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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2020

Single-cell RNA sequencing of Tocilizumab-treated peripheral blood mononuclear cells as an in vitro model of inflammation

COVID-19 has posed a significant threat to global health. Early data has revealed that IL-6, a key regulatory cytokine, plays an important role in the cytokine storm of COVID-19. Multiple trials are therefore looking at the effects of Tocilizumab, an IL-6 receptor antibody that inhibits IL-6 activity, on treatment of COVID-19, with promising findings. As part of a clinical trial looking at the effects of Tocilizumab treatment on kidney transplant recipients with subclinical rejection, we performed single-cell RNA sequencing of comparing stimulated PBMCs before and after Tocilizumab treatment. We leveraged this data to create an in vitro cytokine storm model, to better understand the effects of Tocilizumab in the presence of inflammation. Tocilizumab-treated cells had reduced expression of inflammatory-mediated genes and biologic pathways, particularly amongst monocytes. These results support the hypothesis that Tocilizumab may hinder the cytokine storm of COVID-19, through a demonstration of biologic impact at the single-cell level.

genomics↗

bioRxiv2024

Single-cell RNA Sequencing Reveals Methamphetamine Inhibits the Liver Immune Response with Involvement of the Dopamine D1 Receptor

Methamphetamine (METH) is a highly addictive psychostimulant that causes physical and psychological damage and immune system disorder, especially in the liver, which contains a significant number of immune cells. Dopamine, which is a key neurotransmitter in METH addiction and immune regulation, plays a crucial role in this process. In this study, we developed a chronic METH administration model and conducted single-cell RNA sequencing to investigate the effect of METH on liver immune cells and the involvement of the dopamine receptor D1 (DRD1) in this process. Our findings revealed that chronic exposure to METH induced an immune cell shift from Ifitm3+Mac and Ccl5+Mac to Cd14+Mac, and from Fyn+CD4+Teff, CD8+T, and NKT to Fos+CD4+T and Rora+ILC2, along with suppression of multiple immune functional pathways. DRD1 was implicated in the regulation of some of these pathways and the shifts of hepatic immune cells. This research provides valuable insights into the development of therapies aimed at mitigating METH-induced immune impairment.

immunology↗

bioRxiv2021

Diversity of receptor expression in central and peripheral mouse neurons estimated from single cell RNA sequencing

Because somatosensory PNS neurons, in particular nociceptors, are specially tuned to be able to detect a wide variety of both exogenous and endogenous signals, it is widely assumed that these neurons express a greater variety of receptor genes. Because cells detect such signals via cell surface receptors, we sought to formally test the hypothesis that PNS neurons might express a broader array of cell surface receptors than CNS neurons using existing single cell RNA sequencing resources from mouse. We focused our analysis on ion channels, G-protein coupled receptors (GPCRS), receptor tyrosine kinase and cytokine family receptors. In partial support of our hypothesis, we found that mouse PNS somatosensory, sympathetic and enteric neurons and CNS neurons have similar receptor expression diversity in families of receptors examined, with the exception of GPCRs and cytokine receptors which showed greater diversity in the PNS. Surprisingly, these differences were mostly driven by enteric and sympathetic neurons, not by somatosensory neurons or nociceptors. Secondary analysis revealed many receptors that are very specifically expressed in subsets of PNS neurons, including some that are unique among neurons for nociceptors. Finally, we sought to examine specific ligand-receptor interactions between T cells and PNS and CNS neurons. Again, we noted that most interactions between these cells are shared by CNS and PNS neurons despite the fact that T cells only enter the CNS under rare circumstances. Our findings demonstrate that both PNS and CNS neurons express an astonishing array of cell surface receptors and suggest that most neurons are tuned to receive signals from other cells types, in particular immune cells.

neuroscience↗

bioRxiv2026

Single-cell RNA sequencing of peripheral blood cells identifies transcriptomic signatures in Parkinson's disease

