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Results for “single cell RNA sequencing”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2019

Single Cell RNA Sequencing Reveals Heterogeneity of Human MSC Chondrogenesis: Lasso Regularized Logistic Regression to Identify Gene and Regulatory Signatures

Bone marrow-derived mesenchymal stem cells (MSCs) exhibit the potential to undergo chondrogenesis in vitro, forming de novo tissues with a cartilage-like extracellular matrix that is rich in glycosaminoglycan and collagen type II. However, it is now apparent that MSCs comprise an inhomogeneous population of cells, and the fate of individual subpopulations during this differentiation process is not well understood. We analyzed the trajectory of MSC differentiation during chondrogenesis using single cell RNA sequencing (scRNA-seq). Using a machine learning technique - lasso regularized logistic regression - we showed that multiple subpopulations of cells existed at all stages during MSC chondrogenesis and were better-defined by transcription factor activity rather than gene expression. Trajectory analysis indicated that subpopulations of MSCs were not intrinsically specified or restricted, but instead remained multipotent and could differentiate into three main cell types: cartilage, hypertrophic cartilage, and bone. Lasso regularized logistic regression showed several advances in scRNA-seq analysis, namely identification of a small number of highly influential genes or transcription factors for downstream validation, and cell type classification with high accuracy. Additionally, we showed that MSC differentiation trajectory may exhibit donor to donor variation, although key influential pathways were comparable between donors. Our data provide an important resource to study gene expression and to deconstruct gene regulatory networks in MSC differentiation.

genomics↗

bioRxiv2020

Single-cell RNA sequencing deconvolutes the in vivo heterogeneity of human bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells, which have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat found in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte cells that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of transcriptomes suggest that osteoblast precursors may induce angiogenesis coupled with osteogenesis, and chondrocyte precursors may have the potential to differentiate into myocytes. We discovered transcripts for several cluster of differentiation (CD) markers that were highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs and could be novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing insight into the extent of their cellular heterogeneity and bone homeostasis.

cell biology↗

bioRxiv2023

Single-cell RNA sequencing reveals characteristics of myeloid cells in pulmonary post-acute sequelae of SARS-CoV-2

BackgroundAlthough our understanding of the immunopathology and subsequent risk and severity of COVID-19 disease is evolving, a detailed account of immune responses that contribute to the long-term consequences of pulmonary complication in COVID-19 infection remain unclear. Few studies have detailed the immune and cytokine profiles associated with post-acute sequalae of SARS-CoV-2 infection with persistent pulmonary symptoms (PPASC). However, the dysregulation of the immune system that drives pulmonary sequelae in COVID-19 survivors and PASC sufferers remains largely unknown. ResultsTo characterize the immunological features of pulmonary PASC (PPASC), we performed droplet-based single-cell RNA sequencing to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from participants naive to SARS-CoV-2 (Control) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC). We analyzed more than 34,139 PBMCs by integrating our dataset with previously reported control datasets (GSM4509024) cell distribution. In total, 11 distinct cell populations were identified based on the expression of canonical markers. The proportion of myeloid-lineage cells ([MLCs]; CD14+/CD16+monocytes and dendritic cells) was increased in PPASC compared to controls. MLCs from PPASC displayed up-regulation of genes associated with pulmonary symptoms/fibrosis, while glycolysis metabolism-related genes were downregulated. Similarly, pathway analysis showed that fibrosis- related (VEGF, WNT, and SMAD) and cell death pathways were up-regulated, but immune pathways were down-regulated in PPASC. In PPASC, we observed interactive VEGF ligand- receptor pairs among MLCs, and network modules in CD14+ (cluster 4) and CD16+ (Cluster 5) monocytes displayed a significant enrichment for biological pathways linked to adverse COVID- 19 outcomes, fibrosis, and angiogenesis. Further analysis revealed a distinct metabolic alteration in MLCs with a down-regulation of glycolysis/gluconeogenesis in PPASC compared to SARS- CoV-2 naive samples. ConclusionThis study offers valuable insights into the immune response and cellular landscape in PPASC. The presence of elevated MLC levels and their corresponding gene signatures associated with fibrosis, immune response suppression, and altered metabolic states suggests their potential role as a driver of PPASC.

