Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

Single-cell multi-omics of human preimplantation embryos shows susceptibility to glucocorticoids.

The preconceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of assisted reproductive technologies (ART; ∼25%), additives and adjuvants, such as glucocorticoids, are used to improve the success rate. Considering the dynamic developmental events that occur during this window, these exposures may alter blastocyst formation at a molecular level, and as such, affect not only the viability of the embryo and the ability of the blastocyst to implant, but also the developmental trajectory of the first three cell lineages, ultimately influencing the physiology of the embryo. In this study, we present a comprehensive single-cell transcriptome, methylome, and small RNA atlas in the day 7 human embryo. We show that, despite no change in morphology and developmental features, preimplantation glucocorticoid exposure reprograms the molecular profile of the TE lineage, and these changes are associated with an altered metabolic and inflammatory response. Our data also suggest that glucocorticoids can precociously mature the TE sublineages, supported by the presence of extravillous trophoblast markers in the polar sublineage and presence of X Chromosome dosage compensation. Further, we have elucidated that epigenetic regulation-DNA methylation and microRNAs (miRNAs)-likely underlies the transcriptional changes observed. This study suggests that exposures to exogenous compounds during preimplantation may unintentionally reprogram the human embryo, possibly leading to suboptimal development and longer-term health outcomes.

Molecular spikes: a gold standard for single-cell RNA counting.

Single-cell sequencing methods rely on molecule-counting strategies to account for amplification biases, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes-RNA spike-ins containing built-in unique molecular identifiers (UMIs) that we use to identify critical experimental and computational conditions for accurate RNA counting in single-cell RNA-sequencing (scRNA-seq). Using molecular spikes, we uncovered impaired RNA counting in methods that were not informative for cellular RNA abundances due to inflated UMI counts. We further leverage molecular spikes to improve estimates of total endogenous RNA amounts in cells, and introduce a strategy to correct experiments with impaired RNA counting. The molecular spikes and the accompanying R package UMIcountR ( https://github.com/cziegenhain/UMIcountR ) will improve the validation of new methods, better estimate and adjust for cellular mRNA amounts and enable more indepth characterization of RNA counting in scRNA-seq.

Genomic encoding of transcriptional burst kinetics.

Mammalian gene expression is inherently stochastic1,2, and results in discrete bursts of RNA molecules that are synthesized from each allele3-7. Although transcription is known to be regulated by promoters and enhancers, it is unclear how cis-regulatory sequences encode transcriptional burst kinetics. Characterization of transcriptional bursting, including the burst size and frequency, has mainly relied on live-cell4,6,8 or single-molecule RNA fluorescence in situ hybridization3,5,8,9 recordings of selected loci. Here we determine transcriptome-wide burst frequencies and sizes for endogenous mouse and human genes using allele-sensitive single-cell RNA sequencing. We show that core promoter elements affect burst size and uncover synergistic effects between TATA and initiator elements, which were masked at mean expression levels. Notably, we provide transcriptome-wide evidence that enhancers control burst frequencies, and demonstrate that cell-type-specific gene expression is primarily shaped by changes in burst frequencies. Together, our data show that burst frequency is primarily encoded in enhancers and burst size in core promoters, and that allelic single-cell RNA sequencing is a powerful model for investigating transcriptional kinetics.

Small noncoding RNA profiling across cellular and biofluid compartments and their implications for multiple sclerosis immunopathology.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system (CNS). Small non-coding RNAs (sncRNAs) and, in particular, microRNAs (miRNAs) have frequently been associated with MS. Here, we performed a comprehensive analysis of all classes of sncRNAs in matching samples of peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF) cells, and cell-free CSF from relapsing-remitting (RRMS, n = 12 in relapse and n = 11 in remission) patients, secondary progressive (SPMS, n = 6) MS patients, and noninflammatory and inflammatory neurological disease controls (NINDC, n = 11; INDC, n = 5). We show widespread changes in miRNAs and sncRNA-derived fragments of small nuclear, nucleolar, and transfer RNAs. In CSF cells, 133 out of 133 and 115 out of 117 differentially expressed sncRNAs were increased in RRMS relapse compared to remission and RRMS compared to NINDC, respectively. In contrast, 65 out of 67 differentially expressed PBMC sncRNAs were decreased in RRMS compared to NINDC. The striking contrast between the periphery and CNS suggests that sncRNA-mediated mechanisms, including alternative splicing, RNA degradation, and mRNA translation, regulate the transcriptome of pathogenic cells primarily in the CNS target organ.

