Single-cell Approaches Research Articles

Single-cell analysis of RNase L-mediated mRNA decay.

Ribonuclease L (RNase L) is a mammalian endoribonuclease that initiates the mass degradation of cellular mRNAs in response to double-stranded RNA or viral infection. The kinetic rate of mRNA decay upon RNase L activation has been elusive because RNase L is heterogeneously activated with respect to time in individual cells. Herein, we describe a method using immunofluorescence combined with single-molecule fluorescence in situ hybridization (smFISH) to determine single-cell mRNA decay rates upon RNase L activation. Using these approaches, we deduce that the rate of mRNA decay upon RNase L activation is extremely rapid, whereby the half-life of stable mRNAs such as GAPDH mRNA is reduced to ∼15minutes in individual cells. This allows for RNase L to degrade nearly every mRNA in a cell in less than 1hour, which is much faster than the decay rate that would be derived using bulk measurement techniques for mRNA levels, such as qRT-PCR. These single-cell approaches can generally be employed to resolve mRNA decay kinetics in additional contexts.

Mass cytometry to characterize the immune lung cancer microenvironment.

The human tumor microenvironment requires use of high-dimensional single-cell tools to uncover its cellular complexity and functional variety. For decades, flow cytometry has been the technology of choice to explore immune cell diversity in different pathological contexts. Recently, a new format for flow cytometry - termed mass cytometry - has been developed. It allows for simultaneous interrogation of more than 40 different molecular markers, without the need for spectral compensation, which significantly augments the ability of cytometry to evaluate complex cellular systems and processes. Currently, different multiparametric single-cell analysis approaches are being widely adopted to interrogate the human tumor microenvironment. However, important challenges must be addressed when solid tissues are analyzed by single-cell technologies. This protocol describes the challenge and better use of single-cell mass cytometry to dissect tumor infiltrating leukocytes from surgically resected tumoral lung tissues.

Consistent analysis of differentially expressed genes across 7 cell types in papillary thyroid carcinoma

Single-cell transcriptome sequencing (scRNA-seq) provides a higher resolution of cellular differences than bulk RNA-seq, enabling the dissection of cell-type-specific responses to perturbations in papillary thyroid carcinoma (PTC). However, cellular genomic features are highly heterogeneous and have a large number of genes without any expression signals, which hinders the statistical power to identify differentially expressed genes and may generate many false-positive results. To overcome this challenge, we conducted an integrative analysis on two PTC scRNA-seq datasets and cross-validated consistent differential expression. By combining results from 32 common cell types in the two studies, we identified 31 consistently differentially expressed genes (DEGs) across seven cell types, including B cells, endothelial cells, epithelial cells, monocytes, NK cells, smooth muscle cells, and T cells. Functional enrichment analysis revealed that these genes are important for the adaptive immune response and autoimmune thyroid diseases. The additional disease-free survival analysis also confirmed that these 31 genes significantly affected patient survival time in large scale thyroid cancer cohort. Furthermore, we experimentally validated one of the top consistent DEGs as a potential biomarker gene of PTC epithelial cells, KRT7, which may be a upstream gene for the NF-κB signaling pathway. The result shows that KRT7 may promote thyroid cancer metastasis through the epithelial-mesenchymal transition and NF-κB signaling pathway. In summary, our single-cell transcriptome integration-based approach may provide insights into the important role of NF-κB in the underlying biology of the PTC.

Immortalized hippocampal astrocytes from 3xTg-AD mice, a new model to study disease-related astrocytic dysfunction: a comparative review.

Alzheimer's disease (AD) is characterized by complex etiology, long-lasting pathogenesis, and cell-type-specific alterations. Currently, there is no cure for AD, emphasizing the urgent need for a comprehensive understanding of cell-specific pathology. Astrocytes, principal homeostatic cells of the central nervous system, are key players in the pathogenesis of neurodegenerative diseases, including AD. Cellular models greatly facilitate the investigation of cell-specific pathological alterations and the dissection of molecular mechanisms and pathways. Tumor-derived and immortalized astrocytic cell lines, alongside the emerging technology of adult induced pluripotent stem cells, are widely used to study cellular dysfunction in AD. Surprisingly, no stable cell lines were available from genetic mouse AD models. Recently, we established immortalized hippocampal astroglial cell lines from amyloid-β precursor protein/presenilin-1/Tau triple-transgenic (3xTg)-AD mice (denominated as wild type (WT)- and 3Tg-iAstro cells) using retrovirus-mediated transduction of simian virus 40 large T-antigen and propagation without clonal selection, thereby maintaining natural heterogeneity of primary cultures. Several groups have successfully used 3Tg-iAstro cells for single-cell and omics approaches to study astrocytic AD-related alterations of calcium signaling, mitochondrial dysfunctions, disproteostasis, altered homeostatic and signaling support to neurons, and blood-brain barrier models. Here we provide a comparative overview of the most used models to study astrocytes in vitro, such as primary culture, tumor-derived cell lines, immortalized astroglial cell lines, and induced pluripotent stem cell-derived astrocytes. We conclude that immortalized WT- and 3Tg-iAstro cells provide a non-competitive but complementary, low-cost, easy-to-handle, and versatile cellular model for dissection of astrocyte-specific AD-related alterations and preclinical drug discovery.

