Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.

Leptin is secreted by adipocytes to regulate appetite and body weight. Recent studies have reported that tanycytes actively transport circulating leptin across the brain barrier into the hypothalamus, and are required for normal levels of hypothalamic leptin signaling. However, direct evidence for leptin receptor (LepR) expression is lacking, and the effect of tanycyte-specific deletion of LepR has not been investigated. In this study, we analyze the expression and function of the tanycytic LepR in mice. Using single-molecule fluorescent in situ hybridization (smfISH), RT-qPCR, single-cell RNA sequencing (scRNA-Seq), and selective deletion of the LepR in tanycytes, we are unable to detect expression of LepR in the tanycytes. Tanycyte-specific deletion of LepR likewise did not affect leptin-induced pSTAT3 expression in hypothalamic neurons, regardless of whether leptin was delivered by intraperitoneal or intracerebroventricular injection. Finally, we use activity-regulated scRNA-Seq (act-Seq) to comprehensively profile leptin-induced changes in gene expression in all cell types in mediobasal hypothalamus. Clear evidence for leptin signaling is only seen in endothelial cells and subsets of neurons, although virtually all cell types show leptin-induced changes in gene expression. We thus conclude that LepR expression in tanycytes is either absent or undetectably low, that tanycytes do not directly regulate hypothalamic leptin signaling through a LepR-dependent mechanism, and that leptin regulates gene expression in diverse hypothalamic cell types through both direct and indirect mechanisms.

Single-cell transcriptomic profiling of human bone marrow non-hematopoietic cells provides the basis for a comprehensive understanding of the cellular complexity of the hematopoietic stem cell (HSC) microenvironment and the intricate stroma-hematopoiesis crosstalk mechanisms.

Single-cell chromatin accessibility analysis reveals the intricate regulatory landscape of mouse testicular development, uncovering novel cell subpopulations and transcription factors, and offering valuable insights into the molecular mechanisms driving germ cell and somatic cell maturation.

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

While many genetic variants have been associated with risk for human diseases, how these variants affect gene expression in various cell types remains largely unknown. To address this gap, the DICE (database of immune cell expression, expression quantitative trait loci (eQTLs), and epigenomics) project was established. Considering all human immune cell types and conditions studied, we identified cis-eQTLs for a total of 12,254 unique genes, which represent 61% of all protein-coding genes expressed in these cell types. Strikingly, a large fraction (41%) of these genes showed a strong cis-association with genotype only in a single cell type. We also found that biological sex is associated with major differences in immune cell gene expression in a highly cell-specific manner. These datasets will help reveal the effects of disease risk-associated genetic polymorphisms on specific immune cell types, providing mechanistic insights into how they might influence pathogenesis (https://dice-database.org).

Impact of Genetic Polymorphisms on Human Immune Cell Gene Expression

Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. Here we show that the utilization of dual, or "shadow", enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P) proteins and basic helix-loop-helix (bHLH) repressor (R) factors (a "P+R" regulatory code), we have identified NP-specific enhancer modules associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, we show that P sites in these modules mediate activation by proneural factors in "proneural cluster" territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. To our knowledge, our results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), we demonstrate that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event.

Interleukin-8 (IL-8) is a cytokine which recruits and activates neutrophils into tissue stroma. It is present in uterine tissues and its concentration increases in the third trimester and with labour. The promoter region of the IL-8 gene contains binding sites for the transcription factors, nuclear factor-kappa B (NF-kappaB), activator protein-1 (AP-1) and CCAAT/enhancer-binding protein (C/EBP). These are in close proximity to each other and to the coding region of the gene. This study used site-directed mutagenesis of each of these sites to examine the relative importance of each site in IL-8 gene expression in a cervical cell line and in amnion cells obtained before and after labour. We found that the NF-kappaB site was essential for basal and IL-1beta-stimulated gene expression in all cell types. Neither of the other binding sites was consistently essential for gene expression but may have an additive role in promoter activity. We conclude that the NF-kappaB binding site is essential for up-regulation of IL-8 gene expression in these uterine cell types. An increase in IL-8 expression has been shown to occur in the uterus in association with parturition and NF-kappaB binding to the promoter may be of importance at this time.

