Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets

Abstract Background Human pancreatic islets are a central focus of research in metabolic studies. Transcriptomics is frequently used to interrogate alterations in cultured human islet cells using single-cell RNA-sequencing (scRNA-seq). We introduce single-nucleus RNA-sequencing (snRNA-seq) as an alternative approach for investigating transplanted human islets. Methods The Nuclei EZ protocol was used to obtain nuclear preparations from fresh and frozen human islet cells. Such preparations were first used to generate snRNA-seq datasets and compared to scRNA-seq output obtained from cells from the same donor. Finally, we employed snRNA-seq to obtain the transcriptomic profile of archived human islets engrafted in immunodeficient animals. Results We observed virtually complete concordance in identifying cell types and gene proportions as well as a strong association of global and islet cell type gene signatures between scRNA-seq and snRNA-seq applied to fresh and frozen cultured or transplanted human islet samples. Conclusions We propose snRNA-seq as a reliable strategy to probe transcriptomic profiles of freshly harvested or frozen sources of transplanted human islet cells especially when scRNA-seq is not ideal.

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GABAA receptor-mediated currents and hormone mRNAs in cells expressing more than one hormone transcript in intact human pancreatic islets

10.1101/635144 ◽

2019 ◽

Author(s):

Sergiy V. Korol ◽

Zhe Jin ◽

Bryndis Birnir

Keyword(s):

Pancreatic Islets ◽

Single Channel ◽

Islet Cells ◽

Cell Types ◽

Cell Type ◽

Expression Ratio ◽

Human Pancreatic Islets ◽

The Difference ◽

Type 2 Diabetic

AbstractIn pancreatic islets the major cell-types are α, β and δ cells, secreting the hormones glucagon (GCG), insulin (INS) and somatostatin (SST), respectively. The GABA (γ-aminobutyric acid) signalling system is expressed in human pancreatic islets. We have previously used single-cell RT-PCR in combination with current recordings to correlate expression of single hormone transcript with functional GABAA receptor (iGABAAR) properties in islets. Here we extended these studies to islet cells from non-diabetic and type 2 diabetic donors that express mRNAs for more than one hormone. We detected cells expressing double (α/β, α/δ, β/δ cell-types) and triple (α/β/δ cell-type) hormone transcripts. The most common mixed-identity cell-type was the α/β group where the cells could be grouped into β- and α-like subgroups. The β-like cells had low GCG/INS expression ratio (< 0.6) and significantly higher frequency of single-channel iGABAAR openings than the α-like cells where the GCG/INS expression ratio was high (> 1.2). The difference in expression levels and single channel iGABAAR characteristics varied in the α/β/δ cell-type. No correlation was observed between the cell-types identity with time in culture or cell size. Clearly, multiple hormone transcripts can be expressed in islet cells whereas iGABAAR functional properties appear α or β cell specific.

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Persistent Infection of Human Pancreatic Islets by Coxsackievirus B Is Associated with Alpha Interferon Synthesis in β Cells

Journal of Virology ◽

10.1128/jvi.74.21.10153-10164.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 10153-10164 ◽

Cited By ~ 127

Author(s):

Wassim Chehadeh ◽

Julie Kerr-Conte ◽

François Pattou ◽

Gunar Alm ◽

Jean Lefebvre ◽

...

Keyword(s):

Pancreatic Islets ◽

Islet Cells ◽

Human Islets ◽

Adult Brain ◽

Viral Rna ◽

Alpha Interferon ◽

Β Cells ◽

Human Pancreatic Islets ◽

Reverse Transcription Pcr ◽

Vp1 Capsid Protein

ABSTRACT The interactions of coxsackievirus B3 (CVB3), CVB4E2 (diabetogenic), and CVB4JBV (nondiabetogenic) strains with human pancreatic islets from eight adult brain-dead donors were investigated. Persistent replication of viruses in human islets was proved by detection of viral RNA by in situ hybridization, VP1 capsid protein by immunofluorescence (IF) staining, negative-strand viral RNA by reverse transcription-PCR in extracted RNA from islets, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double IF staining, glucagon-containing α cells and insulin-containing β cells were shown to be susceptible to CVB. The persistence of CVB3 and CVB4 in islet cells was associated with the chronic synthesis of alpha interferon (IFN-α), as evidenced by the detection of IFN-α mRNA and immunoreactive IFN-α with antiviral activity. By double IF staining, IFN-α was detected in insulin-producing β cells only. Experiments with neutralizing anti-coxsackievirus and adenovirus receptor (CAR) antibodies provided evidence that CAR was expressed by α and β cells and that it played a role in the infection of these cells with CVB and the consecutive IFN-α expression in β cells. The viral replication and the expression of IFN-α in islets were not restricted to the CVB4E2 diabetogenic strain and did not depend on the genetic background of the host. The neutralization of endogenous IFN-α significantly enhanced the CVB replication in islet cells and resulted in rapid destruction of islets. Thus, human β cells can harbor a persistent CVB infection, and CVB-induced IFN-α plays a role in the initiation and/or maintenance of chronic CVB infection in human islets.