Methods for Single Cell Transcriptomic Analysis of Adipose Tissue.

The development of single cell approaches has facilitated the investigation of cellular heterogeneity and cell type-specific gene expression in complex tissues. Adipose tissue depots contain lipid storing adipocytes as well as a diverse array of cell types that form the adipocyte niche and regulate adipose tissue function. Here, I describe two protocols for the isolation of single cells and nuclei from white and brown adipose tissue. Additionally, I provide a detailed workflow for isolation of cell type- or lineage-specific single nuclei using nuclear tagging and translating ribosome affinity purification (NuTRAP) mouse models.

Single-cell technologies uncover intra-tumor heterogeneity in childhood cancers.

Childhood cancer is the second leading cause of death in children aged 1 to 14. Although survival rates have vastly improved over the past 40years, cancer resistance and relapse remain a significant challenge. Advances in single-cell technologies enable dissection of tumors to unprecedented resolution. This facilitates unraveling the heterogeneity of childhood cancers to identify cell subtypes that are prone to treatment resistance. The rapid accumulation of single-cell data from different modalities necessitates the development of novel computational approaches for processing, visualizing, and analyzing single-cell data. Here, we review single-cell approaches utilized or under development in the context of childhood cancers. We review computational methods for analyzing single-cell data and discuss best practices for their application. Finally, we review the impact of several studies of childhood tumors analyzed with these approaches and future directions to implement single-cell studies into translational cancer research in pediatric oncology.

High-frequency and functional mitochondrial DNA mutations at the single-cell level

Decline in mitochondrial function underlies aging and age-related diseases, but the role of mitochondrial DNA (mtDNA) mutations in these processes remains elusive. To investigate patterns of mtDNA mutations, it is particularly important to quantify mtDNA mutations and their associated pathogenic effects at the single-cell level. However, existing single-cell mtDNA sequencing approaches remain inefficient due to high cost and low mtDNA on-target rates. In this study, we developed a cost-effective mtDNA targeted-sequencing protocol called single-cell sequencing by targeted amplification of multiplex probes (scSTAMP) and experimentally validated its reliability. We then applied our method to assess single-cell mtDNA mutations in 768 B lymphocytes and 768 monocytes from a 76-y-old female. Across 632 B lymphocyte and 617 monocytes with medium mtDNA coverage over >100×, our results indicated that over 50% of cells carried at least one mtDNA mutation with variant allele frequencies (VAFs) over 20%, and that cells carried an average of 0.658 and 0.712 such mutation for B lymphocytes and monocytes, respectively. Surprisingly, more than 20% of the observed mutations had VAFs of over 90% in either cell population. In addition, over 60% of the mutations were in protein-coding genes, of which over 70% were nonsynonymous, and more than 50% of the nonsynonymous mutations were predicted to be highly pathogenic. Interestingly, about 80% of the observed mutations were singletons in the respective cell populations. Our results revealed mtDNA mutations with functional significance might be prevalent at advanced age, calling further investigation on age-related mtDNA mutation dynamics at the single-cell level.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Single-Cell Approach Reveals Intercellular Heterogeneity in Phage-Producing Capacities.

