Single-cell analysis of the human pancreas in type 2 diabetes using multi-spectral imaging mass cytometry.
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ABSTRACT:
SUBMITTER: Wu M
PROVIDER: S-EPMC8609965 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The interaction between the immune system and endocrine cells in the pancreas is crucial for the initiation and progression of type 1 diabetes (T1D). Imaging mass cytometry (IMC) enables multiplexed assessment of the abundance and localization of more than 30 proteins on the same tissue section at 1-μm resolution. Herein, we have developed a panel of 33 antibodies that allows for the quantification of key cell types including pancreatic exocrine cells, islet cells, immune cells, and stromal components. We employed this panel to analyze 12 pancreata obtained from donors with clinically diagnosed T1D and 6 pancreata from non-diabetic controls. In the pancreata from donors with T1D, we simultaneously visualized significant alterations in islet architecture, endocrine cell composition, and immune cell presentation. Indeed, we demonstrate the utility of IMC to investigate complex events on the cellular level that will provide new insights on the pathophysiology of T1D.
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Project description:The human endocrine pancreas consists of multiple cell types and plays a critical role in glucose homeostasis. Here, we apply mass cytometry technology to measure all major islet hormones, proliferative markers, and readouts of signaling pathways involved in proliferation at single-cell resolution. Using this innovative technology, we simultaneously examined baseline proliferation levels of all endocrine cell types from birth through adulthood, as well as in response to the mitogen harmine. High-dimensional analysis of our marker protein expression revealed three major clusters of beta cells within individuals. Proliferating beta cells are confined to two of the clusters.
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Project description:Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing β cells. A comprehensive picture of the changes during T1D development is lacking due to limited sample availability, inability to sample longitudinally, and the paucity of technologies enabling comprehensive tissue profiling. Here, we analyzed 1,581 islets from 12 human donors, including eight with T1D, using imaging mass cytometry (IMC). IMC enabled simultaneous measurement of 35 biomarkers with single-cell and spatial resolution. We performed pseudotime analysis of islets through T1D progression from snapshot data to reconstruct the evolution of β cell loss and insulitis. Our analyses revealed that β cell destruction is preceded by a β cell marker loss and by recruitment of cytotoxic and helper T cells. The approaches described herein demonstrate the value of IMC for improving our understanding of T1D pathogenesis, and our data lay the foundation for hypothesis generation and follow-on experiments.
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Project description:Macrophages and dendritic cells are phagocytes present in almost all tissues in mammals and play a pivotal role for tissue homeostasis and during immune responses. Liver phagocytes play a pivotal role in host immune responses and exquisite mechanisms are necessary to govern the density and the location of the different hepatic leukocytes. However in catastrophic events including trauma, infections or toxin ingestion, many of the liver phagocytes can be wiped out leaving large areas devoid of these cells. Here we used a unique combination of mass cytometry (CyTOF), gene expression and liver intravital approaches to precisely determine phagocytic populations within the liver and the functional consequences of their replenishment by myeloid precursors. While Kupffer cells were exclusively located in the sinusoidal lumen, we identified a population of dendritic cells that was mainly located under the liver capsule. After full depletion of dendritic and Kupffer cells, intravascular myeloid precursors replenished location, density and function of both populations. However, these emergency repopulated livers were dysfunctional in their ability to respond to injury and to clear bacteria for at least 30 days. After this âeducational periodâ, new phagocytes returned to normal response to injury and bacterial trapping. Conclusions: Our data shed light on the liverâs ability to locally shape phagocyte precursors into two vastly different immune cells localized to two fundamentally different tissue compartments. 4 different cell types were isolated from mice, RNA was extracted, and gene expression was measured using the Nanostring nCounter Mouse Immunology Panel
2016-06-12 | E-GEOD-81645 | biostudies-arrayexpress
Project description:Background and study aimTo develop a molecular imaging endoscopic system that eliminates tissue autofluorescence and distinguishes multiple fluorescent markers specifically on the cancerous lesions.MethodsNewly developed multi-spectral fluorescence endoscope device has the potential to eliminate signal interference due to autofluorescence and multiplex fluorophores in fluorescent probes. The multiplexing capability of the multi-spectral endoscope device was demonstrated in the phantom studies and multi-spectral imaging with endoscopy and macroscopy was performed to analyze fluorescence signals after administration of fluorescent probe that targets cancer in the colon. Because of the limitations in the clinical application using rigid-type small animal endoscope, we developed a flexible channel insert-type fluorescence endoscope, which was validated on the colonoscopy of dummy and porcine model.ResultsWe measured multiple fluorescent signals simultaneously, and the fluorescence spectra were unmixed to separate the fluorescent signals of each probe, in which multiple fluorescent probes clearly revealed spectral deconvolution at the specific targeting area in the mouse colon. The positive area of fluorescence signal for each probe over the whole polyp was segmented with analyzing software, and showed distinctive patterns and significantly distinguishable values: 0.46?±?0.04, 0.39?±?0.08 and 0.73?±?0.12 for HMRG, CET-553 and TRA-675 probes, respectively. The spectral unmixing was finally demonstrated in the dummy and porcine model, corroborating the targeted multi-spectral fluorescence imaging of colon dysplasia.ConclusionThe multi-spectral endoscopy system may allow endoscopists to clearly identify cancerous lesion that has different patterns of various target expression using multiple fluorescent probes.
