Single-cell, single-nucleus, and spatial transcriptomics characterization of the immunological landscape in the healthy and PSC human liver.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which there is an unmet need to understand the cellular composition of the affected liver and how it underlies disease pathogenesis. We aimed to generate a comprehensive atlas of the PSC liver using multi-omic modalities and protein-based functional validation. METHODS: We employed single-cell and single-nucleus RNA sequencing (47,156 cells and 23,000 nuclei) and spatial transcriptomics (one sample by 10x Visium and five samples with Nanostring GeoMx DSP) to profile the cellular ecosystem in 10 PSC livers. Transcriptomic profiles were compared to 24 neurologically deceased donor livers (107,542 cells) and spatial transcriptomics controls, as well as 18,240 cells and 20,202 nuclei from three PBC livers. Flow cytometry was performed to validate PSC-specific differences in immune cell phenotype and function. RESULTS: PSC explants with parenchymal cirrhosis and prominent periductal fibrosis contained a population of cholangiocyte-like hepatocytes that were surrounded by diverse immune cell populations. PSC-associated biliary, mesenchymal, and endothelial populations expressed chemokine and cytokine transcripts involved in immune cell recruitment. Additionally, expanded CD4+ T cells and recruited myeloid populations in the PSC liver expressed the corresponding receptors to these chemokines and cytokines, suggesting potential recruitment. Tissue-resident macrophages, by contrast, were reduced in number and exhibited a dysfunctional and downregulated inflammatory response to lipopolysaccharide and interferon-γ stimulation. CONCLUSIONS: We present a comprehensive atlas of the PSC liver and demonstrate an exhaustion-like phenotype of myeloid cells and markers of chronic cytokine expression in late-stage PSC lesions. This atlas expands our understanding of the cellular complexity of PSC and has potential to guide the development of novel treatments. IMPACT AND IMPLICATIONS: Primary sclerosing cholangitis (PSC) is a rare liver disease characterized by chronic inflammation and irreparable damage to the bile ducts, which eventually results in liver failure. Due to a limited understanding of the underlying pathogenesis of disease, treatment options are limited. To address this, we sequenced healthy and diseased livers to compare the activity, interactions, and localization of immune and non-immune cells. This revealed that hepatocytes lining PSC scar regions co-express cholangiocyte markers, whereas immune cells infiltrate the scar lesions. Of these cells, macrophages, which typically contribute to tissue repair, were enriched in immunoregulatory genes and demonstrated a lack of responsiveness to stimulation. These cells may be involved in maintaining hepatic inflammation and could be a target for novel therapies.

4R-Tau seeding activity unravels molecular subtypes in patients with Progressive Supranuclear Palsy.

Progressive Supranuclear palsy (PSP) is a 4-repeat (4-R) tauopathy. We hypothesized that the molecular diversity of tau could explain the heterogeneity seen in PSP disease progression. To test this hypothesis, we performed an extensive biochemical characterisation of the high molecular weight tau species (HMW-Tau) in 20 different brain regions of 25 PSP patients. We found a correlation between the HMW-Tau species and tau seeding capacity in the primary motor cortex, where we confirmed that an elevated 4R-Tau seeding activity correlates with a shorter disease duration. To identify factors that contribute to these differences, we performed proteomic and spatial transcriptomic analysis that revealed key mechanistic pathways, in particular those involving the immune system, that defined patients demonstrating high and low tau seeding capacity. These observations suggest that differences in the tau seeding activity may contribute to the considerable heterogeneity seen in disease progression of patients suffering from PSP.

A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

Interferon-stimulated and metallothionein-expressing macrophages are associated with acute and chronic allograft dysfunction after lung transplantation.

