Robust temporal map of human in vitro myelopoiesis using single-cell genomics.

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

Locus-specific expression of transposable elements in single cells with CELLO-seq.

Transposable elements (TEs) regulate diverse biological processes, from early development to cancer. Expression of young TEs is difficult to measure with next-generation, single-cell sequencing technologies because their highly repetitive nature means that short complementary DNA reads cannot be unambiguously mapped to a specific locus. Single CELl LOng-read RNA-sequencing (CELLO-seq) combines long-read single cell RNA-sequencing with computational analyses to measure TE expression at unique loci. We used CELLO-seq to assess the widespread expression of TEs in two-cell mouse blastomeres as well as in human induced pluripotent stem cells. Across both species, old and young TEs showed evidence of locus-specific expression with simulations demonstrating that only a small number of very young elements in the mouse could not be mapped back to the reference with high confidence. Exploring the relationship between the expression of individual elements and putative regulators revealed large heterogeneity, with TEs within a class showing different patterns of correlation and suggesting distinct regulatory mechanisms.

Cell reprogramming shapes the mitochondrial DNA landscape.

Individual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Comprehensively analysing the mitochondrial genome (mtDNA) in 146 iPSC and fibroblast lines from 151 donors, we show that most age-related fibroblast mtDNA mutations are lost during reprogramming. However, iPSC-specific mutations are seen in 76.6% (108/141) of iPSC lines at a mutation rate of 8.62 × 10-5/base pair. The mutations observed in iPSC lines affect a higher proportion of mtDNA molecules, favouring non-synonymous protein-coding and tRNA variants, including known disease-causing mutations. Analysing 11,538 single cells shows stable heteroplasmy in sub-clones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. Thus, the dynamic mtDNA landscape contributes to the heterogeneity of human iPSCs and should be considered when using reprogrammed cells experimentally or as a therapy.

Signatures of TSPAN8 variants associated with human metabolic regulation and diseases.

Here, with the example of common copy number variation (CNV) in the TSPAN8 gene, we present an important piece of work in the field of CNV detection, that is, CNV association with complex human traits such as 1H NMR metabolomic phenotypes and an example of functional characterization of CNVs among human induced pluripotent stem cells (HipSci). We report TSPAN8 exon 11 (ENSE00003720745) as a pleiotropic locus associated with metabolomic regulation and show that its biology is associated with several metabolic diseases such as type 2 diabetes (T2D) and cancer. Our results further demonstrate the power of multivariate association models over univariate methods and define metabolomic signatures for variants in TSPAN8.

A map of transcriptional heterogeneity and regulatory variation in human microglia.

Microglia, the tissue-resident macrophages of the central nervous system (CNS), play critical roles in immune defense, development and homeostasis. However, isolating microglia from humans in large numbers is challenging. Here, we profiled gene expression variation in primary human microglia isolated from 141 patients undergoing neurosurgery. Using single-cell and bulk RNA sequencing, we identify how age, sex and clinical pathology influence microglia gene expression and which genetic variants have microglia-specific functions using expression quantitative trait loci (eQTL) mapping. We follow up one of our findings using a human induced pluripotent stem cell-based macrophage model to fine-map a candidate causal variant for Alzheimer's disease at the BIN1 locus. Our study provides a population-scale transcriptional map of a critically important cell for human CNS development and disease.

Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation.

Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.

Genome-wide meta-analysis, fine-mapping and integrative prioritization implicate new Alzheimer's disease risk genes.

Genome-wide association studies have discovered numerous genomic loci associated with Alzheimer's disease (AD); yet the causal genes and variants are incompletely identified. We performed an updated genome-wide AD meta-analysis, which identified 37 risk loci, including new associations near CCDC6, TSPAN14, NCK2 and SPRED2. Using three SNP-level fine-mapping methods, we identified 21 SNPs with >50% probability each of being causally involved in AD risk and others strongly suggested by functional annotation. We followed this with colocalization analyses across 109 gene expression quantitative trait loci datasets and prioritization of genes by using protein interaction networks and tissue-specific expression. Combining this information into a quantitative score, we found that evidence converged on likely causal genes, including the above four genes, and those at previously discovered AD loci, including BIN1, APH1B, PTK2B, PILRA and CASS4.