Chronic Obstructive Pulmonary Disease Is Associated with Epigenome-Wide Differential Methylation in BAL Lung Cells.

DNA methylation patterns in chronic pulmonary obstructive disease (COPD) might offer new insights into disease pathogenesis. To assess methylation profiles in the main COPD target organ, we performed an epigenome-wide association study on BAL cells. Bronchoscopies were performed in 18 subjects with COPD and 15 control subjects (ex- and current smokers). DNA methylation was measured using the Illumina MethylationEPIC BeadChip Kit, covering more than 850,000 CpGs. Differentially methylated positions (DMPs) were examined for 1) enrichment in pathways and functional gene relationships using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology, 2) accelerated aging using Horvath's epigenetic clock, 3) correlation with gene expression, and 4) colocalization with genetic variation. We found 1,155 Bonferroni-significant (P < 6.74 × 10-8) DMPs associated with COPD, many with large effect sizes. Functional analysis identified biologically plausible pathways and gene relationships, including enrichment for transcription factor activity. Strong correlation was found between DNA methylation and chronological age but not between COPD and accelerated aging. For 79 unique DMPs, DNA methylation correlated significantly with gene expression in BAL cells. Thirty-nine percent of DMPs were colocalized with COPD-associated SNPs. To the best of our knowledge, this is the first epigenome-wide association study of COPD on BAL cells, and our analyses revealed many differential methylation sites. Integration with mRNA data showed a strong functional readout for relevant genes, identifying sites where DNA methylation might directly affect expression. Almost half of DMPs were colocated with SNPs identified in previous genome-wide association studies of COPD, suggesting joint genetic and epigenetic pathways related to disease.

Transcriptional bursts explain autosomal random monoallelic expression and affect allelic imbalance.

Transcriptional bursts render substantial biological noise in cellular transcriptomes. Here, we investigated the theoretical extent of allelic expression resulting from transcriptional bursting and how it compared to the amount biallelic, monoallelic and allele-biased expression observed in single-cell RNA-sequencing (scRNA-seq) data. We found that transcriptional bursting can explain the allelic expression patterns observed in single cells, including the frequent observations of autosomal monoallelic gene expression. Importantly, we identified that the burst frequency largely determined the fraction of cells with monoallelic expression, whereas the burst size had little effect on monoallelic observations. The high consistency between the bursting model predictions and scRNA-seq observations made it possible to assess the heterogeneity of a group of cells as their deviation in allelic observations from the expected. Finally, both burst frequency and size contributed to allelic imbalance observations and reinforced that studies of allelic imbalance can be confounded from the inherent noise in transcriptional bursting. Altogether, we demonstrate that allele-level transcriptional bursting renders widespread, although predictable, amounts of monoallelic and biallelic expression in single cells and cell populations.

Methylome and transcriptome signature of bronchoalveolar cells from multiple sclerosis patients in relation to smoking.