Bacteriophage burst size is the average number of phage virions released from infected bacterial cells, and its magnitude depends on the duration of an intracellular progeny accumulation phase. Burst size is often measured at the population level, not the single-cell level, and consequently, statistical moments are not commonly available. In this study, we estimated the bacteriophage lambda (λ) single-cell burst size mean and variance following different intracellular accumulation period durations by employing Escherichia coli lysogens bearing lysis-deficient λ prophages. Single lysogens can be isolated and chemically lysed at desired times following prophage induction to quantify progeny intracellular accumulation within individual cells. Our data showed that λ phage burst size initially increased exponentially with increased lysis time (i.e., period between induction and chemical lysis) and then saturated at longer lysis times. We also demonstrated that cell-to-cell variation, or "noise," in lysis timing did not contribute significantly to burst size noise. The burst size noise remained constant with increasing mean burst size. The most likely explanation for the experimentally observed constant burst size noise was that cell-to-cell differences in burst size originated from intercellular heterogeneity in cellular capacities to produce phages. The mean burst size measured at different lysis times was positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicated that there are other factors in addition to cell size that determine this cellular capacity. IMPORTANCE Phages produce offspring by hijacking a cell's replicative machinery. Previously, it was noted that the variation in the number of phages produced by single infected cells far exceeded cell size variation. It was hypothesized that this variation is a consequence of variation in the timing of host cell lysis. Here, we show that cell-to-cell variation in lysis timing does not significantly contribute to the burst size variation. We suggest that the constant burst size variation across different host lysis times results from cell-to-cell differences in capacity to produce phages. We found that the mean burst size measured at different lysis times was positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicated that there are other factors in addition to cell size that determine this cellular capacity.

Compensability of an enhanced incidence of spermatozoa with cytoplasmic droplets in boar semen for use in artificial insemination: a single cell approach

This single cell study aimed to clarify whether an elevated incidence of sperm with a retained cytoplasmic droplet (CD) can be compensated by a higher sperm number in boar semen doses to maintain fertility. Cluster analysis of motile spermatozoa (ten boars) revealed that spermatozoa with a CD are underrepresented in the fast, linearly moving sperm cohort compared to morphologically normal sperm. Nonetheless, the response to the motility stimulator procaine was barely affected in spermatozoa with distal CD (Cramer’s V = 0.14), but moderately affected in sperm with proximal CD (V = 0.28). Viability was lower in sperm with distal CD (p < 0.05) but not with proximal CD compared to normal sperm during 168 h storage of extended semen samples (n = 11) and subsequent thermic stress. Morphologically normal sperm from normospermic samples (n = 10) or samples with a high incidence (≥ 15%) of sperm with CD (n = 9) had similar motility patterns and responses to procaine. The origin of morphologically normal sperm had no effect on sperm viability (p > 0.05; n = 26). In conclusion, a moderately enhanced prevalence of sperm with CD seems to be compensable by an increase in sperm numbers in boar semen doses.

Isolation of Nuclei from Flash-frozen Liver Tissue for Single-cell Multiomics.

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease. This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Inter and intra-tumor heterogeneity of paediatric type diffuse high-grade gliomas revealed by single-cell mass cytometry.

Paediatric-type diffuse high-grade gliomas (PDHGG) are aggressive tumors affecting children and young adults, with no effective treatment. These highly heterogeneous malignancies arise in different sites of the Central Nervous System (CNS), carrying distinctive molecular alterations and clinical outcomes (inter-tumor heterogeneity). Moreover, deep cellular and molecular profiling studies highlighted the coexistence of genetically and phenotypically different subpopulations within the same tumor mass (intra-tumor heterogeneity). Despite the recent advances made in the field, the marked heterogeneity of PDHGGs still impedes the development of effective targeted therapies and the identification of suitable biomarkers. In order to fill the existing gap, we used mass cytometry to dissect PDHGG inter- and intra-heterogeneity. This is one of the most advanced technologies of the "-omics" era that, using antibodies conjugated to heavy metals, allows the simultaneous measurement of more than 40 markers at single-cell level. To this end, we analyzed eight PDHGG patient-derived cell lines from different locational and molecular subgroups. By using a panel of 15 antibodies, directly conjugated to metals or specifically customized to detect important histone variants, significant differences were highlighted in the expression of the considered antigens. The single-cell multiparametric approach realized has deepened our understanding of PDHGG, confirming a high degree of intra- and inter-tumoral heterogeneity and identifying some antigens that could represent useful biomarkers for the specific PDHGG locational or molecular subgroups.

Immune landscape of viral cancers: Insights from single-cell sequencing.

Viral infections trigger a wide range of immune responses thought to drive tumorigenesis and malignant progression. Dissecting virus-induced changes in the tumor immune microenvironment (TIME) is therefore crucial to identify key leukocyte populations that may represent novel targets for cancer therapy. Single-cell sequencing approaches have now been widely applied to the analysis of various tumors, thus enabling multiomics characterization of the highly heterogeneous TIME that bulk-sequencing cannot fully elucidate. In this review, we summarized key recent findings from sequencing studies of the immune infiltrate and antitumor response in virus-associated cancers at single cell resolution. Additionally, we also reviewed recent developments in immunotherapy for virus-associated cancers. We anticipate that the strategic use of single-cell sequencing will advance our understanding of the TIME of viral cancers, leading to the development of more potent novel treatments.

