Single-cell RNA-seq Studies Research Articles

Kidney organoids in translational medicine: Disease modeling and regenerative medicine.

The kidney is one of the most complex organs composed of multiple cell types, functioning to maintain homeostasis by means of the filtering of metabolic wastes, balancing of blood electrolytes, and adjustment of blood pressure. Recent advances in 3D culture technologies in vitro enabled the generation of "organoids" which mimic the structure and function of in vivo organs. Organoid technology has allowed for new insights into human organ development and human pathophysiology, with great potential for translational research. Increasing evidence shows that kidney organoids are a useful platform for disease modeling of genetic kidney diseases when derived from genetic patient iPSCs and/or CRISPR-mutated stem cells. Although single cell RNA-seq studies highlight the technical difficulties underlying kidney organoid generation reproducibility and variation in differentiation protocols, kidney organoids still hold great potential to understand kidney pathophysiology as applied to kidney injury and fibrosis. In this review, we summarize various studies of kidney organoids, disease modeling, genome-editing, and bioengineering, and additionally discuss the potential of and current challenges to kidney organoid research.

Telomere Damage Maintains Hematopoietic Stem Cells (HSCs) in an Activated Metabolic State, Which Compromises Their Self-Renewal Capability

DNA damage and the attendant cellular responses of apoptosis, senescence, and altered differentiation are major drivers of hematopoietic stem cell (HSC) aging. A reservoir of persistent DNA damage signaling can derive from progressive telomere erosion, which occurs over the lifespan of humans. However, the molecular mechanisms by which telomere damage compromises HSC functions are largely unknown.Here, though combined single-cell RNA-seq and functional studies of highly-purified c-Kit+Sca+Lin-CD34-flk2-CD150+CD48-CD41-HSCs, we show that persistent telomeric damage does not activate programs of apoptosis or senescence but maintains HSCs in an activated metabolic state, which directly compromises their self-renewal capability.To dissect the biological and molecular mechanisms by which persistent DNA damage affects HSC function we analyzed the HSC compartment of mice with short telomeres (G5/G6 TERTER/ER), which developed age-related defects. Immunophenotypic analysis of the HSC compartment showed that, compared with G0 TERTER/+ (G0) mice with intact telomeres (n=12), 2 month-old G5/G6 mice (n=17), had a significantly decreased number of HSCs (p<10-3) that was associated with a decreased number of the lymphoid-biased MPP4 cells, and an increased number of both megakaryocyte-biased MPP2 and myeloid-biased MPP3 cells. HSC exhaustion and increased myeloid-to lymphoid output were reminiscent of stressed hematopoiesis and premature aging. G5/G6 HSCs exhibited a significant accumulation of telomere dysfunction-induced foci (p<10-5) but did not display increased levels of apoptosis in steady-state conditions.HSC exhaustion could result from apoptosis and/or senescence induced by telomere damage in HSCs entering the cell cycle or from an altered balance between self-renewal and differentiation. To distinguish between these two possibilities, we first investigated the effect of inducing young G5/G6 HSCs out of a homeostatic quiescent state. By tracking the real-time changes in the expression level of annexin V on HSCs induced to differentiate towards the myeloid lineage, we found that apoptosis was not the primary fate of G5/G6 HSCs upon entry into the cell cycle. Similarly, in vivo treatment with poly I:C induced the G5/G6 HSCs to enter into the cell cycle at the same rate as that of the G0 mice without inducing apoptosis. Transcriptomic analysis of poly I:C-treated G0 and G5/G6 HSCs, compared with vehicle-treated controls, revealed a significant enrichment of genes involved in the regulation of the cell cycle and platelet production, which is consistent with previous findings showing that megakaryocyte differentiation of HSCs occurs in response to poly I:C to replenish platelets that are lost during inflammatory insult. Importantly, we did not observe any significant change in gene expression between G0 and G5/G6 HSCs isolated from poly I:C-treated mice, which confirmed that telomeric damage did not limit HSCs' proliferation potential by activating programs of senescence or apoptosis.Next, we evaluated the capability of single HSCs isolated from G0 or G5/G6 mice to either self-renew or differentiate. An evaluation of Numb inheritance and expression in G0 and G5/G6 HSCs (n=133 and n=113, respectively) induced to proliferate in vitro showed that G5/G6 HSCs had a 2-fold lower frequency of symmetric self-renewal division (p<10-3) and a concomitant 2-fold higher frequency of symmetric commitment (p<10-4). Accordingly, PB analysis revealed that the CD45.2-derived reconstitution was severely compromised in mice competitively transplanted with G5/G6 HSCs (0.26% vs 77%; p<10-3). Single cell RNA-seq analysis of G0 and G5/G6 HSCs followed by the differential analysis of the clusters showed that 40% of G5/G6 HSCs were in an activated metabolic state associated with hyperactive OXPHOS and ROS signaling pathways, which are directly involved in HSC functional decline. Finally, we reactivated telomerase to investigate the possibility of restoring normal HSC function upon elimination of damage. Single cell RNA-seq and functional studies are ongoing to evaluate whether HSCs' activated metabolic state and compromised self-renewal capability are reversible processes.This study challenges the concept that telomeric damage limits HSC's proliferative potential and offers unparalleled opportunities for unraveling regenerative strategies to ameliorate their decline. DisclosuresColla:Abbvie: Research Funding.