Single-cell transcriptomic analysis reveals novel regenerative endothelial cell populations that mediate remarkably rapid and complete microvascular repair in a novel model of acute lung injury induced by endothelial cell ablation.

BackgroundGenome-wide association studies have identified many single nucleotide polymorphisms (SNPs) associated with increased risk for intracranial aneurysm (IA). However, how such variants affect gene expression within IA is poorly understood. We used publicly-available ChIP-Seq data to study chromatin landscapes surrounding risk loci to determine whether IA-associated SNPs affect functional elements that regulate gene expression in cell types comprising IA tissue.MethodsWe mapped 16 significant IA-associated SNPs to linkage disequilibrium (LD) blocks within human genome. Using ChIP-Seq data, we examined these regions for presence of H3K4me1, H3K27ac, and H3K9ac histone marks (typically associated with latent/active enhancers). This analysis was conducted in several cell types that are present in IA tissue (endothelial cells, smooth muscle cells, fibroblasts, macrophages, monocytes, neutrophils, T cells, B cells, NK cells). In cell types with significant histone enrichment, we used HiC data to investigate topologically associated domains (TADs) encompassing the LD blocks to identify genes that may be affected by IA-associated variants. Bioinformatics were performed to determine the biological significance of these genes. Genes within HiC-defined TADs were also compared to differentially expressed genes from RNA-seq/microarray studies of IA tissues.ResultsWe found that endothelial cells and fibroblasts, rather than smooth muscle or immune cells, have significant enrichment for enhancer marks on IA risk haplotypes (p < 0.05). Bioinformatics demonstrated that genes within TADs subsuming these regions are associated with structural extracellular matrix components and enzymatic activity. The majority of histone marked TADs (83% fibroblasts [IMR90], 77% HUVEC) encompassed at least one differentially expressed gene from IA tissue studies.ConclusionsThese findings provide evidence that genetic variants associated with IA risk act on endothelial cells and fibroblasts. There is strong circumstantial evidence that this may be mediated through altered enhancer function, as genes in TADs encompassing enhancer marks have also been shown to be differentially expressed in IA tissue. These genes are largely related to organization and regulation of the extracellular matrix. This study builds upon our previous (Poppenberg et al., BMC Med Genomics, 2019) by including a more diverse set of data from additional cell types and by identifying potential affected genes (i.e. those in TADs).

