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Adherent reformed islets: a long-term primary cell-based platform for exploring mouse and human islet biology

ABSTRACT: Pancreatic islets are 3-dimensional micro-organs that maintain β-cell functionality via cell-cell and cell-matrix communication. Isolated primary islets are the gold standard for in vitro models. However, native islets present experimental challenges for long-term mechanistic studies owing to their short culture life (approximately 1 week). We developed a novel long-term protocol to study the function of primary islets. The protocol employed reformed islets following dispersion and a fine-tuned culture environment. Reformed islets are highly similar to their primary counterparts across various physiological characteristics. Long-term culture of reformed islets enables high-resolution imaging, repeated functional assessment, and the study of cell-cell communication. Unlike other platforms such as stem cell-derived organoids, reformed islets retain their resident immune populations, making them ideal for studying both resident and infiltrating immune cells and their interactions with hormone-producing islet cells. Qualitative and quantitative analyses revealed that the composition and cytoarchitecture of the reformed islets mimicked those found in primary islets, including the presence of macrophages and CD4+ and CD8+ T cells, which are the key resident immune cell types. Reformed islets secrete insulin and are glucose-responsive, and their β-cells can be stimulated to proliferate using GLP-1 receptor agonism. Furthermore, a comparison of the transcriptomic landscape of isolated human islets and reformed islets generated from the same donor demonstrated a high degree of similarity. Our reformed islets provide an ideal platform to study diabetes pathology. We recapitulated both the T1DM and T2DM disease milieu and validated our model for studying islet immune trafficking and invasion using activated macrophages and T cells. Our data illustrates that reformed islets are an anatomical and functional alternative to native human and mouse islets. Moreover, reformed islets have an advantage over mouse and human β-cell lines, including MIN6 and EndoC-βH1cells, that lack the signalling input of non-β-endocrine cells and immune cell crosstalk. In this study, we showed that reformed islets are a durable paradigm (cell-based model) for islet-based exploration and a means of target discovery/validation for diabetes research.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248341 | GEO | 2023/12/25

REPOSITORIES: GEO

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Project description:Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis. Examination of the miRNA profiles in 3 preparations of isolated pancreatic islets and 3 preparations of FACS-enriched pancreatic beta-cells

Project description:Pancreatic islet (dys)function is central to glucose homeostasis and type 2 diabetes (patho)physiology. Human islets consist of multiple endocrine (alpha, beta, delta, gamma), endothelial, and resident/inflitrating immune cells whose coordinated functions modulate glucose mobilization or disposal. Single cell transcriptome profiling (scRNA-seq) studies have been applied to dissect human islet cellular heterogeneity, identify islet cell (sub)populations, and define their molecular repertoire. However, precise understanding of cell type-specific alterations in type 2 diabetic vs. non-diabetic individuals is lacking, due in part to the limited number of individuals or single cell transcriptomes per individual profiled for comparison. Here, we create a comprehensive single cell transcriptome atlas of 245,878 human islet cells from 48 individuals spanning non-diabetic (ND), pre-diabetic (PD), and type 2 diabetic (T2D) states and matched for sex, age, and ancestry and define marker gene sets that are robustly expressed across disease states for each of the 14 cell types identified. We observe significant decreases in the number of beta cells sampled from T2D vs. ND or PD donors. Two of eight putative beta cell subpopulations, with ‘high functioning’ and ‘senescent’ cell gene signatures, increase and decrease in T2D donor islets, respectively. Importantly, we identify 511 differentially expressed genes in beta cells from T2D vs. ND donors. This includes monogenic and type 2 diabetes effector genes, such as HNF1A, DGKB, ST6GAL1, and FXYD2, for which genetic and environmental effects on their expression is concordant. Human beta cell and islet knockdown of selected newly-identified down-regulated genes impairs beta cell viability or function. Together, this study provides new and robust, cell type-resolved insights on the cellular and molecular changes in healthy vs. diabetic human islets and represents a valuable resource to the islet biology and type 2 diabetes communities.

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Adherent reformed islets: a long-term primary cell-based platform for exploring mouse and human islet biology

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