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Project description:Macrophages and dendritic cells are phagocytes present in almost all tissues in mammals and play a pivotal role for tissue homeostasis and during immune responses. Liver phagocytes play a pivotal role in host immune responses and exquisite mechanisms are necessary to govern the density and the location of the different hepatic leukocytes. However in catastrophic events including trauma, infections or toxin ingestion, many of the liver phagocytes can be wiped out leaving large areas devoid of these cells. Here we used a unique combination of mass cytometry (CyTOF), gene expression and liver intravital approaches to precisely determine phagocytic populations within the liver and the functional consequences of their replenishment by myeloid precursors. While Kupffer cells were exclusively located in the sinusoidal lumen, we identified a population of dendritic cells that was mainly located under the liver capsule. After full depletion of dendritic and Kupffer cells, intravascular myeloid precursors replenished location, density and function of both populations. However, these emergency repopulated livers were dysfunctional in their ability to respond to injury and to clear bacteria for at least 30 days. After this “educational period”, new phagocytes returned to normal response to injury and bacterial trapping. Conclusions: Our data shed light on the liver’s ability to locally shape phagocyte precursors into two vastly different immune cells localized to two fundamentally different tissue compartments.
2016-06-12 | GSE81645 | GEO
Project description:Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer's software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay's ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.
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Project description:Despite the recent advances in our understanding of the role of lipids, metabolites and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving human kidney damage (KD). The limited availability of kidney biopsies from living donors with kidney disease has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study kidney disease in humans and characterized these kidneys using imaging and multi-omics approaches. We demonstrated that changes in kidney injury and inflammatory markers following KD were consistent with the changes in pre-donation renal function in donors. Neighborhood and correlation analyses of imaging mass cytometry data showed that a subset of renal cells (e.g., fibroblasts) are associated with the expression profile of renal immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that renal arachidonic acid metabolism and seven other metabolic pathways were upregulated following KD. To validate the therapeutic potential of targeting the arachidonic acid pathway, we demonstrated increased levels of cytosolic phospholipase A2 (cPLA2) protein and related lipid mediators (e.g., prostaglandin E2) in the injured kidneys. The inhibition of cPLA2 reduced injury and inflammation in human renal proximal tubular epithelial cells (RPTEC) in vitro. This study identifies cell types and metabolic pathways that may be critical for controlling inflammation associated with KD in humans.
2022-11-10 | GSE217427 | GEO
Project description:The study of skeletal muscle continues to support the accurate diagnosis of mitochondrial disease and remains important in delineating molecular disease mechanisms. The heterogeneous expression of oxidative phosphorylation proteins and resulting respiratory deficiency are both characteristic findings in mitochondrial disease, hence the rigorous assessment of these at a single cell level is incredibly powerful. Currently, the number of proteins that can be assessed in individual fibres from a single section by immunohistochemistry is limited but imaging mass cytometry (IMC) enables the quantification of further, discrete proteins in individual cells. We have developed a novel workflow and bespoke analysis for applying IMC in skeletal muscle biopsies from patients with genetically-characterised mitochondrial disease, investigating the distribution of nine mitochondrial proteins in thousands of single muscle fibres. Using a semi-automated analysis pipeline, we demonstrate the accurate quantification of protein levels using IMC, providing an accurate measure of oxidative phosphorylation deficiency for complexes I-V at the single cell level. We demonstrate signatures of oxidative phosphorylation deficiency for common mtDNA variants and nuclear-encoded complex I variants and a compensatory upregulation of unaffected oxidative phosphorylation components. This technique can now be universally applied to evaluate a wide range of skeletal muscle disorders and protein targets.
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Project description:BackgroundVisualizing and quantifying cellular heterogeneity is of central importance to study tissue complexity, development, and physiology and has a vital role in understanding pathologies. Mass spectrometry-based methods including imaging mass cytometry (IMC) have in recent years emerged as powerful approaches for assessing cellular heterogeneity in tissues. IMC is an innovative multiplex imaging method that combines imaging using up to 40 metal conjugated antibodies and provides distributions of protein markers in tissues with a resolution of 1 μm2 area. However, resolving the output signals of individual cells within the tissue sample, i.e., single cell segmentation, remains challenging. To address this problem, we developed MATISSE (iMaging mAss cyTometry mIcroscopy Single cell SegmEntation), a method that combines high-resolution fluorescence microscopy with the multiplex capability of IMC into a single workflow to achieve improved segmentation over the current state-of-the-art.ResultsMATISSE results in improved quality and quantity of segmented cells when compared to IMC-only segmentation in sections of heterogeneous tissues. Additionally, MATISSE enables more complete and accurate identification of epithelial cells, fibroblasts, and infiltrating immune cells in densely packed cellular areas in tissue sections. MATISSE has been designed based on commonly used open-access tools and regular fluorescence microscopy, allowing easy implementation by labs using multiplex IMC into their analysis methods.ConclusionMATISSE allows segmentation of densely packed cellular areas and provides a qualitative and quantitative improvement when compared to IMC-based segmentation. We expect that implementing MATISSE into tissue section analysis pipelines will yield improved cell segmentation and enable more accurate analysis of the tissue microenvironment in epithelial tissue pathologies, such as autoimmunity and cancer.
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