BACKGROUND: Despite compelling evidence that cigarette smoking impacts the risk of developing multiple sclerosis (MS), little is known about smoking-associated changes in the primary exposed lung cells of patients. OBJECTIVES: We aimed to examine molecular changes occurring in bronchoalveolar lavage (BAL) cells from MS patients in relation to smoking and in comparison to healthy controls (HCs). METHODS: We profiled DNA methylation in BAL cells from female MS (n = 17) and HC (n = 22) individuals, using Illumina Infinium EPIC and performed RNA-sequencing in non-smokers. RESULTS: The most prominent changes were found in relation to smoking, with 1376 CpG sites (adjusted P < 0.05) differing between MS smokers and non-smokers. Approximately 30% of the affected genes overlapped with smoking-associated changes in HC, leading to a strong common smoking signature in both MS and HC after gene ontology analysis. Smoking in MS patients resulted in additional discrete changes related to neuronal processes. Methylome and transcriptome analyses in non-smokers suggest that BAL cells from MS patients display very subtle (not reaching adjusted P < 0.05) but concordant changes in genes connected to reduced transcriptional/translational processes and enhanced cellular motility. CONCLUSIONS: Our study provides insights into the impact of smoking on lung inflammation and immunopathogenesis of MS.

Expression of MATE1, P-gp, OCTN1 and OCTN2, in epithelial and immune cells in the lung of COPD and healthy individuals.

BACKGROUND: Several inhaled drugs are dependent on organic cation transporters to cross cell membranes. To further evaluate their potential to impact on inhaled drug disposition, the localization of MATE1, P-gp, OCTN1 and OCTN2 were investigated in human lung. METHODS: Transporter proteins were analysed by immunohistochemistry in lung tissue from healthy subjects and COPD patients. Transporter mRNA was analysed by qPCR in lung tissue and in bronchoalveolar lavage (BAL) cells from smokers and non-smokers. RESULTS: We demonstrate for the first time MATE1 protein expression in the lung with localization to the apical side of bronchial and bronchiolar epithelial cells. Interestingly, MATE1 was strongly expressed in alveolar macrophages as demonstrated both in lung tissue and in BAL cells, and in inflammatory cells including CD3 positive T cells. P-gp, OCTN1 and OCTN2 were also expressed in the alveolar epithelial cells and in inflammatory cells including alveolar macrophages. In BAL cells from smokers, MATE1 and P-gp mRNA expression was significantly lower compared to cells from non-smokers whereas no difference was observed between COPD patients and healthy subjects. THP-1 cells were evaluated as a model for alveolar macrophages but did not reflect the transporter expression observed in BAL cells. CONCLUSIONS: We conclude that MATE1, P-gp, OCTN1 and OCTN2 are expressed in pulmonary lung epithelium, in alveolar macrophages and in other inflammatory cells. This is important to consider in the development of drugs treating pulmonary disease as the transporters may impact drug disposition in the lung and consequently affect pharmacological efficacy and toxicity.

T-cell activation and HLA-regulated response to smoking in the deep airways of patients with multiple sclerosis.

Cigarette smoking is a risk factor for multiple sclerosis (MS), and the risk is further multiplied for HLA-DRB1*15(+) smokers. To define the smoke-induced immune responses in the lung we performed bronchoscopy with bronchoalveolar lavage (BAL) on smokers and non-smokers, both MS-patients and healthy volunteers. In the BAL, non-smokers with MS showed an increased preformed CD40L expression in CD4(+) T-cells while smokers displayed an increase in proliferating (Ki-67(+)) T-cells. In addition, our results confirm that smoking induces an increase of alveolar macrophages in BAL, and further defined a significant attenuation of this response in carriers of the HLA-DRB1*15 allele, in both MS patients and healthy controls. This first systematic investigation of the immune response in the lungs of smokers and non-smokers diagnosed with MS, thus suggests an MS-associated lung T-cell phenotype, involvement of a specific T-cell response to smoke, and a genetic regulation of the macrophage response.

The NKG2D ligand ULBP2 is specifically regulated through an invariant chain-dependent endosomal pathway.

Soluble ULBP2 is a marker for poor prognosis in several types of cancer. In this study we demonstrate that both soluble and cell surface-bound ULBP2 is transported via a so far unrecognized endosomal pathway. ULBP2 surface expression, but not MICA/B, could specifically be targeted and retained by affecting endosomal/lysosomal integrity and protein kinase C activity. The invariant chain was further essential for endosomal transport of ULBP2. This novel pathway was identified through screening experiments by which methylselenic acid was found to possess notable NKG2D ligand regulatory properties. The protein kinase C inhibitor methylselenic acid induced MICA/B surface expression but dominantly blocked ULBP2 surface transport. Remarkably, by targeting this novel pathway we could specifically block the production of soluble ULBP2 from different, primary melanomas. Our findings strongly suggest that the endosomal transport pathway constitutes a novel therapeutic target for ULBP2-producing tumors.

