new target genes, we will prioritize ten enhancers with strong molecular phenotypes for functional analysis by CRISPR/Cas9-mediated deletion in hPSCs. The proposed studies will provide key insight into the molecular and cellular functions of T1D-risk variants identified through GWAS.

DESCRIPTION (provided by applicant): The epigenome, a term referring to the state of DNA methylation and histone modifications, plays a central role in regulating the functional output of the genome in response to environmental signals. Changes in the epigenome can determine future patterns of gene regulation, allowing cells to adapt cellular responses over time. Such adaptation is important during ?-cell development, when differentiation cues need to be read in a context-specific manner. Similarly, during adulthood, ?-cells need to adapt insulin secretion to lasting changes in the nutrient environment. How ?-cells and their precursors read environmental signals and translate these signals into context-specific responses is poorly understood. Preliminary unpublished evidence from our laboratory suggests that by modifying the epigenome, the histone demethylase LSD1 plays an important role in determining future cellular responses to environmental signals in the context of both ?-cell development and mature ?-cells. We have found that during ?-cell development, LSD1 removes activating epigenetic marks from early pancreatic enhancers, thereby limiting the duration during which retinoic acid (RA) can activate early pancreatic genes. Furthermore, we have obtained evidence that in ?-cells, LSD1 modifies the epigenetic state of enhancers linked to ?-cell metabolism genes, thereby modulating future insulin secretory responses. We hypothesize that LSD1 functions as an integration hub between the cell's environment and transcriptional output, and by regulating the epigenome, LSD1 determines how ?-cells and their precursors respond to environmental stimuli. To determine the mechanisms by which LSD1 regulates ?-cell differentiation during development and insulin secretion in mature ?-cells, we will employ state-of-the-art approaches, encompassing novel mouse models, a human embryonic stem cell (hESC)-based in vitro differentiation system of ?-cells, human islet experiments, genome-wide profiling of chromatin state and gene expression, and cutting-edge computational analysis. In Aim 1, we will determine how LSD1 controls ?-cell differentiation. To accomplish this, we will manipulate LSD1 activity and RA exposure in a hESC-based ?-cell differentiation system, and investigate the link between RA signaling, LSD1, chromatin, and ?-cell differentiation. In Aim 2, we will assess the role of LSD1 in adapting ?-cell insulin secretion to nutrient deprivation using mouse genetic models and human islets. Here, we will manipulate LSD1 activity and the nutrient environment and study how these manipulations affect insulin secretory responses, chromatin state, and ?-cell gene transcription. Finally, in Aim 3, we will exam ?-cell chromatin state in overnutrition models to determine the role of LSD1 in adaptation of ?-cells to chronically increased workload. By unveiling fundamental mechanisms by which environmental signals adapt cellular responses through modification of the epigenome, this proposal will prove critical for developing ?-cell programming strategies and for understanding how ?-cells respond to metabolic challenges, as in obesity and type 2 diabetes.