BACKGROUND: Lung transplant recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). ALAD episodes are a risk factor for chronic lung allograft dysfunction (CLAD), the major cause of death after lung transplantation. Our objective was to determine key cellular elements in dysfunctional lung allografts, with a focus on macrophages. METHODS: We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage cells from stable and ALAD patients and to cells from explanted CLAD lung tissue. RESULTS: We identified 2 alveolar macrophage (AM) subsets uniquely represented in ALAD. Using pathway analysis and differentially expressed genes, we annotated these as pro-inflammatory interferon-stimulated gene (ISG) and metallothionein-mediated inflammatory (MT) AMs. Functional analysis of an independent set of AMs in vitro revealed that ALAD AMs exhibited a higher expression of CXCL10, a marker of ISG AMs, and increased secretion of pro-inflammatory cytokines compared to AMs from stable patients. Using publicly available bronchoalveolar lavage scRNAseq datasets, we found that ISG and MT AMs are associated with more severe inflammation in COVID-19 patients. Analysis of cells from 4 explanted CLAD lungs revealed similar macrophage populations. Donor and recipient cells were identified using expressed single nucleotide variations. We demonstrated contributions of donor and recipient cells to all AM subsets early post-transplant, with loss of donor-derived cells over time. CONCLUSIONS: Our data reveal extensive heterogeneity among lung macrophages after lung transplantation and indicates that specific sub-populations may be associated with allograft dysfunction, raising the possibility that these cells may represent important therapeutic targets.

Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity.

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 a single-cell resolution, revealed the presence of rare subtypes of liver mesenchymal cells, and facilitated the detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and natural killer 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 mesenchymal cell populations by an independent spatial transcriptomics data set and immunohistochemistry. Conclusion: 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.

Tutorial: guidelines for annotating single-cell transcriptomic maps using automated and manual methods.

Single-cell transcriptomics can profile thousands of cells in a single experiment and identify novel cell types, states and dynamics in a wide variety of tissues and organisms. Standard experimental protocols and analysis workflows have been developed to create single-cell transcriptomic maps from tissues. This tutorial focuses on how to interpret these data to identify cell types, states and other biologically relevant patterns with the objective of creating an annotated map of cells. We recommend a three-step workflow including automatic cell annotation (wherever possible), manual cell annotation and verification. Frequently encountered challenges are discussed, as well as strategies to address them. Guiding principles and specific recommendations for software tools and resources that can be used for each step are covered, and an R notebook is included to help run the recommended workflow. Basic familiarity with computer software is assumed, and basic knowledge of programming (e.g., in the R language) is recommended.

Tutorial: guidelines for the computational analysis of single-cell RNA sequencing data.

Single-cell RNA sequencing (scRNA-seq) is a popular and powerful technology that allows you to profile the whole transcriptome of a large number of individual cells. However, the analysis of the large volumes of data generated from these experiments requires specialized statistical and computational methods. Here we present an overview of the computational workflow involved in processing scRNA-seq data. We discuss some of the most common tasks and the tools available for addressing central biological questions. In this article and our companion website ( https://scrnaseq-course.cog.sanger.ac.uk/website/index.html ), we provide guidelines regarding best practices for performing computational analyses. This tutorial provides a hands-on guide for experimentalists interested in analyzing their data as well as an overview for bioinformaticians seeking to develop new computational methods.

Single-cell atlas of the first intra-mammalian developmental stage of the human parasite Schistosoma mansoni.

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.

The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle.

Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites.

EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data.

Droplet-based single-cell RNA sequencing protocols have dramatically increased the throughput of single-cell transcriptomics studies. A key computational challenge when processing these data is to distinguish libraries for real cells from empty droplets. Here, we describe a new statistical method for calling cells from droplet-based data, based on detecting significant deviations from the expression profile of the ambient solution. Using simulations, we demonstrate that EmptyDrops has greater power than existing approaches while controlling the false discovery rate among detected cells. Our method also retains distinct cell types that would have been discarded by existing methods in several real data sets.

Challenges in unsupervised clustering of single-cell RNA-seq data.

Single-cell RNA sequencing (scRNA-seq) allows researchers to collect large catalogues detailing the transcriptomes of individual cells. Unsupervised clustering is of central importance for the analysis of these data, as it is used to identify putative cell types. However, there are many challenges involved. We discuss why clustering is a challenging problem from a computational point of view and what aspects of the data make it challenging. We also consider the difficulties related to the biological interpretation and annotation of the identified clusters.

Publisher Correction: Challenges in unsupervised clustering of single-cell RNA-seq data.

During typesetting of this article, errors were inadvertently introduced to the hyperlinked URLs of some of the clustering tools in table 1 (Seurat, CIDR, pcaReduce and mpath), as well as to the numbering of the bold-text annotations in the reference list. The article has now been corrected online. The editors apologize for this error.

M3Drop: dropout-based feature selection for scRNASeq.