The selenium metabolite methylselenol regulates the expression of ligands that trigger immune activation through the lymphocyte receptor NKG2D.

For decades, selenium research has been focused on the identification of active metabolites, which are crucial for selenium chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include increased formation of reactive oxygen species, induction of DNA damage, triggering of apoptosis, and inhibition of angiogenesis. Here we reveal that CH3SeH modulates the cell surface expression of NKG2D ligands. The expression of NKG2D ligands is induced by stress-associated pathways that occur early during malignant transformation and enable the recognition and elimination of tumors by activating the lymphocyte receptor NKG2D. CH3SeH regulated NKG2D ligands both on the transcriptional and the posttranscriptional levels. CH3SeH induced the transcription of MHC class I polypeptide-related sequence MICA/B and ULBP2 mRNA. However, the induction of cell surface expression was restricted to the ligands MICA/B. Remarkably, our studies showed that CH3SeH inhibited ULBP2 surface transport through inhibition of the autophagic transport pathway. Finally, we identified extracellular calcium as being essential for CH3SeH regulation of NKG2D ligands. A balanced cell surface expression of NKG2D ligands is considered to be an innate barrier against tumor development. Therefore, our work indicates that the application of selenium compounds that are metabolized to CH3SeH could improve NKG2D-based immune therapy.

2-deoxy D-glucose prevents cell surface expression of NKG2D ligands through inhibition of N-linked glycosylation.

NKG2D ligand surface expression is important for immune recognition of stressed and neotransformed cells. In this study, we show that surface expression of MICA/B and other NKG2D ligands is dependent on N-linked glycosylation. The inhibitor of glycolysis and N-linked glycosylation, 2-deoxy-D-glucose (2DG), potently inhibited surface expression of MICA/B after histone deacetylase inhibitor treatment; the inhibition occurred posttranscriptionally without affecting MICA promoter activity. Transient overexpression of MICA surface expression was also inhibited by 2DG. 2DG blocks N-linked glycosylation of MICA/B by a reversible mechanism that can be alleviated by addition of d-mannose; this does not, however, affect the inhibition of glycolysis. Addition of d-mannose restored MICA/B surface expression after 2DG treatment. In addition, specific pharmacological or small interfering RNA-mediated targeting of glycolytic enzymes did not affect MICA/B surface expression, strongly suggesting that N-linked glycosylation, and not glycolysis, is essential for MICA/B surface expression. Corroborating this, tunicamycin, a selective inhibitor of N-linked glycosylation, abolished MICA/B surface expression without compromising activation of MICA promoter activity. NK cell-mediated killing assay and staining with a recombinant NKG2D-Fc fusion protein showed that all functional NKG2D ligands induced by histone deacetylase inhibitor treatment were abolished by 2DG treatment and fully reconstituted by further addition of d-mannose. Our data suggest that posttranslational N-linked glycosylation is strictly required for NKG2D ligand surface expression. Cancer and infection often result in aberrant glycosylation, which could likely be involved in modulation of NKG2D ligand expression. Our data further imply that chemotherapeutic use of 2DG may restrict NKG2D ligand surface expression and inhibit secretion of immunoinhibitory soluble NKG2D ligands.

Single-Cell mRNA-sncRNA Co-sequencing of Preimplantation Embryos.

The development of single-cell multiomics has provided the ability to systematically investigate cellular diversity and heterogeneity in different biological systems via comprehensive delineations of individual cellular states. Single-cell RNA sequencing in particular has served as a powerful tool to the study of the molecular circuitries underlying preimplantation embryonic development in both the mouse and human. Here we describe a method to elucidate the cellular dynamics of the embryo further by performing both single-cell RNA sequencing (Smart-Seq2) and single-cell small non-coding RNA sequencing (Small-Seq) on the same individual embryonic cell.