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is an autoimmune disease thought to be caused by immune-mediated destruction of the insulin-producing ?-cells in the pancreatic islets. Studying the mechanisms that underlie ?-cell destruction in humans with T1D has been challenging because most of the important immunological events occur before diagnosis. Furthermore, while rodent models have been informative in defining some aspects of T1D etiology, there are fundamental differences between the rodent and human pancreas with respect to islet architecture and vasculature, as well as between rodent and human immune systems. Additionally, important aspects of human T1D pathology are not replicated in the rodent models. Therefore, to fully understand human T1D pathophysiology, it is critical to develop a human model, where the interactions of all cells involved in the disease process (e.g. ?-cells, endothelial cells (EC), innate and adaptive immune cells) can be studied in the context of normal islet architecture, including vasculature, stromal cells, and native islet matrix. Over the past three years through the NIH ?Consortium on Human Islet Biomimetics?, our team (co-PI Sander: human induced pluripotent stem cells (hiPSC) and diabetes; co-PI Hughes: vascular biology and bioengineering; co-I Christman biomaterials and tissue engineering; co-I George microfluidics and transport) has developed a microfluidic-based platform in which primary human islets or hiPSC-derived islet-like clusters are supported by a network of perfused human microvessels. Our 3D vascularized islet micro-organ (VMO-I) platform allows for physiologic, microvessel-mediated delivery of nutrients, disease-relevant stimuli, or immune cells to the islets. We propose to leverage the unique features of our VMO-I platform to model the cell-cell interactions that occur in the islet niche during T1D pathogenesis, namely immune cell extravasation, tissue penetration, and migration as well as ?-cell killing. For these studies co-I Teyton will provide expertise in T1D immunology. We propose to employ two distinct in vitro models: The first, developed in the UG3 phase, is non-autologous and comprised of primary human islets and vasculature from primary EC. Here, we will introduce either allogeneic lymphocytes (Aim G1) or islet donor-matched ?-cell-reactive T cell clones (Aim G2) to establish parameters for modeling T cell extravasation and T cell-mediated ?-cell killing. We will also work towards the goal of generating a VMO-I model entirely derived from hiPSC (Aim G3). The second model, developed in the UH3 phase, will be fully autologous, comprising ?-cells, vasculature, and stromal cells derived from T1D patient hiPSC, which will be combined with autoreactive T cells isolated from blood of the same patient. By combining live-sensors and real-time imaging with molecular and biochemical assays, we will use these models to study how cells in the islet respond to T1D- relevant stressors, such as pro-inflammatory cytokines, hyperglycemia, and hypoxia, how immune cells and ?- cells interact, and how ?-cells are killed. Finally, we will demonstrate that the platform can be used to assess candidate therapies for efficacy with the long-term goal to utilize the platform to screen for new therapeutics.

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin-producing beta cells in pancreatic islets. In T1D the interplay between immune, endothelial, and endocrine cells in the islet niche leads to beta cell dysfunction and/or destruction; however, there is limited knowledge of the molecular blueprint that initiates and drives immune-mediated beta cell destruction. Recent single cell RNA-seq (scRNA-seq) profiling studies of human pancreas and islets from non-diabetic donors lack the resolution to characterize immune cells. Furthermore, T1D-related changes in the islet cell repertoire have not been comprehensively analyzed, and cell type-resolved epigenomic maps of gene regulatory elements remain to be generated for T1D-relevant cell types. When intersected with genetic variants from genome-wide T1D association studies, such maps could help pinpoint cells and genes with causal roles in T1D. To fill these knowledge gaps, we have assembled a team of highly accomplished researchers in islet biology and diabetes (Sander), genetics and genomics of diabetes (Gaulton) and functional genomics (Ren, UCSD Center for Epigenomics). The proposed project will apply novel single nuclei (sn) technologies to characterize the epigenomic (Aim 1) and transcriptomic (Aim 2) profiles of individual T1D-relevant cells in the pancreas of non-diabetic and T1D individuals. To enrich cell types most relevant for T1D pathogenesis (i.e. endocrine, immune and endothelial cells), we will deplete acinar cells from whole pancreas preparations. From these enriched cell preparations, we will generate maps of accessible chromatin (snATAC-seq) and gene expression (snRNA-seq). First, we will generate reference maps using fresh pancreatic tissue from non-diabetic donors, and then employ our recent adaptions of snATAC-seq and snRNA- seq technology to profile frozen, archived pancreata from non-diabetic, T1D antibody-positive, and T1D donors in the Network for Pancreatic Organ Donors with Diabetes (nPOD) biorepository. In Aim 3, we will integrate snATAC-seq and snRNA-seq data generated in Aims 1 and 2 with T1D genetic association data to identify pancreatic cell types and regulatory programs involved in T1D pathogenesis. This analysis will 1) define cell types and subtypes and their regulatory programs in the non-diabetic pancreas, 2) identify T1D-dependent changes in the existence, composition, regulation and inter-connectivity of pancreatic cell types, and 3) identify cells, networks and genes with likely causal roles in T1D by integrating snATAC-seq and snRNA-seq with T1D genetic association data. By generating reference maps of chromatin and gene expression in pancreatic cells from non-diabetic and T1D individuals, this proposal will identify resident immune and other cells that arise and change during T1D that can serve as novel biomarkers of disease and which will inform strategies for early intervention. Further integration with genetic data will reveal cells, networks and genes that are on the causal pathway to disease, which will inform therapeutic target discovery. Together our findings will provide novel insight into the pathogenic processes of cells in the pancreatic micro-environment that lead to beta cells loss in T1D.