Souporcell: robust clustering of single-cell RNA-seq data by genotype without reference genotypes.

Methods to deconvolve single-cell RNA-sequencing (scRNA-seq) data are necessary for samples containing a mixture of genotypes, whether they are natural or experimentally combined. Multiplexing across donors is a popular experimental design that can avoid batch effects, reduce costs and improve doublet detection. By using variants detected in scRNA-seq reads, it is possible to assign cells to their donor of origin and identify cross-genotype doublets that may have highly similar transcriptional profiles, precluding detection by transcriptional profile. More subtle cross-genotype variant contamination can be used to estimate the amount of ambient RNA. Ambient RNA is caused by cell lysis before droplet partitioning and is an important confounder of scRNA-seq analysis. Here we develop souporcell, a method to cluster cells using the genetic variants detected within the scRNA-seq reads. We show that it achieves high accuracy on genotype clustering, doublet detection and ambient RNA estimation, as demonstrated across a range of challenging scenarios.

Cardelino: computational integration of somatic clonal substructure and single-cell transcriptomes.

Bulk and single-cell DNA sequencing has enabled reconstructing clonal substructures of somatic tissues from frequency and cooccurrence patterns of somatic variants. However, approaches to characterize phenotypic variations between clones are not established. Here we present cardelino (https://github.com/single-cell-genetics/cardelino), a computational method for inferring the clonal tree configuration and the clone of origin of individual cells assayed using single-cell RNA-seq (scRNA-seq). Cardelino flexibly integrates information from imperfect clonal trees inferred based on bulk exome-seq data, and sparse variant alleles expressed in scRNA-seq data. We apply cardelino to a published cancer dataset and to newly generated matched scRNA-seq and exome-seq data from 32 human dermal fibroblast lines, identifying hundreds of differentially expressed genes between cells from different somatic clones. These genes are frequently enriched for cell cycle and proliferation pathways, indicating a role for cell division genes in somatic evolution in healthy skin.

Correction to: Niacin-mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system.

The article Niacin­mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system, written by Khalil S. Rawji, Adam M.H. Young, Tanay Ghosh, Nathan J. Michaels, Reza Mirzaei, Janson Kappen, Kathleen L. Kolehmainen, Nima Alaeiilkhchi, Brian Lozinski, Manoj K. Mishra, Annie Pu, Weiwen Tang, Salma Zein, Deepak K. Kaushik, Michael B. Keough, Jason R. Plemel, Fiona Calvert, Andrew J. Knights, Daniel J. Gaffney, Wolfram Tetzlaff, Robin J. M. Franklin and V. Wee Yong, was originally published electronically on the publisher's internet.

Niacin-mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system.

Remyelination following CNS demyelination restores rapid signal propagation and protects axons; however, its efficiency declines with increasing age. Both intrinsic changes in the oligodendrocyte progenitor cell population and extrinsic factors in the lesion microenvironment of older subjects contribute to this decline. Microglia and monocyte-derived macrophages are critical for successful remyelination, releasing growth factors and clearing inhibitory myelin debris. Several studies have implicated delayed recruitment of macrophages/microglia into lesions as a key contributor to the decline in remyelination observed in older subjects. Here we show that the decreased expression of the scavenger receptor CD36 of aging mouse microglia and human microglia in culture underlies their reduced phagocytic activity. Overexpression of CD36 in cultured microglia rescues the deficit in phagocytosis of myelin debris. By screening for clinically approved agents that stimulate macrophages/microglia, we have found that niacin (vitamin B3) upregulates CD36 expression and enhances myelin phagocytosis by microglia in culture. This increase in myelin phagocytosis is mediated through the niacin receptor (hydroxycarboxylic acid receptor 2). Genetic fate mapping and multiphoton live imaging show that systemic treatment of 9-12-month-old demyelinated mice with therapeutically relevant doses of niacin promotes myelin debris clearance in lesions by both peripherally derived macrophages and microglia. This is accompanied by enhancement of oligodendrocyte progenitor cell numbers and by improved remyelination in the treated mice. Niacin represents a safe and translationally amenable regenerative therapy for chronic demyelinating diseases such as multiple sclerosis.

