Pancreatic ß-Cell Identity Change through the Lens of Single-Cell Omics Research.

The main hallmark in the development of both type 1 and type 2 diabetes is a decline in functional ß-cell mass. This decline is predominantly attributed to ß-cell death, although recent findings suggest that the loss of ß-cell identity may also contribute to ß-cell dysfunction. This phenomenon is characterized by a reduced expression of key markers associated with ß-cell identity. This review delves into the insights gained from single-cell omics research specifically focused on ß-cell identity. It highlights how single-cell omics based studies have uncovered an unexpected level of heterogeneity among ß-cells and have facilitated the identification of distinct ß-cell subpopulations through the discovery of cell surface markers, transcriptional regulators, the upregulation of stress-related genes, and alterations in chromatin activity. Furthermore, specific subsets of ß-cells have been identified in diabetes, such as displaying an immature, dedifferentiated gene signature, expressing significantly lower insulin mRNA levels, and expressing increased ß-cell precursor markers. Additionally, single-cell omics has increased insight into the detrimental effects of diabetes-associated conditions, including endoplasmic reticulum stress, oxidative stress, and inflammation, on ß-cell identity. Lastly, this review outlines the factors that may influence the identification of ß-cell subpopulations when designing and performing a single-cell omics experiment.

Single-cell transcriptomics reveals a role for pancreatic duct cells as potential mediators of inflammation in diabetes mellitus.

Introduction: Inflammation of the pancreas contributes to the development of diabetes mellitus. Although it is well-accepted that local inflammation leads to a progressive loss of functional beta cell mass that eventually causes the onset of the disease, the development of islet inflammation remains unclear. Methods: Here, we used single-cell RNA sequencing to explore the cell type-specific molecular response of primary human pancreatic cells exposed to an inflammatory environment. Results: We identified a duct subpopulation presenting a unique proinflammatory signature among all pancreatic cell types. Discussion: Overall, the findings of this study point towards a role for duct cells in the propagation of islet inflammation, and in immune cell recruitment and activation, which are key steps in the pathophysiology of diabetes mellitus.

Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system.

One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC) tests specific for hiPSCs, which are required for GMP batch release. This study presents a comprehensive description of the validation process for hiPSC-specific GMP-compliant QC assays; more specifically, the validation of assays to assess the potential presence of residual episomal vectors (REVs), the expression of markers of the undifferentiated state and the directed differentiation potential of hiPSCs. Critical aspects and specific acceptance criteria were formulated in a validation plan prior to assay validation. Assay specificity, sensitivity and reproducibility were tested, and the equipment used for each assay was subjected to performance qualification. A minimum input of 20 000 cells (120 ng of genomic DNA) was defined for accurate determination of the presence of REVs. Furthermore, since vector loss in hiPSC lines is a passage-dependent process, we advocate screening for REVs between passages eight and 10, as testing at earlier passages might lead to unnecessary rejection of hiPSC lines. The cutoff value for assessment of markers of the undifferentiated state was set to the expression of at least three individual markers on at least 75% of the cells. When multi-color flow cytometry panels are used, a fluorescence minus one control is advised to ensure the control for fluorescent spread. For the assay to assess the directed differentiation potential, the detection limit was set to two of three positive lineage-specific markers for each of the three individual germ layers. All of our assays proved to be reproducible and specific. Our data demonstrate that our implemented analytical procedures are suitable as QC assays for the batch release of GMP-compliant hiPSCs.

Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

BACKGROUND AIMS: Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. METHODS: Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). RESULTS: Two MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). CONCLUSIONS: We successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

Apolipoprotein L genes are novel mediators of inflammation in beta cells.

AIMS/HYPOTHESIS: Inflammation induces beta cell dysfunction and demise but underlying molecular mechanisms remain unclear. The apolipoprotein L (APOL) family of genes has been associated with innate immunity and apoptosis in non-pancreatic cell types, but also with metabolic syndrome and type 2 diabetes mellitus. Here, we hypothesised that APOL genes play a role in inflammation-induced beta cell damage. METHODS: We used single-cell transcriptomics datasets of primary human pancreatic islet cells to study the expression of APOL genes upon specific stress conditions. Validation of the findings was carried out in EndoC-ßH1 cells and primary human islets. Finally, we performed loss- and gain-of-function experiments to investigate the role of APOL genes in beta cells. RESULTS: APOL genes are expressed in primary human beta cells and APOL1, 2 and 6 are strongly upregulated upon inflammation via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. APOL1 overexpression increases endoplasmic reticulum stress while APOL1 knockdown prevents cytokine-induced beta cell death and interferon-associated response. Furthermore, we found that APOL genes are upregulated in beta cells from donors with type 2 diabetes compared with donors without diabetes mellitus. CONCLUSIONS/INTERPRETATION: APOLs are novel regulators of islet inflammation and may contribute to beta cell damage during the development of diabetes. DATA AVAILABILITY: scRNAseq data generated by our laboratory and used in this study are available in the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/ ), accession number GSE218316.