microbiology↗

bioRxiv2019

Allele-specific single-cell RNA sequencing reveals different architectures of intrinsic and extrinsic gene expression noises

Gene expression noise refers to the variation of the expression level of a gene among isogenic cells in the same environment, and has two sources: extrinsic noise arising from the disparity of the cell state and intrinsic noise arising from the stochastic process of gene expression in the same cell state. Due to the low throughput of the existing method for measuring the two noise components, the architectures of intrinsic and extrinsic expression noises remain elusive. Using allele-specific single-cell RNA sequencing, we here estimate the two noise components of 3975 genes in mouse fibroblast cells. Our analyses verify predicted influences of several factors such as the TATA-box and microRNA targeting on intrinsic and extrinsic noises and reveal gene function-associated noise trends implicating the action of natural selection. These findings unravel differential regulations, optimizations, and biological consequences of intrinsic and extrinsic noises and can aid the construction of desired synthetic circuits.

bioinformatics↗

bioRxiv2020

Single cell RNA sequence analysis of human bone marrow samples reveals new targets for isolation of skeletal stem cells using DNA-coated gold nanoparticles

There is a wealth of data indicating human bone marrow derived stromal cells (HBMSCs) contain the skeletal stem cell (SSC) with the potential to differentiate along the stromal osteogenic, adipogenic and chondrogenic lineages. However, despite these advances, current methods to isolate skeletal stem cells (SSCs) from human tissues have proved challenging as no single specific marker has been identified limiting understanding of SSC fate, immunophenotype and the widespread clinical application of these cells. While a number of cell surface markers can enrich for SSCs, none of the proposed markers, alone, provide a platform to isolate single cells with the ability to form bone, cartilage, and adipose tissue in humans. The current study details the application of oligonucleotide-coated nanoparticles, spherical nucleic acids (SNAs), to rapidly isolate human cells using mRNAs signatures detected in SSCs in real time, to identify stem and progenitor skeletal populations using single cell RNA sequencing. Based on scRNA-seq of samples from 11 patients, this method was able to identify novel targets for SSC enrichment, which were assessed in a total of 80 patients. This methodology was able to isolate potential SSCs found at a frequency of <1 in 1,000,000 in human bone marrow, with a capacity for tri-lineage differentiation in vitro. The current approach provides new targets and a platform to advance SSC isolation, enrichment with significant therapeutic impact therein.

cell biology↗

bioRxiv2025

Single-cell RNA sequencing identifies progenitor dysfunction, inflammation and premature aging in ex vivo airway epithelium-derived from transplant recipients

Pulmonary dysfunction is a common complication following hematopoietic stem cell transplantation (HSCT) or lung transplantation (LT). Bronchiolitis obliterans syndrome (BOS), an alloimmune complication of transplantation, with complex pathophysiology, contributes substantially to morbidity and mortality in these settings. We aimed to identify common epithelial features predisposing to BOS by comparing epithelia from HSCT and LT without BOS and non-transplant (NT) individuals. We developed an ex vivo model of human airway epithelia (HAE) reconstituted from bronchial biopsies from 6 patients of each group. Using single-cell RNA sequencing, we identified two distinct epithelial profiles among transplant recipients: one resembling NT epithelium and another displaying altered cellular composition and transcriptional signatures across basal, suprabasal, club and submucosal basal duct cells, which were even more pronounced in a patient who subsequently developed BOS. This latter subgroup exhibited dysregulation of epithelial-mesenchymal transition, TNF/NFB signaling and inflammatory pathways, suggesting impaired epithelial function. Moreover, these HAE demonstrated premature epithelial aging and increased expression of genes encoding damage associated molecular patterns. Together, these findings indicate that epithelial abnormalities specific to certain transplant recipients may contribute to BOS development. Although causality cannot yet be definitively established, our data highlight the airway epithelium as a site of sustained post-transplant injury and reveal potential molecular mechanisms underlying BOS pathogenesis.