Clonally heritable gene expression imparts a layer of diversity within cell types.

Cell types can be classified according to shared patterns of transcription. Non-genetic variability among individual cells of the same type has been ascribed to stochastic transcriptional bursting and transient cell states. Using high-coverage single-cell RNA profiling, we asked whether long-term, heritable differences in gene expression can impart diversity within cells of the same type. Studying clonal human lymphocytes and mouse brain cells, we uncovered a vast diversity of heritable gene expression patterns among different clones of cells of the same type in vivo. We combined chromatin accessibility and RNA profiling on different lymphocyte clones to reveal thousands of regulatory regions exhibiting interclonal variation, which could be directly linked to interclonal variation in gene expression. Our findings identify a source of cellular diversity, which may have important implications for how cellular populations are shaped by selective processes in development, aging, and disease. A record of this paper's transparent peer review process is included in the supplemental information.

Erythroid Differentiation Enhances RNA Mis-Splicing in SF3B1-Mutant Myelodysplastic Syndromes with Ring Sideroblasts.

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly develop from hematopoietic stem cells (HSC) bearing mutations in the splicing factor SF3B1 (SF3B1mt). Direct studies into MDS-RS pathobiology have been limited by a lack of model systems that fully recapitulate erythroid biology and RS development and the inability to isolate viable human RS. Here, we combined successful direct RS isolation from patient samples, high-throughput multiomics analysis of cells encompassing the SF3B1mt stem-erythroid continuum, and functional assays to investigate the impact of SF3B1mt on erythropoiesis and RS accumulation. The isolated RS differentiated, egressed into the blood, escaped traditional nonsense-mediated decay (NMD) mechanisms, and leveraged stress-survival pathways that hinder wild-type hematopoiesis through pathogenic GDF15 overexpression. Importantly, RS constituted a contaminant of magnetically enriched CD34+ cells, skewing bulk transcriptomic data. Mis-splicing in SF3B1mt cells was intensified by erythroid differentiation through accelerated RNA splicing and decreased NMD activity, and SF3B1mt led to truncations in several MDS-implicated genes. Finally, RNA mis-splicing induced an uncoupling of RNA and protein expression, leading to critical abnormalities in proapoptotic p53 pathway genes. Overall, this characterization of erythropoiesis in SF3B1mt RS provides a resource for studying MDS-RS and uncovers insights into the unexpectedly active biology of the "dead-end" RS. SIGNIFICANCE: Ring sideroblast isolation combined with state-of-the-art multiomics identifies survival mechanisms underlying SF3B1-mutant erythropoiesis and establishes an active role for erythroid differentiation and ring sideroblasts themselves in SF3B1-mutant myelodysplastic syndrome pathogenesis.

Smoking affects epigenetic ageing of lung bronchoalveolar lavage cells in Multiple Sclerosis.