IFNÉ£ but not IFNα increases recognition of insulin defective ribosomal product-derived antigen to amplify islet autoimmunity.

AIMS/HYPOTHESIS: The inflammatory milieu characteristic of insulitis affects translation fidelity and generates defective ribosomal products (DRiPs) that participate in autoimmune beta cell destruction in type 1 diabetes. Here, we studied the role of early innate cytokines (IFNα) and late immune adaptive events (IFNÉ£) in insulin DRiP-derived peptide presentation to diabetogenic CD8+ T cells. METHODS: Single-cell transcriptomics of human pancreatic islets was used to study the composition of the (immuno)proteasome. Specific inhibition of the immunoproteasome catalytic subunits was achieved using siRNA, and antigenic peptide presentation at the cell surface of the human beta cell line EndoC-ßH1 was monitored using peptide-specific CD8 T cells. RESULTS: We found that IFNγ induces the expression of the PSMB10 transcript encoding the ß2i catalytic subunit of the immunoproteasome in endocrine beta cells, revealing a critical role in insulin DRiP-derived peptide presentation to T cells. Moreover, we showed that PSMB10 is upregulated in a beta cell subset that is preferentially destroyed in the pancreases of individuals with type 1 diabetes. CONCLUSIONS/INTERPRETATION: Our data highlight the role of the degradation machinery in beta cell immunogenicity and emphasise the need for evaluation of targeted immunoproteasome inhibitors to limit beta cell destruction in type 1 diabetes. DATA AVAILABILITY: The single-cell RNA-seq dataset is available from the Gene Expression Omnibus (GEO) using the accession number GSE218316 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218316 ).

EndoC-ßH5 cells are storable and ready-to-use human pancreatic beta cells with physiological insulin secretion.

OBJECTIVES: Readily 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-ßH5 cells, the latest in the EndoC-ßH cell family. METHODS: EndoC-ßH5 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-ßH5 cells. We performed transcriptome, immunological and extensive functional assays. RESULTS: Ready to use EndoC-ßH5 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-ßH5 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-ßH5 cells elicit specific A2-alloreactive CD8 T cell activation. CONCLUSIONS: EndoC-ßH5 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.

Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells.

AIMS/HYPOTHESIS: Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. METHODS: Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1ß release. RESULTS: We identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells. CONCLUSIONS/INTERPRETATION: Our data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity. DATA AVAILABILITY: The full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/ ). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas . The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474).

ER stress promotes mitochondrial DNA mediated type-1 interferon response in beta-cells and interleukin-8 driven neutrophil chemotaxis.

Beta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. Accumulating evidence suggests the engagement of cellular stress during the initial stage of the disease, preceding destruction and triggering immune cell infiltration. While the role of the endoplasmic reticulum (ER) in this process has been largely described, the participation of the other cellular organelles, particularly the mitochondria which are central mediator for beta-cell survival and function, remains poorly investigated. Here, we have explored the contribution of ER stress, in activating type-I interferon signaling and innate immune cell recruitment. Using human beta-cell line EndoC-ßH1 exposed to thapsigargin, we demonstrate that induction of cellular stress correlates with mitochondria dysfunction and a significant accumulation of cytosolic mitochondrial DNA (mtDNA) that triggers neutrophils migration by an IL8-dependent mechanism. These results provide a novel mechanistic insight on how ER stress can cause insulitis and may ultimately facilitate the identification of potential targets to protect beta-cells against immune infiltration.

Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the degradation of the low-density lipoprotein receptor. PCSK9 also targets proteins involved in lipid metabolism (very low-density lipoprotein receptor), immunity (major histocompatibility complex I), and viral infection (cluster of differentiation 81). Recent studies have also indicated that PCSK9 loss-of-function mutations are associated with an increased incidence of diabetes; however, the expression and function of PCSK9 in insulin-producing pancreatic beta cells remain unclear. Here, we studied PCSK9 regulation and function by performing loss- and gain-of-function experiments in the human beta cell line EndoC-ßH1. We demonstrate that PCSK9 is expressed and secreted by EndoC-ßH1 cells. We also found that PCSK9 expression is regulated by cholesterol and sterol regulatory element-binding protein transcription factors, as previously demonstrated in other cell types such as hepatocytes. Importantly, we show that PCSK9 knockdown using siRNA results in deregulation of various elements of the transcriptome, proteome, and secretome, and increases insulin secretion. We also observed that PCSK9 decreases low-density lipoprotein receptor and very low-density lipoprotein receptor levels via an extracellular signaling mechanism involving exogenous PCSK9, as well as levels of cluster of differentiation 36, a fatty acid transporter, through an intracellular signaling mechanism. Finally, we found that PCSK9 regulates the cell surface expression of PDL1 and HLA-ABC, proteins involved in cell-lymphocyte interaction, also via an intracellular mechanism. Collectively, these results highlight PCSK9 as a regulator of multiple cell surface receptors in pancreatic beta cells.

Organization and dynamics of the cortical complexes controlling insulin secretion in ß-cells.

Insulin secretion in pancreatic ß-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 LL5ß (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5ß 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 exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

Stem cell-based islet replacement therapy in diabetes: A road trip that reached the clinic.

One hundred years after the discovery of insulin, Kieffer and colleagues (Ramzy et al., 2021) and Foyt and colleagues (Shapiro et al., 2021) report interim results from a multicenter clinical trial showing insulin secretion from engrafted pluripotent stem cell-derived endocrine progenitor cells in patients with type 1 diabetes.

Single-Cell Transcriptomics Links Loss of Human Pancreatic ß-Cell Identity to ER Stress.

The maintenance of pancreatic islet architecture is crucial for proper ß-cell function. We previously reported that disruption of human islet integrity could result in altered ß-cell identity. Here we combine ß-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of ß-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and ß-cell phenotype, and strengthen the concept of altered ß-cell identity as a mechanism underlying the loss of functional ß-cell mass.

Oxidative Stress Leads to ß-Cell Dysfunction Through Loss of ß-Cell Identity.

Pancreatic ß-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. ß-cells have low anti-oxidant capacity, making them more susceptible to oxidative stress. In type 1 diabetes (T1D), reactive oxygen species (ROS) are associated with pro-inflammatory conditions at the onset of the disease. Here, we investigated the effects of hydrogen peroxide-induced oxidative stress on human ß-cells. We show that primary human ß-cell function is decreased. This reduced function is associated with an ER stress response and the shuttling of FOXO1 to the nucleus. Furthermore, oxidative stress leads to loss of ß-cell maturity genes MAFA and PDX1, and to a concomitant increase in progenitor marker expression of SOX9 and HES1. Overall, we propose that oxidative stress-induced ß-cell failure may result from partial dedifferentiation. Targeting antioxidant mechanisms may preserve functional ß-cell mass in early stages of development of T1D.

Long RNA Sequencing and Ribosome Profiling of Inflamed ß-Cells Reveal an Extensive Translatome Landscape.

Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of the insulin-producing pancreatic ß-cells. Increasing evidence suggest that the ß-cells themselves contribute to their own destruction by generating neoantigens through the production of aberrant or modified proteins that escape central tolerance. We recently demonstrated that ribosomal infidelity amplified by stress could lead to the generation of neoantigens in human ß-cells, emphasizing the participation of nonconventional translation events in autoimmunity, as occurring in cancer or virus-infected tissues. Using a transcriptome-wide profiling approach to map translation initiation start sites in human ß-cells under standard and inflammatory conditions, we identify a completely new set of polypeptides derived from noncanonical start sites and translation initiation within long noncoding RNA. Our data underline the extreme diversity of the ß-cell translatome and may reveal new functional biomarkers for ß-cell distress, disease prediction and progression, and therapeutic intervention in T1D.

Dichotomic role of heparanase in a murine model of metabolic syndrome.