molecular biology↗

bioRxiv2023

Single-cell RNA sequencing reveals aortic cellular heterogeneity in MSC_ITGB3-treated ApoE-/- mice on a high-fat diet

The aorta contains various cell types that are involved in the development of vascular inflammation and atherosclerosis. However, the cellular atlas of heterogeneous aorta cells, cellular responses, and intercellular communication has not been investigated in the background of a high-fat diet (HFD) and treatment with integrin beta 3-modified mesenchymal stem cells (MSC_ITGB3). In this study, 33,782 individual cells from mouse aortas under HFD with or without MSC_ITGB3 treatment were subjected to single-cell RNA sequencing as an unbiased analysis strategy. We generated a compendium of 30 different clusters, mainly smooth muscle cells (SMCs), endothelial cells, and immune cells. The proportion of the different cell types was considerably influenced by HFD and MSC_ITGB3. In the HFD group, genes associated with proliferation, migration, and collagen were highly expressed in the major SMC subpopulations. However, the expression of contraction-related genes in SMC subpopulations was significantly higher in the MSC_ITGB3 group than in the HFD group. After HFD consumption, subpopulations of ECs with active PI3K-Akt signaling pathway, ECM-receptor interaction, and contraction-related genes were significantly enriched. In the MSC_ITGB3 group, the number of dendritic cells (DCs), which are positively correlated with atherosclerotic lesion progression and contribute to lipid accumulation, and levels of inflammatory factors notably decreased. Our findings provide data on the composition, signaling pathways, and cellular communication of the aorta cells following stem cell treatment as well as on the evolution and progression of atherosclerotic disease. The findings may help in improving the treatment of atherosclerosis.

bioinformatics↗

bioRxiv2025

Single-Cell RNA Sequencing Reveals Hemocyte Heterogeneity, Differentiation Trajectories, and Viral Tropism in Shrimp (Macrobrachium rosenbergii) Infected with Decapod Iridescent Virus 1

Decapod Iridescent Virus 1 (DIV1) is a highly pathogenic virus threatening crustacean aquaculture. Hemocytes, the primary immune cells in shrimp, play crucial roles in host defense, yet their transcriptional heterogeneity and differentiation dynamics under viral infection remain poorly understood. Here, we characterize hemocyte subpopulations in Macrobrachium rosenbergii before and after DIV1 infection at single-cell resolution. We identified 12 distinct hemocyte clusters with unique molecular signatures. DIV1 infection induced significant shifts in hemocyte composition, with clusters 0, 5, 6, and 8 expanding, while clusters 9 and 10 exhibited resistance. Clusters 0, 5, and 8 were highly susceptible, showing elevated viral gene expression. Pseudo-time analysis revealed that DIV1 accelerates hemocyte differentiation, driving prohemocytes and semi-granulocytes toward terminally differentiated granulocytes and hyalinocytes, particularly the immune-active HC4 subpopulation. Functional enrichment analysis showed that susceptible hemocytes were associated with viral processes, oxidative stress, and phagocytosis, while resistant clusters exhibited distinct immune signatures. Furthermore, knockdown experiments confirmed the antiviral roles of PPO2, RAB11B, LAMP1, and Dorsal, as their silencing led to increased viral loads, higher MCP protein levels, and reduced shrimp survival. Taken together, our study provides the first single-cell resolution atlas of shrimp hemocytes under DIV1 infection, revealing their transcriptional heterogeneity, differentiation trajectories, and immune responses. These findings offer novel insights into shrimp antiviral immunity and lay the foundation for disease-resistant breeding and immunostimulatory strategies in aquaculture. ImportancesDecapod Iridescent Virus 1 (DIV1) is a highly pathogenic virus threatening crustacean aquaculture, causing severe economic losses. Hemocytes, the primary immune cells in shrimp, play crucial roles in antiviral defense, yet their diversity, differentiation, and responses to viral infection remain poorly understood. In this study, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively characterize hemocyte subpopulations in Macrobrachium rosenbergii before and after DIV1 infection. We identified 12 transcriptionally distinct hemocyte clusters and revealed significant changes in hemocyte composition following infection. Notably, we discovered that specific clusters (0, 5 and 8) were highly susceptible to DIV1, while others (9 and 10) exhibited resistance. Additionally, we reconstructed hemocyte differentiation trajectories and found that DIV1 infection drives hematopoiesis, accelerating the transition of progenitor cells into terminally differentiated immune-active hemocytes. Functional analysis highlighted key antiviral effectors, including PPO2, RAB11B, LAMP1, and Dorsal, which were significantly upregulated in response to infection. These findings provide novel insights into shrimp antiviral immunity and viral pathogenesis, offering potential targets for disease-resistant breeding and immunostimulatory interventions.