Parkinsons disease (PD) is a progressive age-related neurodegenerative disorder characterized by both motor and non-motor symptoms. The poorly understood prodromal period, decades-long progression, and disease-phenotype heterogeneity continue to impede the development of preventive and curative therapies. A growing appreciation of immune system changes during the progression of PD suggests that evaluating peripheral immune cells may help identify signatures relevant to disease etiology. We employed single-cell RNA sequencing to profile the transcriptomes of peripheral blood mononuclear cells (PBMCs) from a cohort of 12 patients with PD and 12 healthy controls, equally distributed by sex. Analysis identified gene expression signatures specific to immune cell lineages in PD when compared with healthy controls. Analysis of the dataset indicated that aspects of the PD-related changes were associated with sex, including metabolic and inflammatory changes. Further analysis of myeloid and T cell subsets identified additional pathways and gene expression profiles associated with PD. Trajectory analysis of the myeloid and T cell datasets indicated significant changes in the distribution of cells across states of gene expression in PD compared with controls. This work provides new evidence of peripheral immune cell changes in PD utilizing high-resolution transcriptomics in a cohort powered to analyze sex as a variable. HighlightsTranscriptomic dataset in a cohort powered to analyze immune phenotype in Parkinsons disease Parkinsons disease-specific gene expression signatures in peripheral immune cell lineages Identification of sex differences in the immune cell transcriptome in Parkinsons disease Trajectory analysis identifies changes in immune cell phenotypic distribution in Parkinsons monocytes

immunology↗

bioRxiv2020

Single cell RNA sequencing redefines the mesenchymal cell landscape of mouse endometrium

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration and remodelling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. In the current study we used the Pdgfrb-BAC-eGFP transgenic reporter mouse in combination with bulk and single cell RNA sequencing (scRNAseq) to redefine the endometrial mesenchyme. Contrary to previous reports we show that CD146 is expressed in both PDGFR{beta}+ perivascular cells as well as CD31+ endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, Abcc9). scRNAseq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFR+/CD34+ but were distinct in their expression of Spon2/Angptl7 (fibroblast 1), Smoc2/Rgs2 (fibroblast 2) and Clec3b/Col14a1/Mmp3 (fibroblast 3), with potential functions in regulation of immune responses, response to wounding and organisation of extracellular matrix respectively. In conclusion, these data are the first to provide a single cell atlas of the mesenchymal cell landscape in mouse endometrium. By identifying novel markers for subpopulations of mesenchymal cells we can use mouse models investigate their contribution to endometrial function, compare with other tissues and apply these findings to further our understanding of human endometrium. HighlightsO_LIGFP expression in the mouse endometrium, under the control of the Pdgfrb promoter, is restricted to two cell populations based on the intensity of GFP with GFPbright cells close to the vasculature C_LIO_LISingle cell RNAseq identified five subpopulations of GFP+ mesenchymal cells: pericytes, vascular smooth muscle cells (vSMC) and three closely related but distinct populations of fibroblasts C_LIO_LIBioinformatics revealed that pericytes and vSMC share functions associated with the circulatory system, actin-filament process and cell adhesion, and an apparent role for pericytes in smooth muscle cell migration and response to interferons C_LIO_LIComparisons between the fibroblast subpopulations suggest distinct roles in regulation of immune response, response to wound healing and collagen organisation. C_LI Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

physiology↗

bioRxiv2019

Characterization of human-iPSCs derived spinal motor neurons by single-cell RNA sequencing

Human induced pluripotent stem cells (iPSCs) offer the opportunity to generate specific cell types from healthy and diseased individuals, allowing the study of mechanisms of early human development, modelling a variety of human diseases, and facilitating the development of new therapeutics. Human iPSC-based applications are often limited by the variability among iPSC lines originating from a single donor, as well as the heterogeneity among specific cell types that can be derived from iPSCs. The ability to deeply phenotype different iPSC-derived cell types is therefore of primary importance to the successful and informative application of this technology. Here we describe a combination of motor neuron (MN) derivation and single-cell RNA sequencing approaches to generate and characterize specific MN subtypes obtained from human iPSCs. Our studies provide evidence for rapid and robust generation of MN progenitor cells that can give rise to a heterogenous population of brainstem and spinal cord MNs. Approximately 58% of human iPSC-derived MNs display molecular characteristics of lateral motor column MNs, [~]19% of induced MNs resemble hypaxial motor column MNs, while [~]6% of induced MNs have features of medial motor column MNs. The present study has the potential to improve our understanding of iPSC-derived MN subtype function and dysfunction, possibly leading to improved iPSC-based applications for the study of human MN biology and diseases.

neuroscience↗

bioRxiv2021

Single-cell RNA sequencing reveals a role for reactive oxygen species and peroxiredoxins in fatty-acid-induced rat β-cell proliferation