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GABAA Receptor-Mediated Currents and Hormone mRNAs in Cells Expressing More Than One Hormone Transcript in Intact Human Pancreatic Islets

International Journal of Molecular Sciences ◽

10.3390/ijms21020600 ◽

2020 ◽

Vol 21 (2) ◽

pp. 600

Author(s):

Sergiy V. Korol ◽

Zhe Jin ◽

Bryndis Birnir

Keyword(s):

Pancreatic Islets ◽

Functional Properties ◽

Single Channel ◽

Islet Cells ◽

Cell Types ◽

Activity Level ◽

Cell Type ◽

Expression Ratio ◽

Human Pancreatic Islets ◽

Channel Characteristics

In pancreatic islets, the major cell-types are α, β and δ cells. The γ-aminobutyric acid (GABA) signalling system is expressed in human pancreatic islets. In single hormone transcript-expressing cells, we have previously characterized the functional properties of islet GABAA receptors (iGABAARs). Here, we extended these studies to islet cells expressing mRNAs for more than one hormone and sought for correlation between iGABAAR activity level and relative mRNA expression ratio. The single-cell RT-PCR in combination with the patch-clamp current recordings was used to examine functional properties of iGABAARs in the multiple hormone mRNA-expressing cells. We detected cells expressing double (α/β, α/δ, β/δ cell-types) and triple (α/β/δ cell-type) hormone transcripts. The most common mixed-identity cell-type was the α/β group where the cells could be grouped into β- and α-like subgroups. The β-like cells had low GCG/INS expression ratio (<0.6) and significantly higher frequency of iGABAAR single-channel openings than the α-like cells where the GCG/INS expression ratio was high (>1.2). The hormone expression levels and iGABAAR single-channel characteristics varied in the α/β/δ cell-type. Clearly, multiple hormone transcripts can be expressed in islet cells whereas iGABAAR single-channel functional properties appear to be α or β cell specific.

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Asymmetrical distribution of δ and PP cells in human pancreatic islets

Journal of Endocrinology ◽

10.1530/joe-15-0542 ◽

2016 ◽

Vol 229 (2) ◽

pp. 123-132 ◽

Cited By ~ 4

Author(s):

Charlotte Barbieux ◽

Géraldine Parnaud ◽

Vanessa Lavallard ◽

Estelle Brioudes ◽

Jérémy Meyer ◽

...

Keyword(s):

Endothelial Cells ◽

Pancreatic Islets ◽

Islet Cells ◽

Human Islets ◽

Human Pancreatic Islets ◽

Asymmetrical Distribution ◽

Computerized Morphometry ◽

Vascular Channels ◽

Type 2 Diabetic

The aim of this study was to evaluate the location of PP and δ cells in relation to the vascularization within human pancreatic islets. To this end, pancreas sections were analysed by immunofluorescence using antibodies against endocrine islet and endothelial cells. Staining in different islet areas corresponding to islet cells adjacent or not to peripheral or central vascular channels was quantified by computerized morphometry. As results, α, PP and δ cells were preferentially found adjacent to vessels. In contrast to α cells, which were evenly distributed between islet periphery and intraislet vascular channels, PP and δ cells had asymmetric and opposite distributions: PP staining was higher and somatostatin staining was lower in the islet periphery than in the area around intraislet vascular channels. Additionally, frequencies of PP and δ cells were negatively correlated in the islets. No difference was observed between islets from the head and the tail of the pancreas, and from type 2 diabetic and non-diabetic donors. In conclusion, the distribution of δ cells differs from that of PP cells in human islets, suggesting that vessels at the periphery and at the centre of islets drain different hormonal cocktails.