immunology↗

bioRxiv2021

Single-cell RNA sequencing uncovers the excitatory/inhibitory synaptic unbalance in the retrosplenial cortex after peripheral nerve injury

Nerve injury in the somatosensory pathway may induce maladaptive changes at the transcriptional or protein level, contributing to the development and maintenance of neuropathic pain. In contrast to the retrosplenial cortex (RSC), which processes nociceptive information and exhibits structural and molecular changes after nerve injury, detailed transcriptional changes in the RSC are not yet known. Here we confirm the involvement of the RSC in regulating pain sensation and observe that the same peripheral stimulation activates more retrosplenial neurons after nerve injury; reducing the activities of CaMKII+ splenial cells relieves peripheral pain hypersensitivity after nerve injury. Using a single-cell RNA sequencing (scRNA-seq) approach, we identified cell-type-specific gene expression changes after nerve injury, and the gene set enrichment analysis results revealed suppressed ion homeostasis in CaMKII+ neurons. Furthermore, examination of the expression of genes encoding ligand-gated ion channels showed a decrease in Gabar1a but an increase in Gria1 in CaMKII+ neurons; consistently, we confirmed the unbalanced excitatory/inhibitory synaptic transmission by using the electrophysiological recording approach. Moreover, micro-infusion of 1-Naphthyl acetyl spermine in the RSC to reduce excitatory synaptic transmission alleviated peripheral pain hypersensitivity. Our data confirm the involvement of the RSC in pain regulation and provide information on cell type-dependent transcriptomic changes after nerve injury, which will contribute to the understanding of the mechanisms mediating neuropathic pain.

neuroscience↗

bioRxiv2022

Single cell RNA sequencing reveals mechanisms underlying a senescence-like phenotype of Alveolar Macrophages during Aging

Alveolar Macrophages (AMs) are unique innate immune cells that reside in the alveolar space and accommodate the ever-changing needs of the lungs against internal and external challenges. During homeostasis, AMs maintain themselves through self-renewal without input from adult hematopoietic stem cells. Currently, little is known regarding the influence of aging on AM dynamics, heterogeneity and transcriptional profiles. Here, we identified CBF{beta} as an indispensable transcription factor that ensures AM self-renewal. Deficiency in CBF{beta} led to decreased proliferation and self-renewal ability of AMs. Moreover, with single cell RNA sequencing analysis of AMs from young and aged mice, we discovered that despite similarities in the transcriptome of proliferating cells, AMs from the aged mice exhibited reduced embryotic stem cell-like features. Aged AMs also showed diminished capacity for DNA repair, potentially contributing to impaired cell cycle progression and elevation of senescence markers. In accordance with the analysis, we observed reduced number of AMs in aged mice, which had defective self-renewal ability and were more sensitive to the reduction of GM-CSF. Interestingly, decreased CBF{beta} was observed in the cytosol of AMs from aged mice. A similar senescence-like phenotype was also found in human AMs. Taken together, we conclude that AMs in the aged host harbor a senescence-like phenotype, potentially mediated by the activity of CBF{beta}. HighlightsO_LIscRNAseq revealed Alveolar Macrophage (AM) heterogeneity and self-renewal C_LIO_LICBF{beta} is associated with AM cell cycle and facilitate AM self-renewal C_LIO_LIAMs displayed a senescence-like phenotype during physiological Aging C_LIO_LIAging impairs CBF{beta} expression in mouse and human AMs C_LI

immunology↗

bioRxiv2023

Single-cell RNA sequencing reveals HIF1A as a severity-sensitive immunological scar in circulating monocytes of convalescent comorbidity-free COVID-19 patients