Thyroid hormone regulators in human cerebral cortex development.

Brain development is critically dependent on the timely supply of thyroid hormones. The thyroid hormone transporters are central to the action of thyroid hormones in the brain, facilitating their passage through the blood-brain barrier. Mutations of the monocarboxylate transporter 8 (MCT8) cause the Allan-Herndon-Dudley syndrome, with altered thyroid hormone concentrations in the blood and profound neurological impairment and intellectual deficit. Mouse disease models have revealed interplay between transport, deiodination, and availability of T3 to receptors in specific cells. However, the mouse models are not satisfactory, given the fundamental differences between the mouse and human brains. The goal of the present work is to review human neocortex development in the context of thyroid pathophysiology. Recent developments in single-cell transcriptomic approaches aimed at the human brain make it possible to profile the expression of thyroid hormone regulators in single-cell RNA-Seq datasets of the developing human neocortex. The data provide novel insights into the specific cellular expression of thyroid hormone transporters, deiodinases, and receptors.

Heterogeneity and Evolutionary Tunability of Escherichia coli Resistance against Extreme Acid Stress.

Since acidic environments often serve as an important line of defense against bacterial pathogens, it is important to fully understand how the latter manage to mount and evolve acid resistance mechanisms. Escherichia coli, a species harboring many pathovars, is typically equipped with the acid fitness island (AFI), a genomic region encoding the GadE master regulator together with several GadE-controlled functions to counter acid stress. This study reveals that gadE and consequently AFI functions are heterogeneously expressed even in the absence of any prior acid stress, thereby preemptively creating acid-resistant subpopulations within a clonal E. coli population. Directed evolution efforts selecting for modulated gadE expression confirm that a gain-of-function mutation in the EvgS sensor kinase can constitutively upregulate gadE expression and concomitant acid resistance. However, we reveal that such upregulation of EvgS also causes cross-resistance to heat stress because of SafA-mediated cross-activation of the PhoPQ regulon. Surprisingly, loss of function of the serC gene (encoding phosphoserine/phosphohydroxythreonine aminotransferase) can also significantly upregulate gadE expression, acid resistance, and heat cross-resistance, although via a currently cryptic mechanism. As such, our data reveal a noisy expression of gadE in E. coli that is functional for the survival of sudden acid stress and that can readily be genetically tuned. IMPORTANCE Acidic environments constitute one of the most important stresses for enteric bacteria and can be encountered in both natural (e.g., host gastrointestinal tract) and manmade (e.g., food processing) environments. The enteric species Escherichia coli harbors many pathovars and is well known for its ability to cope with acid stress. In this study, we uncover that E. coli's acid fitness island (AFI), a genomic region that encodes important functions to deal with acid stress, is by default expressed in a heterogeneous manner. In fact, using microfluidics-based single-cell approaches, we further demonstrate that this heterogeneity preemptively creates a clonal subpopulation that is much better equipped to survive a sudden acid shock. In addition, we reveal that environments with recurring acid stress can readily select for mutants displaying a higher fraction of AFI-expressing cells. These new insights are important to properly understand and anticipate the survival characteristics of E. coli.

Magnetron-sputtered thin-film catalyst with low-Ir-Ru content for water electrolysis: Long-term stability and degradation analysis

Proton Exchange Membrane Water Electrolyzers (PEM-WEs) are entering the phase of commercial mass production. However, the issue of an iridium catalyst for the anode remains. This work presents an iridium-ruthenium-based catalyst (25% Ir = 158 μg cm−2, 75% Ru) prepared as a thin film on the surface-enhanced-anode of PEM-WE via magnetron sputtering. Using a strictly practical single-cell approach, we show its excellent activity – 1 A cm−2 at 1.606 V, 80 °C, and stability – 1.3 μV h−1 at 1 A cm−2 over the period of 500 h. Together with a purely Ir-based catalyst (158 μg cm−2), we subject it to a massive electrochemical and material analysis, showing that the thickness and interconnectivity are essential for the catalyst's stability. Consequently, we believe that magnetron sputtering is currently the most perspective method for preparing low-Ir-loading catalysts. Compared to the nanoparticle approach, it allows the creation of very thin films with unprecedented interconnectivity.

Transcriptional glucocorticoid effects in the brain: Finding the relevant target genes.