It was an honor to give the Giles F. Filley Lecture. Dr. Filley was cofounder of the Aspen Lung Conference and a master clinician and scientist with major contributions to the field of acid base balance. He developed “caribicarb,” a combination of calcium carbonate and sodium bicoarbonate. He also contributed to our understanding of the pathophysiology of chronic obstructive pulmonary disease and emphysema through his many publications including one in which he described the elastic properties of the different lobes of the human lung.[1] This manuscript describes using induced pluripotent stem cells (iPSCs) combined with next generation sequencing to gain new information about the pathobiology of pulmonary arterial hypertension (PAH) and to discover ways in which future therapies for this disorder might be personalized. The studies we propose represent a team effort between our laboratory, that of Dr. Joseph Wu in the Department of Medicine at Stanford University who pioneered the derivation and characterization of endothelial cells from iPSCs,[2] and that of Dr. Michael Snyder in the Department of Genetics, who developed next generation sequencing approaches for personalized medicine.[3] Our plan is to harvest pulmonary artery endothelial cells (PAECs) from idiopathic PAH (IPAH) patients and unused donor controls whose lungs come to explant through the Pulmonary Hypertension Breakthrough Initiative (PHBI) Network and to compare these cells to ECs differentiated from iPSCs derived either from fibroblasts obtained from the skin incision at the time of transplant or from the PAECs as the starting cell type (Fig. 1). The skin biopsies were provided to us by the Network investigators under a separate IRB agreement independent of PHBI. We plan to compare the three cell lines to each other and in patients versus controls in terms of gene variants using a combination of exome and low coverage whole genome sequencing analysis[3] and then we will assess methylation changes by Methyl-Seq[4] and gene expression changes by RNA-Seq.[5] Differences will then be related to function as assessed by angiogenesis and signaling using a novel high throughput methodology that uses isotope labeling of antibodies to phosphoproteins and cell surface antigens.[6] Schema of proposed harvest of pulmonary artery (PA) endothelial cells and skin fibroblasts, generation of induced pluripotent stem cells (iPSC), transformation of both into endothelial cells and subsequent studies. Our hypotheses are that ECs derived from fibroblast iPSCs will reveal functional abnormalities related to gene variants in IPAH patients and that ECs derived from PAEC-iPSCs will be informative of methylation changes that are maintained as memory from the tissue that is the site of the pathology. We predict that the native PAECs will show further methylation and gene expression changes that are reflective of end-stage disease. The next phase of the study involves comparison of all three cell types from IPAH patients after correction of a gene variant using the zinc finger nuclease technology[7] to determine the extent to which this restores normal gene expression as assessed by RNA-Seq and normal function, as reflected in normal properties of angiogenesis and signaling. We will also compare the three cell types in terms of their abilities to respond to novel therapies by correction of gene expression and function. Signaling studies are carried out according to a new methodology that involves labeling antibodies to phosphorylated or other proteins with isotopes instead of fluorescent probes, because this allows mass spectrometry detection of numerous molecules, not just those limited by the fluorescence spectra utilized by conventional flow cytometry. Mass spectrometry can thus be used to interrogate changes in signaling molecules in response to different agonists in single cells and so heat maps can be established, or 2D plots of a surface molecule and a signaling molecule, or spade analysis of a hierarchy of cells. The fidelity of the analysis is exceptionally good as previously reported and compares favorably with fluorescence flow cytometry, but the capacity of assessing more molecules and better defining signaling networks related to cell type is much greater.[6] Our initial studies are focused on IPAH and on ECs, although we may incorporate APAH and differentiation of iPSCs to other vascular cell types in future studies. We chose to begin with ECs as the differentiated cells because of experience in differentiating these cells from iPSCS, but mostly because ECs from PAH patients have functional abnormalities associated with pulmonary vascular pathology. These include propensity to apoptosis that leads to loss of small distal microvessels[8] as well as later apoptosis resistance in plexogenic lesions,[9] aberrant activation of matrix elastase and other proteinases that cause release of paracrine growth factors that contribute to the exuberant proliferation of the underlying cells of the vessel wall that culminate in occlusion of the lumen.[10] In addition, EC dysfunction promotes inflammation through production of chemokines such as IL-6.[11] Moreover, PAECs from PAH patients do not form normal tubes in culture and are highly glycolytic.[9] Pulmonary arterial endothelial cells from a normal patient form a well-established network of interconnecting tubes when grown on a collagen gel as compared to the poor formation of a tubular network using PAECs from IPAH patients[9] (Fig. 2). Representative frame of collagen gel assay shows that pulmonary arterial endothelial cells from representative idiopathic pulmonary arterial hypertension patient (IPAH PAECs) show impaired tubular network formation comared to PAECs from a donor control lung Reprogramming to iPSC involves transfection of the four transcription factors described by Yamanaka (Oct 4, Sox 2 KLF 4 and c-myc) either by lentivirus or by minicircle.[12] The procedure takes up to four weeks. Pluripotency is assessed both by teratoma formation and by specific markers of pluripotency like NANOG. Differentiation of ECs is accomplished following embryoid body formation by a combination of activin, bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF).[2] The ECs are evident at the edge of the embryoid body and are then expanded and harvested either by FACS or by CD31 (PECAM) antibody-coated immunobeads or by a combination of sorting with CD144 (VE-cadherin) and CD31 antibodies. Expression profiling shows that embryonic stem cell-derived and fibroblast-derived ECs have a similar expression profile by microarray analysis when compared to human umbilical vein endothelial cells that is markedly in contrast to the fibroblast.[2] Challenges in this field remain the propensity of the differentiated ECs to dedifferentiate or to senesce. As we are accumulating sets of PAECs, fibroblasts, iPSC-ECs differentiated from fibroblasts, and endothelial cells, we have been carrying out RNA-Seq analyses on PAECs to determine which changes in gene expression might be of interest when interrogating the iPSC-ECs. RNA-seq involved extraction of RNA conversion to cDNA and random fragmentation of DNA to prepare libraries with fragments ligated by specific adapters.[5] The fragments are incubated on a flowcell that is covered by a lawn of primers that attach to the adapters ligated to the DNA fragments. Polymerase chain reaction (PCR) is used to replicate the fragments, and fluorescently labeled nucleotides are added in each cycle. Sequencing records the fluorescence at each cycle and then sequences are aligned to a reference so that SNPs deletions and insertions can be identified. Sequencing depth refers to the number of times a base or region has been sequenced. The depth of reads can then be related to the length of the sequence to give an assessment of expression level.[5] We found that comparison of two runs gave a very similar distribution of genes expressed at high frequency and gene expressed at low frequency. Then we compared each RNA-Seq for genes that were expressed by a greater than one-fold increase or decrease in expression and were able to correlate our findings with quantitative reverse transcription (qRT)-PCR quite faithfully. Preliminary studies suggest that some of these genes are related to expression of bone morphogenetic protein receptor (BMPR2), the gene mutated in the overwhelming majority of hereditary PAH. Nil None declared.