COVID-19, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is characterized by a wide range of clinical symptoms and a poorly predictable disease course. Although in-depth transcriptomic investigations of peripheral blood samples from COVID-19 patients have been performed, the detailed molecular mechanisms underlying an asymptomatic, mild or severe disease course, particularly in patients without relevant comorbidities, remain poorly understood. While previous studies have mainly focused on the cellular and molecular dissection of ongoing COVID-19, we set out to characterize transcriptomic immune cell dysregulation at the single-cell level at different time points in patients without comorbidities after disease resolution to identify signatures of different disease severities in convalescence. With single-cell RNA sequencing we reveal a role for hypoxia-inducible factor 1-alpha (HIF1A) as a severity-sensitive long-term immunological scar in circulating monocytes of convalescent COVID-19 patients. Additionally, circulating complexes formed by monocytes with either T cells or NK cells represent a characteristic cellular marker in convalescent COVID-19 patients irrespective of their preceding symptom severity. Together, these results provide cellular and molecular correlates of recovery from COVID-19 and could help in immune monitoring and in the design of new treatment strategies.

immunology↗

bioRxiv2024

Investigating Subpopulation Dynamics in Clonal CHO-K1 Cells with Single-Cell RNA Sequencing

Chinese Hamster Ovary (CHO) cells are used to produce monoclonal antibodies and other biotherapeutics at industrial scale. Despite their ubiquitous nature in the biopharmaceutical industry, little is known about the behaviors of individual transfected clonal CHO cells. Most CHO cells are assessed on their ability to produce the protein of interest over time, known as their stability. But these CHO cells have primarily been studied in bulk, working under the assumption that these bulk samples are identical because of genetic clonality across the sample; however, this does not address other forms of cellular heterogeneity in these ostensibly clonal cells. It is possible these variable stability phenotypes reflect heterogeneity within the clonal samples. In this study, we performed single-cell RNA sequencing on two clonal CHO-K1 cell populations with different stability phenotypes over a 90 day culture period. Our data showed that the instability of the unstable clone was due in part to the emergence of a low-producing subpopulation in the aged samples. This low-producing subpopulation did not exhibit markers of cellular stress which were expressed in the higher-producing populations. Further multiomic investigation should be performed to better characterize this heterogeneity.

genomics↗

bioRxiv2025

Transcriptome comparison between the cultured and in vivo Chick Primordial Germ Cells by SMART-seq-based single cell RNA sequencing

Primordial germ cells (PGC), the precursors of the germline, have unique cellular characteristics to undergo long-distance migration to the embryonic gonads and have the potential to differentiate into somatic cells. Among the animal models studying PGC development, the chicken PGCs are an ideal model, since it is a rare model in which long-term PGC cultivation is applicable. Although the cultural applicability of chicken PGC makes it attractive for revealing the PGC character and its developmental processes, some differences from endogenous PGCs are known, such as the remarkable up-regulation of cell proliferation and a lesser ability to reach the gonads. Understanding these differences at the molecular level is crucial. To this end, we first performed SMART-seq-based single-cell RNA sequencing to compare transcriptomes between endogenous PGCs and cultivated PGCs. Our results revealed that PGC cultivation causes a shift from a MYC-dependent to a MYCN-dependent gene regulatory network in PGCs, suggesting that this reprogramming contributes to the acquisition of proliferation ability and stem cell characteristics in cultivated PGCs. Additionally, our results suggest that the MYCN-dependent gene regulatory network increases the risk of somatic differentiation, particularly in neural fate, in cultivated PGCs. In addition, our transcriptome analysis identified new cell populations that show molecular character as intermediate cell states between germline and pluripotent cells from the early embryonic stage. Thus, our study provides fundamental molecular information to understand both the effects of PGC cultivation and the developmental process of chicken PGCs.

developmental biology↗

bioRxiv2021

Single-cell RNA Sequencing Reveals Immunosuppressive Myeloid Cell Diversity and Restricted Cytotoxic Effector Cell Trafficking and Activation During Malignant Progression in Glioma