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

Publisher Correction: Molecular and functional variation in iPSC-derived sensory neurons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic effects on promoter usage are highly context-specific and contribute to complex traits.

Genetic variants regulating RNA splicing and transcript usage have been implicated in both common and rare diseases. Although transcript usage quantitative trait loci (tuQTLs) have been mapped across multiple cell types and contexts, it is challenging to distinguish between the main molecular mechanisms controlling transcript usage: promoter choice, splicing and 3' end choice. Here, we analysed RNA-seq data from human macrophages exposed to three inflammatory and one metabolic stimulus. In addition to conventional gene-level and transcript-level analyses, we also directly quantified promoter usage, splicing and 3' end usage. We found that promoters, splicing and 3' ends were predominantly controlled by independent genetic variants enriched in distinct genomic features. Promoter usage QTLs were also 50% more likely to be context-specific than other tuQTLs and constituted 25% of the transcript-level colocalisations with complex traits. Thus, promoter usage might be an underappreciated molecular mechanism mediating complex trait associations in a context-specific manner.

High-resolution genetic mapping of putative causal interactions between regions of open chromatin.

Physical interaction of regulatory elements in three-dimensional space poses a challenge for studies of disease because non-coding risk variants may be great distances from the genes they regulate. Experimental methods to capture these interactions, such as chromosome conformation capture, usually cannot assign causal direction of effect between regulatory elements, an important component of fine-mapping studies. We developed a Bayesian hierarchical approach that uses two-stage least squares and applied it to an ATAC-seq (assay for transposase-accessible chromatin using sequencing) data set from 100 individuals, to identify over 15,000 high-confidence causal interactions. Most (60%) interactions occurred over <20 kb, where chromosome conformation capture-based methods perform poorly. For a fraction of loci, we identified a single variant that alters accessibility across multiple regions, and experimentally validated the BLK locus, which is associated with multiple autoimmune diseases, using CRISPR genome editing. Our study highlights how association genetics of chromatin state is a powerful approach for identifying interactions between regulatory elements.

Shared genetic effects on chromatin and gene expression indicate a role for enhancer priming in immune response.

Regulatory variants are often context specific, modulating gene expression in a subset of possible cellular states. Although these genetic effects can play important roles in disease, the molecular mechanisms underlying context specificity are poorly understood. Here, we identified shared quantitative trait loci (QTLs) for chromatin accessibility and gene expression in human macrophages exposed to IFNγ, Salmonella and IFNγ plus Salmonella. We observed that ~60% of stimulus-specific expression QTLs with a detectable effect on chromatin altered the chromatin accessibility in naive cells, thus suggesting that they perturb enhancer priming. Such variants probably influence binding of cell-type-specific transcription factors, such as PU.1, which can then indirectly alter the binding of stimulus-specific transcription factors, such as NF-κB or STAT2. Thus, although chromatin accessibility assays are powerful for fine-mapping causal regulatory variants, detecting their downstream effects on gene expression will be challenging, requiring profiling of large numbers of stimulated cellular states and time points.

Molecular and functional variation in iPSC-derived sensory neurons.

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools for modeling biological processes, particularly in cell types that are difficult to obtain from living donors. Here we present a map of regulatory variants in iPSC-derived neurons, based on 123 differentiations of iPSCs to a sensory neuronal fate. Gene expression was more variable across cultures than in primary dorsal root ganglion, particularly for genes related to nervous system development. Using single-cell RNA-sequencing, we found that the number of neuronal versus contaminating cells was influenced by iPSC culture conditions before differentiation. Despite high differentiation-induced variability, our allele-specific method detected thousands of quantitative trait loci (QTLs) that influenced gene expression, chromatin accessibility, and RNA splicing. On the basis of these detected QTLs, we estimate that recall-by-genotype studies that use iPSC-derived cells will require cells from at least 20-80 individuals to detect the effects of regulatory variants with moderately large effect sizes.