MOTIVATION: Most genomes contain thousands of genes, but for most functional responses, only a subset of those genes are relevant. To facilitate many single-cell RNASeq (scRNASeq) analyses the set of genes is often reduced through feature selection, i.e. by removing genes only subject to technical noise. RESULTS: We present M3Drop, an R package that implements popular existing feature selection methods and two novel methods which take advantage of the prevalence of zeros (dropouts) in scRNASeq data to identify features. We show these new methods outperform existing methods on simulated and real datasets. AVAILABILITY AND IMPLEMENTATION: M3Drop is freely available on github as an R package and is compatible with other popular scRNASeq tools: https://github.com/tallulandrews/M3Drop. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Phosphorylation switches Bax from promoting to inhibiting apoptosis thereby increasing drug resistance.

Akt is a pro-survival kinase frequently activated in human cancers and is associated with more aggressive tumors that resist therapy. Here, we connect Akt pathway activation to reduced sensitivity to chemotherapy via Akt phosphorylation of Bax at residue S184, one of the pro-apoptotic Bcl-2 family proteins required for cells to undergo apoptosis. We show that phosphorylation by Akt converts the pro-apoptotic protein Bax into an anti-apoptotic protein. Mechanistically, we show that phosphorylation (i) enables Bax binding to pro-apoptotic BH3 proteins in solution, and (ii) prevents Bax inserting into mitochondria. Together, these alterations promote resistance to apoptotic stimuli by sequestering pro-apoptotic activator BH3 proteins. Bax phosphorylation correlates with cellular resistance to BH3 mimetics in primary ovarian cancer cells. Further, analysis of the TCGA database reveals that 98% of cancer patients with increased BAX levels also have an upregulated Akt pathway, compared to 47% of patients with unchanged or decreased BAX levels. These results suggest that in patients, increased phosphorylated anti-apoptotic Bax promotes resistance of cancer cells to inherent and drug-induced apoptosis.

Single-cell transcriptomics reveals a new dynamical function of transcription factors during embryonic hematopoiesis.

Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.

False signals induced by single-cell imputation.

Background: Single-cell RNASeq is a powerful tool for measuring gene expression at the resolution of individual cells.  A significant challenge in the analysis of this data is the large amount of zero values, representing either missing data or no expression. Several imputation approaches have been proposed to deal with this issue, but since these methods generally rely on structure inherent to the dataset under consideration they may not provide any additional information. Methods: We evaluated the risk of generating false positive or irreproducible results when imputing data with five different methods. We applied each method to a variety of simulated datasets as well as to permuted real single-cell RNASeq datasets and consider the number of false positive gene-gene correlations and differentially expressed genes. Using matched 10X Chromium and Smartseq2 data from the Tabula Muris database we examined the reproducibility of markers before and after imputation. Results: The extent of false-positive signals introduced by imputation varied considerably by method. Data smoothing based methods, MAGIC and knn-smooth, generated a very high number of false-positives in both real and simulated data. Model-based imputation methods typically generated fewer false-positives but this varied greatly depending on how well datasets conformed to the underlying model. Furthermore, only SAVER exhibited reproducibility comparable to unimputed data across matched data. Conclusions: Imputation of single-cell RNASeq data introduces circularity that can generate false-positive results. Thus, statistical tests applied to imputed data should be treated with care. Additional filtering by effect size can reduce but not fully eliminate these effects. Of the methods we considered, SAVER was the least likely to generate false or irreproducible results, thus should be favoured over alternatives if imputation is necessary.

Identifying cell populations with scRNASeq.

Single-cell RNASeq (scRNASeq) has emerged as a powerful method for quantifying the transcriptome of individual cells. However, the data from scRNASeq experiments is often both noisy and high dimensional, making the computational analysis non-trivial. Here we provide an overview of different experimental protocols and the most popular methods for facilitating the computational analysis. We focus on approaches for identifying biologically important genes, projecting data into lower dimensions and clustering data into putative cell-populations. Finally we discuss approaches to validation and biological interpretation of the identified cell-types or cell-states.

SC3: consensus clustering of single-cell RNA-seq data.

Single-cell RNA-seq enables the quantitative characterization of cell types based on global transcriptome profiles. We present single-cell consensus clustering (SC3), a user-friendly tool for unsupervised clustering, which achieves high accuracy and robustness by combining multiple clustering solutions through a consensus approach (http://bioconductor.org/packages/SC3). We demonstrate that SC3 is capable of identifying subclones from the transcriptomes of neoplastic cells collected from patients.