Low grade gliomas (LGG) account for about two-thirds of all glioma diagnoses in adolescents and young adults (AYA) and malignant progression of these patients leads to dismal outcomes. Recent studies have shown the importance of the dynamic tumor microenvironment in high-grade gliomas (HGG), yet its role is still poorly understood in low-grade glioma malignant progression. Here, we investigated the heterogeneity of the immune microenvironment using a platelet-derived growth factor (PDGF)-driven RCAS (replication-competent ASLV long terminal repeat with a splice acceptor) glioma model that recapitulates the malignant progression of low to high-grade glioma in humans and also provides a model system to characterize immune cell trafficking and evolution. To illuminate changes in the immune cell landscape during tumor progression, we performed single-cell RNA sequencing on immune cells isolated from animals bearing no tumor (NT), LGG and HGG, with a particular focus on the myeloid cell compartment, which is known to mediate glioma immunosuppression. LGGs demonstrated significantly increased infiltrating T cells, CD4 T cells, CD8 T cells, B cells, and natural killer cells in the tumor microenvironment, whereas HGGs significantly abrogated this infiltration. Our study identified two distinct macrophage clusters in the tumor microenvironment; one cluster appeared to be bone marrow-derived while another was defined by overexpression of Trem2, a marker of tumor associated macrophages. Our data demonstrates that these two distinct macrophage clusters show an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG which evolves to an immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG that restricts T cell recruitment and activation. We identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Interestingly, these results were mirrored by our analysis of the TCGA dataset, which demonstrated a statistically significant association between CD74 overexpression and decreased overall survival in AYA patients with grade II gliomas. Targeting immunosuppressive myeloid cells and intra-tumoral macrophages within this therapeutic window may ameliorate mechanisms associated with immunosuppression before and during malignant progression.

cancer biology↗

bioRxiv2024

Single cell RNA sequencing reveals immunomodulatory mechanism of stem cell factor and granulocyte colony-stimulating factor treatment in the brains of aged APP/PS1 mice

Alzheimers disease (AD) leads to progressive neurodegeneration and dementia. AD primarily affects older adults with neuropathological changes including amyloid-beta (A{beta}) deposition, neuroinflammation, and neurodegeneration. We have previously demonstrated that systemic treatment with combined stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF), reduces A{beta} load, increases A{beta} uptake by activated microglia and macrophages, reduces neuroinflammation, and restores dendrites and synapses in the brains of aged APPswe/PS1dE9 (APP/PS1) mice. However, the mechanisms underlying SCF+G-CSF-enhanced brain repair in aged APP/PS1 mice remain unclear. This study used a transcriptomic approach to identify potential mechanisms by which SCF+G-CSF treatment modulates microglia and peripheral myeloid cells to mitigate AD pathology in the aged brain. After injections of SCF+G-CSF for 5 consecutive days, single-cell RNA sequencing was performed on CD11b+ cells isolated from the brains of 28-month-old APP/PS1 mice. The vast majority of cell clusters aligned with transcriptional profiles of microglia in various activation states. However, SCF+G-CSF treatment dramatically increased a cell population showing upregulation of marker genes related to peripheral myeloid cells. Flow cytometry data also revealed an SCF+G-CSF-induced increase of cerebral CD45high/CD11b+ active phagocytes. SCF+G-CSF treatment robustly increased the transcription of genes implicated in immune cell activation, including gene sets that regulate inflammatory processes and cell migration. Expression of S100a8 and S100a9 were robustly enhanced following SCF+G-CSF treatment in all CD11b+ cell clusters. Moreover, the topmost genes differentially expressed with SCF+G-CSF treatment were largely upregulated in S100a8/9-positive cells, suggesting a well-conserved transcriptional profile related to SCF+G-CSF treatment in resident and peripherally derived CD11b+ immune cells. This S100a8/9-associated transcriptional profile contained notable genes related to pro-inflammatory and anti-inflammatory responses, neuroprotection, and A{beta} plaque inhibition or clearance. Altogether, this study reveals immunomodulatory effects of SCF+G-CSF treatment in the aged brain with AD pathology, which will guide future studies to further uncover the therapeutic mechanisms.