BACKGROUND: A compelling body of evidence implicates cigarette smoking and lung inflammation in Multiple Sclerosis (MS) susceptibility and progression. Previous studies have reported epigenetic age (DNAm age) acceleration in blood immune cells and in glial cells of people with MS (pwMS) compared to healthy controls (HC). OBJECTIVES: We aimed to examine biological ageing in lung immune cells in the context of MS and smoking. METHODS: We analyzed age acceleration residuals in lung bronchoalveolar lavage (BAL) cells, constituted of mainly alveolar macrophages, from 17 pwMS and 22 HC in relation to smoking using eight DNA methylation-based clocks, namely AltumAge, Horvath, GrimAge, PhenoAge, Zhang, SkinBlood, Hannum, Monocyte clock as well as two RNA-based clocks, which capture different aspects of biological ageing. RESULTS: After adjustment for covariates, five epigenetic clocks showed significant differences between the groups. Four of them, Horvath (Padj = 0.028), GrimAge (Padj = 4.28 × 10-7), SkinBlood (Padj = 0.001) and Zhang (Padj = 0.02), uncovered the sole effect of smoking on ageing estimates, irrespective of the clinical group. The Horvath, SkinBlood and Zhang clocks showed a negative impact of smoking while GrimAge detected smoking-associated age acceleration in BAL cells. On the contrary, the AltumAge clock revealed differences between pwMS and HC and indicated that, in the absence of smoking, BAL cells of pwMS were epigenetically 5.4 years older compared to HC (Padj = 0.028). Smoking further affected epigenetic ageing in BAL cells of pwMS specifically as non-smoking pwMS exhibited a 10.2-year AltumAge acceleration compared to pwMS smokers (Padj = 0.0049). Of note, blood-derived monocytes did not show any MS-specific or smoking-related AltumAge differences. The difference between BAL cells of pwMS smokers and non-smokers was attributable to the differential methylation of 114 AltumAge-CpGs (Padj < 0.05) affecting genes involved in innate immune processes such as cytokine production, defense response and cell motility. These changes functionally translated into transcriptional differences in BAL cells between pwMS smokers and non-smokers. CONCLUSIONS: BAL cells of pwMS display inflammation-related and smoking-dependent changes associated to epigenetic ageing captured by the AltumAge clock. Future studies examining potential confounders, such as the distribution of distinct BAL myeloid cell types in pwMS compared to control individuals in relation to smoking may clarify the varying performance and DNAm age estimations among epigenetic clocks.

Reconstructing clonal tree for phylo-phenotypic characterization of cancer using single-cell transcriptomics.

Functional characterization of the cancer clones can shed light on the evolutionary mechanisms driving cancer's proliferation and relapse mechanisms. Single-cell RNA sequencing data provide grounds for understanding the functional state of cancer as a whole; however, much research remains to identify and reconstruct clonal relationships toward characterizing the changes in functions of individual clones. We present PhylEx that integrates bulk genomics data with co-occurrences of mutations from single-cell RNA sequencing data to reconstruct high-fidelity clonal trees. We evaluate PhylEx on synthetic and well-characterized high-grade serous ovarian cancer cell line datasets. PhylEx outperforms the state-of-the-art methods both when comparing capacity for clonal tree reconstruction and for identifying clones. We analyze high-grade serous ovarian cancer and breast cancer data to show that PhylEx exploits clonal expression profiles beyond what is possible with expression-based clustering methods and clear the way for accurate inference of clonal trees and robust phylo-phenotypic analysis of cancer.

Spatial transcriptomics of B cell and T cell receptors reveals lymphocyte clonal dynamics.

The spatial distribution of lymphocyte clones within tissues is critical to their development, selection, and expansion. We have developed spatial transcriptomics of variable, diversity, and joining (VDJ) sequences (Spatial VDJ), a method that maps B cell and T cell receptor sequences in human tissue sections. Spatial VDJ captures lymphocyte clones that match canonical B and T cell distributions and amplifies clonal sequences confirmed by orthogonal methods. We found spatial congruency between paired receptor chains, developed a computational framework to predict receptor pairs, and linked the expansion of distinct B cell clones to different tumor-associated gene expression programs. Spatial VDJ delineates B cell clonal diversity and lineage trajectories within their anatomical niche. Thus, Spatial VDJ captures lymphocyte spatial clonal architecture across tissues, providing a platform to harness clonal sequences for therapy.

Scalable single-cell RNA sequencing from full transcripts with Smart-seq3xpress.

Current single-cell RNA sequencing (scRNA-seq) methods with high cellular throughputs sacrifice full-transcript coverage and often sensitivity. Here we describe Smart-seq3xpress, which miniaturizes and streamlines the Smart-seq3 protocol to substantially reduce reagent use and increase cellular throughput. Smart-seq3xpress analysis of peripheral blood mononuclear cells resulted in a granular atlas complete with common and rare cell types. Compared with droplet-based single-cell RNA sequencing that sequences RNA ends, the additional full-transcript coverage revealed cell-type-associated isoform variation.