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Single Cell, Single Nucleus and Spatial RNA Sequencing of the Human Liver Identifies Hepatic Stellate Cell and Cholangiocyte Heterogeneity

10.1101/2021.03.27.436882 ◽

2021 ◽

Author(s):

Tallulah S Andrews ◽

Jawairia Atif ◽

Jeff C Liu ◽

Catia T Perciani ◽

Xue-Zhong Ma ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Human Liver ◽

Stellate Cell ◽

Parenchymal Cell ◽

Cell Types ◽

Cell Populations ◽

Healthy Human ◽

Single Nucleus

The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation related cell perturbation can limit the ability to fully capture the human liver's parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at single-cell resolution, revealed the presence of rare subtypes of hepatic stellate cells previously only seen in disease, and detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and NK cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell-types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte and stellate cell populations by an independent spatial transcriptomics dataset and immunohistochemistry. Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.

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Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers

Nature Communications ◽

10.1038/s41467-020-20063-w ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Michael J. Petrany ◽

Casey O. Swoboda ◽

Chengyi Sun ◽

Kashish Chetal ◽

Xiaoting Chen ◽

...

Keyword(s):

Skeletal Muscle ◽

Neuromuscular Junction ◽

Postnatal Development ◽

Rna Sequencing ◽

Cell Types ◽

Myotendinous Junction ◽

Rna Seq ◽

Muscle Biology ◽

Single Nucleus ◽

Aging Muscle

AbstractWhile the majority of cells contain a single nucleus, cell types such as trophoblasts, osteoclasts, and skeletal myofibers require multinucleation. One advantage of multinucleation can be the assignment of distinct functions to different nuclei, but comprehensive interrogation of transcriptional heterogeneity within multinucleated tissues has been challenging due to the presence of a shared cytoplasm. Here, we utilized single-nucleus RNA-sequencing (snRNA-seq) to determine the extent of transcriptional diversity within multinucleated skeletal myofibers. Nuclei from mouse skeletal muscle were profiled across the lifespan, which revealed the presence of distinct myonuclear populations emerging in postnatal development as well as aging muscle. Our datasets also provided a platform for discovery of genes associated with rare specialized regions of the muscle cell, including markers of the myotendinous junction and functionally validated factors expressed at the neuromuscular junction. These findings reveal that myonuclei within syncytial muscle fibers possess distinct transcriptional profiles that regulate muscle biology.

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Interleukin-22 reverses human islet dysfunction and apoptosis triggered by hyperglycemia and LIGHT

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0182 ◽

2018 ◽

Vol 60 (3) ◽

pp. 171-183

Author(s):

Shadab Abadpour ◽

Bente Halvorsen ◽

Afaf Sahraoui ◽

Olle Korsgren ◽

Pål Aukrust ◽

...

Keyword(s):

Islet Cells ◽

Harmful Effect ◽

Human Islets ◽

Protective Role ◽

Human Islet ◽

Islet Function ◽

Post Transplantation ◽

Interleukin 22 ◽

Glucose Stimulated Insulin Secretion

Interleukin (IL)-22 has recently been suggested as an anti-inflammatory cytokine that could protect the islet cells from inflammation- and glucose-induced toxicity. We have previously shown that the tumor necrosis factor family member, LIGHT, can impair human islet function at least partly via pro-apoptotic effects. Herein, we aimed to investigate the protective role of IL-22 on human islets exposed to the combination of hyperglycemia and LIGHT. First, we found upregulation of LIGHT receptors (LTβR and HVEM) in engrafted human islets exposed to hyperglycemia (>11 mM) for 17 days post transplantation by using a double islet transplantation mouse model as well as in human islets cultured with high glucose (HG) (20 mM glucose) + LIGHT in vitro, and this latter effect was attenuated by IL-22. The effect of HG + LIGHT impairing glucose-stimulated insulin secretion was reversed by IL-22. The harmful effect of HG + LIGHT on human islet function seemed to involve enhanced endoplasmic reticulum stress evidenced by upregulation of p-IRE1α and BiP, elevated secretion of pro-inflammatory cytokines (IL-6, IL-8, IP-10 and MCP-1) and the pro-coagulant mediator tissue factor (TF) release and apoptosis in human islets, whereas all these effects were at least partly reversed by IL-22. Our findings suggest that IL-22 could counteract the harmful effects of LIGHT/hyperglycemia on human islet cells and potentially support the strong protective effect of IL-22 on impaired islet function and survival.

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