Glucocorticoids are powerful modulators of brain function. They act via mineralocorticoid and glucocorticoid receptors (MR and GR). These are best understood as transcription factors. Although many glucocorticoid effects depend on the modulation of gene transcription, it is a major challenge to link gene expression to function given the large-scale, apparently pleiotropic genomic responses. The extensive sets of MR and GR target genes are highly specific per cell type, and the brain contains many different (neuronal and non-neuronal) cell types. Next to the set "trait" of cellular context, the "state" of other active signaling pathways will affect MR and GR transcriptional activity. Here, we discuss receptor specificity and contextual factors that determine the transcriptional outcome of MR/GR signaling, experimental possibilities offered by single-cell transcriptomics approaches, and reflect on how to make sense of lists of target genes in relation to understanding the functional effects of steroid receptor activation.

Comparative single-cell analysis of the adult heart and coronary vasculature

The structure and function of the circulatory system, including the heart, have undergone substantial changes with the vertebrate evolution. Although the basic function of the heart is to pump blood through the body, its size, shape, speed, regeneration capacity, etc. vary considerably across species. Here, we address the differences among vertebrate hearts using a single-cell transcriptomics approach. Published datasets of macaque (Macaca fascicularis), mouse, and zebrafish hearts were integrated and compared to the human heart as a reference. While the three mammalian hearts integrated well, the zebrafish heart showed very little overlap with the other species. Our analysis revealed a mouse-specific cell subpopulation of ventricular cardiomyocytes (CM), represented by strikingly different expression patterns of specific genes related to high-energy metabolism. Interestingly, the observed differences between mouse and human CM coincided with actual biological differences between the two species. Smooth muscle and endothelial cells (EC) exhibited species-specific differences in clustering and gene expression, respectively, which we attribute to the tissues selected for sequencing, given different focuses of the original studies. Finally, we compared human and zebrafish heart-specific fibroblasts (FB) and identified a distinctively high expression of genes associated with heart regeneration following injury in zebrafish. Together, our results show that integration of numerous datasets of different species and different sequencing technologies is feasible and that this approach can identify species-specific differences and similarities in the heart.

Interoperable vocabulary for marine microbial flow cytometry

The recent development of biological sensors has extended marine plankton studies from conducting laboratory bench work to in vivo and real-time observations. Flow cytometry (FCM) has shed new light on marine microorganisms since the 1980s through its single-cell approach and robust detection of the smallest cells. FCM records valuable optical properties of light scattering and fluorescence from cells passing in a single file in front of a narrow-collimated light source, recording tens of thousands of cells within a few minutes. Depending on the instrument settings, the sampling strategy, and the automation level, it resolves the spatial and temporal distribution of microbial marine prokaryotes and eukaryotes. Cells are usually classified and grouped on cytograms by experts and are still lacking standards, reducing data sharing capacities. Therefore, the need to make FCM data sets FAIR (Findability, Accessibility, Interoperability, and Reusability of digital assets) is becoming critical. In this paper, we present a consensus vocabulary for the 13 most common marine microbial groups observed with FCM using blue and red-light excitation. The authors designed a common layout on two-dimensional log-transformed cytograms reinforced by a decision tree that facilitates the characterization of groups. The proposed vocabulary aims at standardising data analysis and definitions, to promote harmonisation and comparison of data between users and instruments. This represents a much-needed step towards FAIRification of flow cytometric data collected in various marine environments.

Glioma Stem Cells: Novel Data Obtained by Single-Cell Sequencing.

Glioma is the most common type of primary CNS tumor, composed of cells that resemble normal glial cells. Recent genetic studies have provided insight into the inter-tumoral heterogeneity of gliomas, resulting in the updated 2021 WHO classification of gliomas. Thorough understanding of inter-tumoral heterogeneity has already improved the prognosis and treatment outcomes of some types of gliomas. Currently, the challenge for researchers is to study the intratumoral cell heterogeneity of newly defined glioma subtypes. Cancer stem cells (CSCs) present in gliomas and many other tumors are an example of intratumoral heterogeneity of great importance. In this review, we discuss the modern concept of glioma stem cells and recent single-cell sequencing-driven progress in the research of intratumoral glioma cell heterogeneity. The particular emphasis was placed on the recently revealed variations of the cell composition of the subtypes of the adult-type diffuse gliomas, including astrocytoma, oligodendroglioma and glioblastoma. The novel data explain the inconsistencies in earlier glioma stem cell research and also provide insight into the development of more effective targeted therapy and the cell-based immunotherapy of gliomas. Separate sections are devoted to the description of single-cell sequencing approach and its role in the development of cell-based immunotherapies for glioma.