PROJECT SUMMARY/ABSTRACT Recent years have brought rapid advances in our understanding of the molecular mechanisms involved in pancreatic development, regeneration, and malignant transformation, as well as our ability to model these processes in mice and recently also human cells. These studies have revealed lineage relationships between the different pancreatic cell types and plasticity in response to environmental influences. At the clinical level, epidemiological studies show a clear association between pancreatic diseases, in particular between diabetes and pancreatic cancer. Yet, the mechanisms that underlie the co-occurrence of pancreatic diseases are poorly understood. This relatively new GRC aims to link the understanding of the biology of the exocrine and endocrine pancreas to applications for human diseases. This GCR is unique in that it is the only conference that convenes experts from all aspects of pancreas biology as well as clinicians working on translational approaches for diabetes, pancreatic cancer and pancreatitis. By providing a forum for the presentation and discussion of cutting edge, unpublished concepts and approaches, the goal of this GRC is to stimulate discussion across fields and disciplines. This exchange will foster interdisciplinary approaches in the field and promote exchange of ideas and experiences to lead to new collaborations, as well as empower young scientists to showcase their work to peers and leaders in the field. Following the success of the previous conferences, the fourth GRC Pancreatic Diseases will take place at the Jordan Hotel, Sunday River, ME, USA from June 16 - 21, 2019. The conference will focus on understanding cell behavior and environmental influences in pancreatic diseases with the goal of gaining insights into how the systemic and local environment impacts the state of endocrine and exocrine cells in health and disease. The program that has been assembled is first-rate, benefitting from the contribution of international world-class scientists engaged in research relevant to different aspects of pancreatic diseases. The ultimate goal of the conference is the identification of novel concepts in pancreatic diseases that might be exploitable for pharmacological and cell-based therapeutic approaches. As for all GRCs, the guiding principle of this conference is the presentation of new, unpublished results and the free, unhampered discussion that follows. Participation of young scientists, including PhD students, postdocs and junior group leaders is especially emphasized, with opportunities of short talks and poster presentations.

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin-producing beta cells in pancreatic islets. While studies of T1D risk mechanisms have largely focused on immune cell function, recent evidence suggests the beta cells themselves actively contribute to the disease process. Beta cells are exposed to different environmental stimuli and stressors in the course of T1D development, such as pro-inflammatory cytokines and hyperglycemia which can contribute to beta cell stress and death. However, the extent to which T1D risk variants affect the beta cell epigenome and gene regulation in response to these external signals is unknown. To gain a deeper understanding of the variants, genes, and pathways that impact beta cell function and survival in T1D pathophysiology, it is critical to map changes in beta cell gene regulation the context of T1D-relevant immune and metabolic stressors. We have generated chromatin accessibility maps from primary pancreatic islet samples exposed to T1D-relevant cytokines and identified thousands of cytokine-responsive sites and transcription factors. Integrating these data with T1D genetic fine-mapping then revealed T1D risk variants with cytokine- dependent effects on islet chromatin accessibility. The proposed project will build on these findings in combining human genetics, islet epigenomics, and genome engineering to map T1D risk variants that affect beta cell chromatin upon in vitro exposure to multiple T1D-relevant stressors and identify target genes of stress-induced T1D variant effects that impact beta cell ER stress and survival. To accomplish this, in Aim 1 we will generate comprehensive maps of changes in beta cell chromatin accessibility and transcription factor binding upon exposure to multiple T1D-relevant stressors. Using these data, we will then fine-map T1D risk variants with stress-induced effects on beta cell chromatin using QTL mapping and validate their allelic effects using reporter assays. In Aim 2, we will identify target genes of stress-induced T1D variants by generating and analyzing changes in beta cell gene expression and 3D chromatin architecture upon exposure to the same stressors, and then validate target genes of stress-induced sites using a CRISPRi regulatory screen. Finally, in Aim 3 we will identify target genes of T1D risk variants that directly modulate beta cell ER stress and survival phenotypes using genome-wide CRISPR-mediated loss-of-function screens. The cellular phenotype of these genes will then be validated using CRISPR-mediated gene deletions in hiPSC-derived beta cells. Together our findings will provide novel insight into the intrinsic role of beta cells in T1D pathophysiology and inform therapeutic intervention through target discovery of T1D risk genes involved in beta cell stress response and survival.