Heparanase is the predominant enzyme that cleaves heparan sulfate, the main polysaccharide in the extracellular matrix. While the role of heparanase in sustaining the pathology of autoimmune diabetes is well documented, its association with metabolic syndrome/type 2 diabetes attracted less attention. Our research was undertaken to elucidate the significance of heparanase in impaired glucose metabolism in metabolic syndrome and early type 2 diabetes. Here, we report that heparanase exerts opposite effects in insulin-producing (i.e., islets) vs. insulin-target (i.e., skeletal muscle) compartments, sustaining or hampering proper regulation of glucose homeostasis depending on the site of action. We observed that the enzyme promotes macrophage infiltration into islets in a murine model of metabolic syndrome, and fosters ß-cell-damaging properties of macrophages activated in vitro by components of diabetogenic/obese milieu (i.e., fatty acids). On the other hand, in skeletal muscle (prototypic insulin-target tissue), heparanase is essential to ensure insulin sensitivity. Thus, despite a deleterious effect of heparanase on macrophage infiltration in islets, the enzyme appears to have beneficial role in glucose homeostasis in metabolic syndrome. The dichotomic action of the enzyme in the maintenance of glycemic control should be taken into account when considering heparanase-targeting strategies for the treatment of diabetes.

ß-Cell Stress Shapes CTL Immune Recognition of Preproinsulin Signal Peptide by Posttranscriptional Regulation of Endoplasmic Reticulum Aminopeptidase 1.

The signal peptide of preproinsulin is a major source for HLA class I autoantigen epitopes implicated in CD8 T cell (CTL)-mediated ß-cell destruction in type 1 diabetes (T1D). Among them, the 10-mer epitope located at the C-terminal end of the signal peptide was found to be the most prevalent in patients with recent-onset T1D. While the combined action of signal peptide peptidase and endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) is required for processing of the signal peptide, the mechanisms controlling signal peptide trimming and the contribution of the T1D inflammatory milieu on these mechanisms are unknown. Here, we show in human ß-cells that ER stress regulates ERAP1 gene expression at posttranscriptional level via the IRE1α/miR-17-5p axis and demonstrate that inhibition of the IRE1α activity impairs processing of preproinsulin signal peptide antigen and its recognition by specific autoreactive CTLs during inflammation. These results underscore the impact of ER stress in the increased visibility of ß-cells to the immune system and position the IRE1α/miR-17 pathway as a central component in ß-cell destruction processes and as a potential target for the treatment of autoimmune T1D.

Tacrolimus-Induced BMP/SMAD Signaling Associates With Metabolic Stress-Activated FOXO1 to Trigger ß-Cell Failure.

Active maintenance of ß-cell identity through fine-tuned regulation of key transcription factors ensures ß-cell function. Tacrolimus, a widely used immunosuppressant, accelerates onset of diabetes after organ transplantation, but underlying molecular mechanisms are unclear. Here we show that tacrolimus induces loss of human ß-cell maturity and ß-cell failure through activation of the BMP/SMAD signaling pathway when administered under mild metabolic stress conditions. Tacrolimus-induced phosphorylated SMAD1/5 acts in synergy with metabolic stress-activated FOXO1 through formation of a complex. This interaction is associated with reduced expression of the key ß-cell transcription factor MAFA and abolished insulin secretion, both in vitro in primary human islets and in vivo in human islets transplanted into high-fat diet-fed mice. Pharmacological inhibition of BMP signaling protects human ß-cells from tacrolimus-induced ß-cell dysfunction in vitro. Furthermore, we confirm that BMP/SMAD signaling is activated in protocol pancreas allograft biopsies from recipients on tacrolimus. To conclude, we propose a novel mechanism underlying the diabetogenicity of tacrolimus in primary human ß-cells. This insight could lead to new treatment strategies for new-onset diabetes and may have implications for other forms of diabetes.

Organoids from the Human Fetal and Adult Pancreas.

PURPOSE OF REVIEW: Novel 3D organoid culture techniques have enabled long-term expansion of pancreatic tissue. This review comprehensively summarizes and evaluates the applications of primary tissue-derived pancreatic organoids in regenerative studies, disease modelling, and personalized medicine. RECENT FINDINGS: Organoids derived from human fetal and adult pancreatic tissue have been used to study pancreas development and repair. Generated adult human pancreatic organoids harbor the capacity for clonal expansion and endocrine cell formation. In addition, organoids have been generated from human pancreatic ductal adenocarcinoma in order to study tumor behavior and assess drug responses. Pancreatic organoids constitute an important translational bridge between in vitro and in vivo models, enhancing our understanding of pancreatic cell biology. Current applications for pancreatic organoid technology include studies on tissue regeneration, disease modelling, and drug screening.