The functional mass of insulin-secreting pancreatic {beta} cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing {beta} cells. To decipher the underlying molecular mechanisms, we assessed {beta}-cell proliferation in isolated rat islets exposed to glucose and oleate or palmitate for 48 h and analyzed the transcriptional response by single-cell RNA sequencing. Unsupervised clustering of pooled {beta} cells identified subpopulations, including proliferating {beta} cells. {beta}-cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved energy metabolism and mitochondrial activity. Comparison of proliferating vs. non-proliferating {beta} cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, the antioxidant N-acetyl cysteine and the peroxiredoxin inhibitor Conoidin A both blocked oleate-induced {beta}-cell proliferation. Our data reveal a key role for ROS signaling in {beta}-cell proliferation in response to nutrients.

cell biology↗

bioRxiv2022

Single-cell RNA sequencing analyses of primary cutaneous B-cell disorders reveal distinct molecular patterns consistent with clinical behavior

Primary cutaneous B-cell lymphomas comprise a heterogeneous group of extranodal non-Hodgkin lymphomas. While primary cutaneous diffuse large B-cell lymphoma leg type (pcDLBCL-LT) is highly aggressive, the two other subtypes, primary cutaneous follicle centre lymphoma (pcFCL) and primary cutaneous marginal zone lymphoma (pcMZL), usually follow an indolent course. To better understand the molecular landscape of these entities, we performed single-cell RNA sequencing of pcFCL, pcMZL, and pcDLBCL-LT skin lesions and compared them to B-cell rich lymphoid proliferation (rB-LP) lesions, gastric MALT lymphoma, nodal FCL, and nodal DLBCL. Our data show that these lymphomas can be clearly distinguished from each other on a transcriptomic level based on scRNA-seq. pcMZL, pcFCL, and rB-LP all exhibited a persistent germinal centre reaction as evidenced by the presence of required support cells and continuous somatic hypermutation within the expanded clone. By contrast, malignant clones of pcDLBCL-LT and gastric MALT lymphoma lesions lacked these features. Further, pcMZL top expanded clones were developing within lesions from naive and not post-germinal centre B cells as currently presumed. Therefore, pcMZL may represent a non-malignant reaction against a yet to be determined antigen. Conversely, in pcFCL, B cells showed a larger amount of clonal expansion. The lack of further differentiation of these B cells may explain its indolent clinical course. In contrast to pcDLCBL-LT, our data thus indicate that pcMZL and pcFCL, similar to rB-LP are characterised by a functional germinal centre reaction likely driven by (a yet unknown) antigen recognition, which supports the classification of pcMZL as a lymphoproliferative disease. Key pointsO_LIIndolent B cell neoplasms are uniquely characterised by ongoing, antigen-driven germinal centre reactions. C_LIO_LIPrimary cutaneous B cell lymphomas are distinct entities compared to their systemic counterparts. C_LI

cancer biology↗

bioRxiv2022

Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor, a candidate mediator of hearing restoration mechanisms.

Hearing loss caused by the death of cochlear hair cells might be restored through regeneration from supporting cells via dedifferentiation and proliferation, as observed in birds. We recently found that in mice, activation of ERBB2 in supporting cells promoted the differentiation of hair cell-like cells. Here we analyze transcriptomes of neonatal mouse cochlear supporting cells with activated ERBB2 using single-cell RNA sequencing. ERBB2 induction in vivo generated a new population of cells expressing de novo SIBLING (small integrin-binding ligand n-linked glycoproteins) proteins and their regulators, particularly Secreted Phosphoprotein 1 (SPP1). In other systems, SIBLINGs promote cell survival, proliferation, and differentiation. ERBB2 signaling induced after noise exposure in young adult mice also up-regulated both SPP1 protein and the SPP1 receptor CD44, and drove formation of proliferating stem-like cell aggregates in the organ of Corti. Our results suggest that ectopic activation of ERBB2 signaling in cochlear supporting cells alters the microenvironment, promoting proliferation and cell rearrangements. Together these results suggest a novel mechanism for inducing stem cell-like activity in the adult mammalian cochlea.

neuroscience↗

bioRxiv2018

Single cell RNA sequencing reveals a novel, microglia-like cell type in cerebrospinal fluid during virologically suppressed HIV