Deficit of beta cell mass and function occurs in all types of diabetes. The efforts to improve beta cell replacement therapy are compromised by persistent islet inflammation that either destructs beta cells or impairs their function. Our long-term goal is to develop novel approaches that can expand beta cells, while simultaneously protect beta cells from inflammatory insults. We have concentrated on pancreatic macrophages to understand their role in regulating islet inflammation and beta cell biology. In this proposal, we will explore a new mechanism to expand beta cells. Our preliminary studies have found that an epigenetic modulation targeting BET protein bromodomain enhances beta cell proliferation in vivo in animal models of type 1 diabetes. Our data strongly suggest that islet macrophages play a critical role in this process. These macrophages exhibit an elevated activation of PPAR-gamma pathway and also are immunosuppressive. Based on these findings, we propose that modulation of BET protein bromodomain reprograms islet macrophages to promote beta cell proliferation in an immunosuppressive islet environment. We will use animal models of type 1 diabetes, as well as human islet cultures to rigorously assess this novel strategy to expand beta cells in vitro and in vivo. The overall objective of this proposal is to gain an in-depth mechanistic view of this novel epigenetic modification strategy in expanding functional beta cells in a ?protective? islet microenvironment. We will address our goal in the framework of three specific aims. Aim 1 will specifically focus on exploring the role of PPAR-gamma pathway activation in macrophages for these cells to promote beta cell proliferation. In Aim 2, we will determine what factors are involved in macrophage-mediated beta cell proliferation, with a focus on PDGF signaling. In Aim 3, we will determine whether macrophage-mediated immunosuppression is a mechanism to promote beta cell functional recovery under autoimmune conditions. Successful completion of these aims will significantly advance our understanding of the novel roles of macrophages in beta cell biology. This study will pave a new way to expand functional beta cells for diabetes treatment.

A decline in functional ?-cell mass and subsequent inability to maintain adequate glycemic control are hallmarks of both type 1 and type 2 diabetes. Innovative therapeutic approaches are aimed at preserving and restoring functional ?-cell mass in diabetes; however, strategies to safely expand ?-cell mass remain to be identified. The predominant mechanism for adapting ?-cell mass to states of increased insulin demand is through modulation of ?-cell replication. Therefore, there has been considerable interest in understanding the mechanisms that regulate ?-cell replication with the goal of discovering new therapeutic targets to promote ?-cell regeneration. Preliminary unpublished evidence from our laboratory suggests that the NAD+-dependent cytoplasmic deacetylase Sirtuin 2 (SIRT2) acts as a nutrient-dependent regulator of mitogenic signaling in rodent and human ?-cells. Using mouse genetic and inhibitor approaches in human islets, we found that loss of SIRT2 activity stimulates ?-cell proliferation and ?-cell mass expansion under hyperglycemic conditions. We have also obtained evidence that mimicking nutrient state changes by manipulating NAD+ availability regulates ?-cell proliferation in a manner consistent with SIRT2-dependent responses. Since intracellular NAD+ levels fluctuate with glucose availability, we hypothesize that SIRT2 couples ?-cell proliferation to glucose metabolism. Furthermore, we have found that SIRT2 inhibits ?-cell proliferation by dampening MAPK signaling and that SIRT2 inhibition in systemic hyperglycemia promotes ?-cell proliferation, while protecting ?-cells from activating pro-apoptotic signaling downstream of the endoplasmic reticulum (ER) stress response. In this proposal, we will explore how SIRT2 regulates mitogenic signaling as well as ER stress responses in ?-cells. To accomplish this, we will pursue three Aims. In Aim 1 we will employ mouse genetic approaches and experiments in human islets to determine how glucose and nutrient state affect SIRT2-dependent regulation of ?