Unique Molecular Identifiers reveal a novel sequencing artefact with implications for RNA-Seq based gene expression analysis

Attaching Unique Molecular Identifiers (UMI) to RNA molecules in the first step of sequencing library preparation establishes a distinct identity for each input molecule. This makes it possible to eliminate the effects of PCR amplification bias, which is particularly important where many PCR cycles are required, for example, in single cell studies. After PCR, molecules sharing a UMI are assumed to be derived from the same input molecule. In our single cell RNA-Seq studies of Physcomitrellapatens, we discovered that reads sharing a UMI, and therefore presumed to be derived from the same mRNA molecule, frequently map to different, but closely spaced locations. This behaviour occurs in all such libraries that we have produced, and in multiple other UMI-containing RNA-Seq data sets in the public domain. This apparent paradox, that reads of identical origin map to distinct genomic coordinates may be partially explained by PCR stutter, which is often seen in low-entropy templates and those containing simple tandem repeats. In the absence of UMI this artefact is undetectable. We show that the common assumption that sequence reads having different mapping coordinates are derived from different starting molecules does not hold. Unless taken into account, this artefact is likely to result in over-estimation of certain transcript abundances, depending on the counting method employed.

Clonal and Functional Analysis Reveals the Capacity of Allograft T cells to Join the Circulating Pool after Human Intestinal Transplantation

Resident memory T cells (Trm) are non-recirculating cells that persist in tissues. It has so far not been possible to assess whether migration between tissues and the circulating pool occurs in humans. Surveillance endoscopic biopsies on intestinal transplant patients permitted longitudinal and spatial analysis of relationships between intestinal T cells in allografts, peripheral blood and bone marrow (BM) using FCM, MLR, TCRβ DNA-seq and single-cell RNA-seq. Our published protocol was used to identify graft-versus-host (GVH) clones from pre-Tx donor lymphoid tissues1.While the circulating donor T cells expressed a “blood” phenotype (CD28+CD69-CD103-), donor ileal lymphocytes in the graft displayed a Trm phenotype (CD28-CD69+CD103+/-). Donor T cells in post-Tx biopsies were greatly enriched for clones detected in both donor spleen and mesenteric lymph nodes (MLN), suggesting that Trm, including GVH clones, transferred in the allograft had a circulating memory counterpart. Clonal diversity analysis suggested that clones detected in both donor spleen and MLN were memory rather than naïve cells1. GVH clones identified in pre-Tx MLR by their CDR3 sequences persisted long-term in both the allograft (at high frequency1) and recipient peripheral blood (at low frequency) (Fig.1). These data suggest that clonal expansion occurred, likely in response to the early entry of recipient myeloid APCs into the graft1, and these expanded GVH Trm emigrated to the recipient circulation and acquired the “blood” phenotype. Single-cell RNA-seq data on GVH and donor-mappable non-GVH clones identifiable by their TCRβ sequences plus unknown TCR clones from a quiescent Day 237 allograft biopsy were divided into 5 clusters in t-SNE plots based on 31 Trm signature genes2 (Fig.2). All 4 “donor-mappable” clones (GVH or non-GVH CD4 or CD8) distributed into multiple clusters, showing no functional distinction between GVH- and non-GVH-reactive donor T cells in the graft. 4 of 5 clusters upregulated RGS1, a Trm retention signal, while other features of Trm such as ITGAE and CXCR6 were limited to certain clusters (Fig.3). Effector molecules including RORA and STAT3 were associated with several clusters.Strikingly, in one multivisceral Tx patient analyzed, donor-HLA+ cells (0.04%) were detected in recipient BM on Day 105. Greater than 70% of these were T cells that were enriched for GVH clones among donor-mappable CD4s. The temporal and spatial tracking of donor T cells and GVH clones derived from memory cells carried in intestinal allografts provides new insight into their migration patterns. Donor-derived Trm may leave the tissue of residence (intestinal graft) and join the circulating T cell pool, possibly after differentiating into effector T cells upon encounter with recipient alloantigen. Some of these cells may eventually reside in the BM niche.1.1. 1. Zuber et al., 2016 Science Immunology 2. Kumar et al., 2017 Cell Reports The study was funded by the National Institute of Allergy and Infectious Diseases grant P01 AI106697. Research reported in this study was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. Single cell RNA-seq study was supported in part by the Immune Tolerance Network supplement grant.