Central nervous system (CNS) immune activation in an important driver of neuronal injury during several neurodegenerative and neuroinflammatory diseases. During HIV infection, CNS immune activation is associated with high rates of neurocognitive impairment, even with sustained long-term suppressive antiretroviral therapy (ART). However, the cellular subsets that drive immune activation and neuronal damage in the CNS during HIV infection and neurological conditions remain unknown, in part because CNS cells are difficult to access in living humans. Using single cell RNA sequencing (scRNA-seq) on cerebrospinal fluid (CSF) and blood from adults with HIV, we identified a rare (<5% of cells) subset of myeloid cells in CSF presenting a gene expression signature consistent with neurodegenerative disease associated microglia. This highlights the power of scRNA-seq of CSF to identify rare CNS immune cell subsets that may perpetuate neuronal injury during HIV infection and other conditions.

immunology↗

bioRxiv2018

Bayesian Inference for a Generative Model of Transcriptome Profiles from Single-cell RNA Sequencing

Transcriptome profiles of individual cells reflect true and often unexplored biological diversity, but are also affected by noise of biological and technical nature. This raises the need to explicitly model the resulting uncertainty and take it into account in any downstream analysis, such as dimensionality reduction, clustering, and differential expression. Here, we introduce Single-cell Variational Inference (scVI), a scalable framework for probabilistic representation and analysis of gene expression in single cells. Our model uses variational inference, stochastic optimization and deep neural networks to approximate the parameters that govern the distribution of expression values of each gene in every cell, using a non-linear mapping between the observations and a low-dimensional latent space.\n\nBy doing so, scVI pools information between similar cells or genes while taking nuisance factors of variation such as batch effects and limited sensitivity into account. To evaluate scVI, we conducted a comprehensive comparative analysis to existing methods for distributional modeling and dimensionality reduction, all of which rely on generalized linear models. We first show that scVI scales to over one million cells, whereas competing algorithms can process at most tens of thousands of cells. Next, we show that scVI fits unseen data more closely and can impute missing data more accurately, both indicative of a better generalization capacity. We then utilize scVI to conduct a set of fundamental analysis tasks - including batch correction, visualization, clustering and differential expression - and demonstrate its accuracy in comparison to the state-of-the-art tools in each task. scVI is publicly available, and can be readily used as a principled and inclusive solution for multiple tasks of single-cell RNA sequencing data analysis.

bioinformatics↗

bioRxiv2024

Immune disease dialogue of chemokine-based cell communications as revealed by single-cell RNA sequencing meta-analysis

Immune-mediated diseases are characterized by aberrant immune responses, posing significant challenges to global health. In both inflammatory and autoimmune diseases, dysregulated immune reactions mediated by tissue-residing immune and non-immune cells precipitate chronic inflammation and tissue damage that is amplified by peripheral immune cell extravasation into the tissue. Chemokine receptors are pivotal in orchestrating immune cell migration, yet deciphering the signaling code across cell types, diseases and tissues remains an open challenge. To delineate disease-specific cell-cell communications involved in immune cell migration, we conducted a meta-analysis of publicly available single-cell RNA sequencing (scRNA-seq) data across diverse immune diseases and tissues. Our comprehensive analysis spanned multiple immune disorders affecting major organs: atopic dermatitis and psoriasis (skin), chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (lung), ulcerative colitis (colon), IgA nephropathy and lupus nephritis (kidney). By interrogating ligand-receptor (L-R) interactions, alterations in cell proportions, and differential gene expression, we unveiled intricate disease- specific and common immune cell chemoattraction and extravasation patterns. Our findings delineate disease-specific L-R networks and shed light on shared immune responses across tissues and diseases. Insights gleaned from this analysis hold promise for the development of targeted therapeutics aimed at modulating immune cell migration to mitigate inflammation and tissue damage. This nuanced understanding of immune cell dynamics at the single-cell resolution opens avenues for precision medicine in immune disease management.

immunology↗

bioRxiv2023

Single Cell RNA Sequencing Reveals Emergent Notochord-Derived Cell Subpopulations in the Postnatal Nucleus Pulposus