neuroscience↗

bioRxiv2018

SymSim: simulating multi-faceted variability in single cell RNA sequencing

The abundance of new computational methods for processing and interpreting transcriptomes at a single cell level raises the need for in-silico platforms for evaluation and validation. Simulated datasets which resemble the properties of real datasets can aid in method development and prioritization as well as in questions in experimental design by providing an objective ground truth. Here, we present SymSim, a simulator software that explicitly models the processes that give rise to data observed in single cell RNA-Seq experiments. The components of the SymSim pipeline pertain to the three primary sources of variation in single cell RNA-Seq data: noise intrinsic to the process of transcription, extrinsic variation that is indicative of different cell states (both discrete and continuous), and technical variation due to low sensitivity and measurement noise and bias. Unlike other simulators, the parameters that govern the simulation process directly represent meaningful properties such as mRNA capture rate, the number of PCR cycles, sequencing depth, or the use of unique molecular identifiers. We demonstrate how SymSim can be used for benchmarking methods for clustering and differential expression and for examining the effects of various parameters on their performance. We also show how SymSim can be used to evaluate the number of cells required to detect a rare population and how this number deviates from the theoretical lower bound as the quality of the data decreases. SymSim is publicly available as an R package and allows users to simulate datasets with desired properties or matched with experimental data.

bioinformatics↗

bioRxiv2022

Single-cell RNA sequencing highlights a reduced function of natural killer and cytotoxic T cells in recovered COVID-19 pregnant women

Pregnancy is a complex phenomenon during which women undergo immense immunological change throughout this period. Having an infection with the SARS-CoV-2 virus leads to an additional burden on the highly stretched immune response. Some studies suggest that age-matched pregnant women are more prone to SARS-CoV-2 infection compared with normal healthy (non-pregnant) women, while alternative evidence proposed that pregnant women are neither susceptible nor develop severe symptoms. This discrepancy in different findings regarding the immune responses of pregnant women infected with SARS-CoV-2 virus is not well understood. In this study, we investigated how SARS-CoV-2 viral infection could modulate the immune landscape during the active infection phase and recovery in pregnant females. Using flow cytometry, we identified that intermediate effector CD8+ T cells were increased in pregnant women who had recovered from COVID-19 as opposed to those currently infected. Similarly, an increase in CD4+ T helper cells (early or late) during the recovered phase was observed during the recovery phase compared with infected pregnant women or healthy pregnant women, whilst infected pregnant women had a reduced number of late effector CD4+ T cells. CD3+CD4- CD8-NKT cells that diminished during active infection in contrast to healthy pregnant women were significant increase in recovered COVID-19 recovered pregnant women. Further, our single-cell RNA sequencing data revealed that infection of SARS-CoV-2 had changed the gene expression profile of monocytes, CD4+ effector cells and antibody producing B cells in convalescent as opposed to healthy pregnant women. Additionally, several genes with cytotoxic function, interferon signalling type I & II, and pro- and anti-inflammatory functions in natural killer cells and CD8+ cytotoxic T cells were compromised in recovered patients compared with healthy pregnant women. Overall, our study highlights that SARS-CoV-2 infection deranged the adaptive immune response in pregnant women and could be implicated in pregnancy complications in ongoing pregnancies.

immunology↗

bioRxiv2020

Molecular determinants and heterogeneity of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of non-circulating tissue-resident memory (TRM) cells that mediate potent protection within non-lymphoid tissues. Here, we utilized single-cell RNA-sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several states of differentiation, representing functionally distinct TRM cell subsets as well as a subset of TRM cell precursors within the tissue early in infection. Taken together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer. One sentence summaryHere, we applied single-cell RNA-sequencing to elucidate the gene expression patterns of individual CD8+ T cells differentiating throughout the course of infection in the spleen and small intestinal epithelium, which revealed previously unidentified molecular determinants of tissue-resident T cell differentiation as well as functional heterogeneity within the tissue-resident CD8+ T cell population.

immunology↗