New Pericytes in the Adult Brain Originate from a Previously Unrecognized Pericyte Precursor

Pericytes are essential for maintaining the stability of CNS blood-vessels and the blood-brain- barrier. They are closely associated with endothelia and can be identified by a set of markers. New pericytes are required during neo-angiogenesis but how pericytes are generated in the adult brain is unknown. Here, we investigated neoplastic vascularization of the adult brain in a transgenic lineage-tracing model. We observed that mature pericytes largely originate from a newly identified precursor celltype, which is distinct from established brain cell-populations as evidenced by single-cell RNAseq studies. These precursors are characterized as pericyte marker-negative cells of non-hematopoietic origin, which are distant from blood-vessels and highly proliferative. Pericyte precursors emerge also in developmental angiogenesis and lineage ablation experiments in brain tumor models showed that they are necessary to generate new, mature pericytes supporting tumor expansion. Overall, pericyte precursors control vascular plasticity and provide an excellent therapeutic target for brain tumors.

Generating tissue-resident macrophages from pluripotent stem cells: Lessons learned from microglia

Over the past decades, the importance of the immune system in a broad scope of pathologies, has drawn attention towards tissue-resident macrophages, such as microglia in the brain. To enable the study of for instance microglia, it is crucial to recreate in vitro (and in vivo) assays. However, very fast loss of tissue-specific features of primary tissue resident macrophages, including microglia, upon in vitro culture has complicated such studies. Moreover, limited knowledge of macrophage developmental pathways and the role of local ‘niche factors’, has hampered the generation of tissue-resident macrophages from pluripotent stem cells (PSC). Recent data on the ontogeny of tissue-resident macrophages, combined with bulk and single cell RNAseq studies have identified the distinct origins and gene profile of microglia compared to other myeloid cells. As a result, over the past years, protocols have been published to create hPSC-derived microglia-‘like’ cells, as these cells are considered potential new therapeutic targets for therapies to treat neurodegenerative diseases. In this review we will provide an overview of different approaches taken to generate human microglia in vitro, taking into account their origin, and resemblance to their in vivo counterpart. Finally, we will discuss cell-extrinsic (culture conditions) and intrinsic factors (transcriptional machinery and epigenetics) that we believe can improve future differentiation protocols of tissue-resident macrophages from stem cells.

Functional genomics of stromal cells in chronic inflammatory diseases.

Stroma is a broad term referring to the connective tissue matrix in which other cells reside. It is composed of diverse cell types with functions such as extracellular matrix maintenance, blood and lymph vessel development, and effector cell recruitment. The tissue microenvironment is determined by the molecular characteristics and relative abundances of different stromal cells such as fibroblasts, endothelial cells, pericytes, and mesenchymal precursor cells. Stromal cell heterogeneity is explained by embryonic developmental lineage, stages of differentiation to other cell types, and activation states. Interaction between immune and stromal cell types is critical to wound healing, cancer, and a wide range of inflammatory diseases. Here, we review recent studies of inflammatory diseases that use functional genomics and single-cell technologies to identify and characterize stromal cell types associated with pathogenesis. High dimensional strategies using mRNA sequencing, mass cytometry, and fluorescence activated cell-sorting with fresh primary tissue samples are producing detailed views of what is happening in diseased tissue in rheumatoid arthritis, inflammatory bowel disease, and cancer. Fibroblasts positive for CD90 (Thy-1) are enriched in the synovium of rheumatoid arthritis patients. Single-cell RNA-seq studies will lead to more discoveries about the stroma in the near future. Stromal cells form the microenvironment of inflamed and diseased tissues. Functional genomics is producing an increasingly detailed view of subsets of stromal cells with pathogenic functions in rheumatic diseases and cancer. Future genomics studies will discover disease mechanisms by perturbing molecular pathways with chemokines and therapies known to affect patient outcomes. Functional genomics studies with large sample sizes of patient tissues will identify patient subsets with different disease phenotypes or treatment responses.