-cell proliferation. Here, we will investigate links between NAD metabolism, activity of the master regulator of cellular energy homeostasis AMPK, SIRT2 activity, and ?-cell proliferation to gain mechanistic insight into the signaling cascades that couple nutrient availability to proliferation in ?-cells. To understand how SIRT2 modulates intracellular signaling to affect glucose-induced proliferative and apoptotic responses in ?-cells, in Aim 2, we will identify the downstream effectors of SIRT2 in the regulation of ?-cell proliferation, employing proteomic as well as in vitro and in vivo approaches. Finally, in Aim 3, we will examine the effects of SIRT2 inhibition on human ?-cell proliferation and function in vivo and explore whether combinatorial targeting of different mitogenic signaling pathways can augment pro-proliferative effects of SIRT2 inhibition. Together, experiments under this proposal will uncover how ?-cells translate nutrient cues into mitogenic signals as well as pave the way for developing pharmacological strategies to safely increase ?-cell mass in humans with diabetes.

PROJECT SUMMARY/ABSTRACT Pancreatic beta cells secrete insulin in order to maintain blood glucose homeostasis. Insulin secretion is tightly regulated by glucose and modulated by numerous environmental signals, including other nutrients, hormones, and inflammatory cytokines. Exposure of beta cells to environmental signals affects gene regulatory programs within hours, and these signal-dependent changes serve to adapt insulin secretion to changes in organismal states. Genetic variants associated with measures of insulin secretion are strongly enriched in genomic elements active in beta cells, and many of these variants are also associated with risk of diabetes. Beta cells therefore possess characteristics that make them an ideal cellular model for studying signal-dependent gene regulatory processes relevant to human health and disease. However, the specific genomic programs that drive signal- induced state changes in beta cells remain poorly characterized. Recent advances in the development of human pluripotent stem cell (hPSC)-derived multi-cellular islet organoid models by us and others provide a genetically tractable beta cell model for linking genomic activity to cellular phenotypes. Our group has further pioneered the development of numerous single cell assays, including chromatin accessibility, ultra-high-throughput paired chromatin accessibility and gene expression, and paired 3D chromatin interactions and DNA methylation; methods that we have successfully applied to both primary human islets and hPSC-islet organoids. We have further developed machine learning and network-based approaches for variant interpretation including from single cell RNA and epigenetic data. In this proposal we will develop novel gene regulatory network (GRN) models to predict network-level relationships among genomic elements, genes, and phenotypes derived from single cell multiomic maps charting signal- and time-dependent changes in hPSC-islet organoids. In Sections B and C we will measure genomic element activity, gene expression, and insulin secretion in hPSC-islet organoids exposed to ten different secretory signals each across four time points using paired single nucleus accessible chromatin and gene expression and paired single cell DNA methylation and 3D chromatin architecture assays. In Section D we will generate a GRN from these data, use machine learning to infer element-gene and element-phenotype relationships and use the trained models to refine the GRN. From the resulting GRN we will predict the effects of genetic variants in specific genomic elements on target gene expression, gene network activity, and cellular phenotype. In Section E we will validate and refine models by using medium-scale CRISPR interference of genomic elements individually and in combination as well as allele-specific gene editing of selected glucose-associated variants in hPSC-islet organoids and measuring gene expression changes in cis and trans. Together, the results, data, and methods from this project using a model of a cell type which both rapidly responds to environmental signals and has a quantifiable phenotypic output will be widely applicable to the community studying the dynamics of genomic regulation.