See related article, pages 200–208 The endothelium is an expansive spatially distributed organ.1 Endothelial cells participate in a large number of physiological processes including the control of vasomotor tone, the trafficking of cells and nutrients, the regulation of permeability, and the maintenance of blood fluidity. In addition, the endothelium mediates new blood vessel formation, contributes to the balance of pro- and antiinflammatory mediators, and may play a role in antigen presentation. In accomplishing these tasks, the endothelium exhibits a remarkable “division of labor”. For example, arteriolar endothelium is primarily responsible for mediating vasomotor tone; endothelium in postcapillary venules regulates leukocyte trafficking; capillary endothelial cells display organ-specific barrier properties (eg, blood brain barrier versus fenestrated, discontinuous endothelium in hepatic sinusoids); and endothelial cells from different vascular beds balance local hemostasis via the expression of site-specific patterns of anticoagulants and procoagulants.2 In recent years, in vivo phage display and direct proteome mapping of the intact vasculature have revealed a rich diversity in endothelial cell surface markers.3,4 Any consideration of the mechanisms underlying endothelial heterogeneity is best framed around the time-honored debate of nature versus nurture (which will be addressed here in reverse order) (Figure). Mechanisms of endothelial cell heterogeneity. Relative importance of epigenetics and microenvironment in mediating site-specific phenotypes is indicated by +. The table is designed to provide a conceptual framework; the scores are largely speculative and will require ongoing experimental validation. ### Nurture Site-specific endothelial cell phenotypes may be initiated and maintained by signals residing in the extracellular environment. The endothelium is analogous to a barcode reader, constantly taking inventory of its surrounding extracellular environment on the luminal side (circulating blood and its constituents), the abluminal side, and at the endothelial junctions. Environmental cues may be classified into biomechanical or biochemical. Biochemical forces include shear stress and strain. …

Decision letter: Single-cell multiomic profiling of human lungs reveals cell-type-specific and age-dynamic control of SARS-CoV2 host genes

The Emerging Role of Valve Interstitial Cell Phenotypes in Regulating Heart Valve Pathobiology

Genome-wide association studies have implicated dozens of genes with migraine susceptibility, but it remains unclear in which nervous system cell types these genes are expressed. Using single-cell RNA sequencing data from the central and peripheral nervous system, including the trigeminal ganglion, the expression of putative migraine-associated genes was compared across neuronal, glial and neurovascular cell types within these tissues. Fifty-four putative migraine-associated genes were expressed in the central nervous system, peripheral nervous system or neurovascular cell types analyzed. Six genes (11.1%) were selectively enriched in central nervous system cell types, three (5.5%) in neurovascular cell types, and two (3.7%) in peripheral nervous system cell types. The remaining genes were expressed in multiple cell types. Single-cell RNA sequencing of the brain and peripheral nervous system localizes each migraine-associated gene to its respective nervous system tissue and the cell types in which it is expressed. While the majority of migraine-associated genes are broadly expressed, we identified several cell-type-specific migraine-associated genes in the central nervous system, peripheral nervous system, and neurovasculature. Trial registration: not applicable.