Going single but not solo with podocytes: potentials, limitations, and pitfalls of single-cell analysis

Single-cell RNA-sequence (RNA-seq) is a widely used tool to study biological questions in single cells. The discussed study identified 92 genes being predominantly expressed in podocytes based on a 5-fold higher expression compared with endothelial and mesangial cells. In addition to technical pitfalls, the question that is discussed in this commentary is whether results of a single-cell RNAseq study are able to deliver expression data that truly characterize a podocyte.

Einzelzell-RNA-Sequenzierung beleuchtet den Infektionsprozess

Single-cell RNA-seq has revolutionized our understanding of cellular heterogeneity in a wide spectrum of biological processes. Pioneering studies suggest complex mechanisms and division of labour employed by the bacterial pathogen Salmonella to subvert host cell functions during infection. Here we describe some of our findings from single-cell RNA-seq studies that have revealed and characterized heterogeneity between individual cells infected with this intracellular pathogen, and outline future challenges in this burgeoning field.

Hematopoietic Stem Cells Respond Heterogeneously to Inflammatory Signals in an Age-Dependent Manner

Hematopoietic stem cells (HSCs) have the unique responsibility to produce balanced immune cell output throughout an organism's life. Importantly, they must do so robustly despite a plethora of external stress, including frequent inflammatory challenge. With age, the accumulation of these stresses leads to impaired HSC function and myeloid-biased output. Aged HSCs are also more prone to pathological hematopoiesis, such as myeloproliferative disorder, leukemia and autoimmune diseases. However, little is known about the subcellular mechanisms that govern the inflammatory response of HSCs with age, which in turn might contribute to pathologic transformation.We show that young hematopoietic stem and progenitor cells (HSPCs) demonstrate a robust transcriptional response to toll-like receptor (TLR) ligands. Interestingly, this response is similar to that of mature immune cell types such as dendritic cells. Using single-cell proteomic assays, we found that young HSPCs secrete a diverse array of myeloid and lymphoid cytokines. However, when challenged with TLR ligands in vivo, young mice acutely increase myeloid-biased output but return rapidly to baseline hematopoietic output of both lymphoid and myeloid cells. Moreover, inflammatory challenge of young long-term HSCs in vitro did not perturb the function and output of these cells in bone marrow reconstitution experiments.In contrast to their counterparts from young mice, we found HSPCs obtained from aged mice have a diminished ability to secrete cytokines in response to TLR ligands. Furthermore, they secrete a homogenous subset of myeloid-biased cytokines. When challenged with TLR ligands in vivo, aged mice acutely increased myeloid output and maintain elevated myeloid output for several months implying memory of the inflammatory challenge. Consistent with this, we also found that pre-stimulation of aged HSCs prior to bone marrow transplant results in a sustained increase in myeloid output compared to unstimulated aged HSCs.To elucidate the differential heterogeneity between young and aged HSPCs in response to TLR signaling, we next performed single-cell RNA sequencing (RNA-seq) experiments. We found that the sustained myeloid output in aged mice after TLR stimulation is largely due to expansion of a myeloid-biased HSC subset in the aged HSC pool. By characterizing the gene expression networks that define these myeloid-biased HSCs under stimulation conditions, we were then able to identify a myeloid-biased HSC subset in both the unperturbed young and aged HSC pools. Moreover, we found that these cells are more abundant in aged mice at steady-state, and that these HSCs demonstrate a unique response to inflammatory challenge. We further identify putative transcriptional regulators, including Klf4, Klf5, Ikzf1 and Stat3, among others, that define gene expression in these myeloid-biased HSCs. We further show that loss of function of these factors can differentially alter myeloid output in young and aged mice both in vitro and in vivo.Our results demonstrate that there is a differential response of young and aged HSCs to inflammatory signals. Using single-cell RNA-seq and protein secretion studies, we elucidate the molecular heterogeneity of the HSC pool at steady state and with TLR stimulation. By resolving heterogeneous subsets of cells in both the young and aged HSC pool, and by uncovering the transcriptional regulators that influence their function, we thus propose a new model of inflammatory hematopoiesis that may have implications to understanding age-related defects in immune development. DisclosuresNo relevant conflicts of interest to declare.

Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

BackgroundViral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line.ResultWe developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins.ConclusionOur results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13742-015-0091-4) contains supplementary material, which is available to authorized users.