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native nucleus pulposus cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived nucleus pulposus cells in the postnatal mouse disc. Specifically, we established the existence of early and late stage nucleus pulposus cells, corresponding to notochordal progenitor and mature cells, respectively. Late stage cells exhibited significantly higher expression levels of extracellular matrix genes including aggrecan, and collagens II and VI, along with elevated TGF-{beta} and PI3K-Akt signaling. Additionally, we identified Cd9 as a novel surface marker of late stage nucleus pulposus cells, and demonstrated that these cells were localized to the nucleus pulposus periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show the Cd9+ nucleus pulposus cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy nucleus pulposus extracellular matrix. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

cell biology↗

bioRxiv2026

Single-cell RNA sequencing reveals that host Glutamine Metabolism Inhibition Enhances Macrophage Phagocytosis of Mycobacterium tuberculosis

Macrophages are crucial for host defense against the pathogen. However, pathogens such as Mycobacterium tuberculosis (Mtb) have evolved mechanisms to alter macrophage physiology and exploit these cells as their primary niche. Mtb-infected macrophages upregulate several metabolic pathways including glutamine metabolism. We previously showed that inhibiting glutamine metabolism with the pleiotropic glutamine metabolism antagonist prodrug JHU083 has dual antibacterial and immunomodulatory effects in a mouse model of tuberculosis. In the present study, using single-cell RNA sequencing and LS-MS/MS metabolomics, we showed that JHU083-mediated glutamine metabolism inhibition increased the population of interstitial macrophages in Mtb-infected lungs. JHU083 treatment also increased inflammatory signatures while lowering immunosuppressive markers on these macrophages. Metabolically, these macrophages exhibited marked depletion of complex lipids, accumulation of free fatty acids, and increased expression of transcripts associated with the {beta}-oxidation pathway. Additionally, JHU083-treatment also improved phagocytic activity of macrophages, as measured by using fluorescent E. coli as a bait. In conclusion, JHU083-mediated glutamine metabolism inhibition metabolically reprograms macrophages, increasing both their lipid utilization as well as phagocytic activity, potentially driving their antimycobacterial activity that we had observed earlier.

microbiology↗

bioRxiv2021

Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at unprecedented resolution. Using our platform, we have corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Our transcriptomic analysis revealed that this neuronal bias is mainly due to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. Such ability to parallelize the characterization of AAV tropism and simultaneously measure the transcriptional response of transduction will facilitate the advancement of safe and precise gene delivery vehicles.

bioengineering↗

bioRxiv2024

Description of Bacterial RNA Transcripts Detected in Mycobacterium tuberculosis Infected Cells from Peripheral Human Granulomas using Single Cell RNA Sequencing

Mycobacterium tuberculosis (Mtb) remains a global human health threat and a significant cause of human morbidity and mortality. We document here the capture of Mtb transcripts in libraries designed to amplify eukaryotic mRNA. These reads are often considered spurious or nuisance and are rarely investigated. Because of early literature suggesting the possible presence of polyadenylated transcripts in Mtb RNA, we included the H37Rv Mtb reference genome when assembling scRNA seq libraries from fine needle aspirate samples from patients presenting at the TB clinic, Port Moresby General Hospital, Papua New Guinea. We used 10X Genomics single-cell RNA sequencing transcriptomics pipeline, which initiates mRNA amplification with poly-T primers on [~]30-micron beads designed to capture, in this case, human mRNA associated with individual cells in the clinical samples. Utilizing the 10X Genomics Cell Ranger tool to align sequencing reads, we consistently detected bacterial small and large ribosomal subunit RNA sequences (rrs and rrl, respectively) and other bacterial gene transcripts in the cell culture and patient samples. We interpret Mtb reads associated with the host cells unique molecular identifier (UMI) and transcriptome to indicate infection of that individual host cell. The Mtb transcripts detected showed frequent sequence variation from the reference genome, with greater than 90% of the rrs or rrl reads from many clinical samples having at least 1 sequence difference compared to the H37Rv reference genome. The data presented includes only bacterial sequences from patients with TB infections that were confirmed by the hospital pathology lab using acid-fast microscopy and/or GeneXpert analysis. The repeated, non-random nature of the sequence variations detected in Mtb rrs and rrl transcripts from multiple patients, suggests that, even though this appears to be a stochastic process, there is possibly some selective pressure that limits the types and locations of sequence variation allowed. The variation does not appear to be entirely artefactual, and it is hypothesized that it could represent an additional mechanism of adaptation to enhance bacterial fitness against host defenses or chemotherapy.

microbiology↗

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↗