We performed RNA sequencing on 40,000 cells to create a high-resolution single-cell gene expression atlas of developing human cortex, providing the first single-cell characterization of previously ...uncharacterized cell types, including human subplate neurons, comparisons with bulk tissue, and systematic analyses of technical factors. These data permit deconvolution of regulatory networks connecting regulatory elements and transcriptional drivers to single-cell gene expression programs, significantly extending our understanding of human neurogenesis, cortical evolution, and the cellular basis of neuropsychiatric disease. We tie cell-cycle progression with early cell fate decisions during neurogenesis, demonstrating that differentiation occurs on a transcriptomic continuum; rather than only expressing a few transcription factors that drive cell fates, differentiating cells express broad, mixed cell-type transcriptomes before telophase. By mapping neuropsychiatric disease genes to cell types, we implicate dysregulation of specific cell types in ASD, ID, and epilepsy. We developed CoDEx, an online portal to facilitate data access and browsing.
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•High-resolution transcriptome map of 40,000 cells from developing human brain•Cell-type-specific transcription factor (TF) expression and TF-gene networks•Defines intermediate cell transition states during early neurogenesis•Implicates specific cell types in neuropsychiatric disorders
An extensive single-cell catalog of cell types in the mid-gestation human neocortex extends our understanding of early cortical development, including subplate neuron transcriptomes, cell-type-specific regulatory networks, brain evolution, and the cellular basis of neuropsychiatric disease.
Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X ...chromosome (Xi) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets Xi abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications.
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•Naive female hPSCs have two active X chromosomes (XaXa) and express XIST RNA•Differentiation of naive hPSCs prompts X inactivation via a state without XIST•Some X chromosome features of the embryo are not captured by naive hPSCs•The naive state erases epigenetic abnormalities of the Xi seen in primed hPSCs
Plath and colleagues study the epigenetic state of the X chromosomes in naive female hPSCs and discover that it closely, but not perfectly, resembles the X chromosome pattern of pre-implantation blastocysts. The naive state enables de novo X inactivation upon differentiation and thus can provide a model for studying X chromosome regulation in human cells.
Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. ...Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.
Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream ...regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1−/− tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.
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•IRF1 expression in tumor cells or the TME governs tumor progression or regression•Tumor cell IRF1 blocks host IFNAR and TLR activation of antitumor adaptive immunity•Tumor cell IRF1 controls only an immunosuppressive gene program induced by IFN-γ•IRF1 controls distinct IFN-γ-induced genes by binding promoters alone or with STAT1
Proper lymphocyte activation is required for antitumor immunity. Purbey et al. show that, while IRF1 in immune cells is required for antitumor immunity, tumor cell IRF1 suppresses Toll-like receptor- and interferon-induced antitumor lymphocyte activation and regulates select immunosuppressive and MHC genes in interferon-stimulated tumor cells.
To determine the role for mutations of MECP2 in Rett syndrome, we generated isogenic lines of human induced pluripotent stem cells, neural progenitor cells, and neurons from patient fibroblasts with ...and without MECP2 expression in an attempt to recapitulate disease phenotypes in vitro. Molecular profiling uncovered neuronal-specific gene expression changes, including induction of a senescence-associated secretory phenotype (SASP) program. Patient-derived neurons made without MECP2 showed signs of stress, including induction of P53, and senescence. The induction of P53 appeared to affect dendritic branching in Rett neurons, as P53 inhibition restored dendritic complexity. The induction of P53 targets was also detectable in analyses of human Rett patient brain, suggesting that this disease-in-a-dish model can provide relevant insights into the human disorder.
•Development of a patient-specific model of human Rett syndrome•Loss of function of MECP2 leads to induction of p53•MECP2 null neurons show evidence of cellular senescence•Inhibition of p53 can restore dendritic branching in MECP2 null neurons
In this report, Lowry and colleagues found that loss of MECP2 has a more profound effect as pluripotent stem cells are terminally differentiated toward neurons. The loss of MECP2 leads to induction of P53 protein and subsequent senescence pathways including an SASP gene program, which appears to be a cause of diminished dendritic branching in Rett neurons.
A dramatic difference in global DNA methylation between male and female cells characterizes mouse embryonic stem cells (ESCs), unlike somatic cells. We analyzed DNA methylation changes during ...reprogramming of male and female somatic cells and in resulting induced pluripotent stem cells (iPSCs). At an intermediate reprogramming stage, somatic and pluripotency enhancers are targeted for partial methylation and demethylation. Demethylation within pluripotency enhancers often occurs at ESC binding sites of pluripotency transcription factors. Late in reprogramming, global hypomethylation is induced in a female-specific manner. Genome-wide hypomethylation in female cells affects many genomic landmarks, including enhancers and imprint control regions, and accompanies the reactivation of the inactive X chromosome. The loss of one of the two X chromosomes in propagating female iPSCs is associated with genome-wide methylation gain. Collectively, our findings highlight the dynamic regulation of DNA methylation at enhancers during reprogramming and reveal that X chromosome dosage dictates global DNA methylation levels in iPSCs.
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•Female iPSCs are globally hypomethylated•Hypomethylation affects multiple genomic features including imprint control regions•Loss of X chromosome restores global methylation but not at imprint control regions•Methylation changes at enhancers in male and female reprogramming intermediates
Somatic cells can be reprogrammed to iPSCs, inducing reactivation of the inactive X chromosome. Using genome-scale DNA methylation analyses, Plath, Pasque, and colleagues show that iPSCs adopt sex-specific differences in global DNA methylation that correlate with the presence of two active X chromosomes. Upon culture, female iPSCs lose one of the two X chromosomes and adopt male-like DNA methylation.
Cells secrete numerous bioactive molecules that are essential for the function of healthy organisms. However, scalable methods are needed to link individual cell secretions to their transcriptional ...state over time. Here, by developing and using secretion-encoded single-cell sequencing (SEC-seq), which exploits hydrogel particles with subnanolitre cavities (nanovials) to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells. Our data indicate that VEGF-A secretion is heterogeneous across the cell population and is poorly correlated with the VEGFA transcript level. The highest VEGF-A secretion occurs in a subpopulation of mesenchymal stromal cells characterized by a unique gene expression signature comprising a surface marker, interleukin-13 receptor subunit alpha 2 (IL13RA2), which allowed the enrichment of this subpopulation. SEC-seq enables the identification of gene signatures linked to specific secretory states, facilitating mechanistic studies, the isolation of secretory subpopulations and the development of means to modulate cellular secretion.
The developmental trajectory of human skeletal myogenesis and the transition between progenitor and stem cell states are unclear. We used single-cell RNA sequencing to profile human skeletal muscle ...tissues from embryonic, fetal, and postnatal stages. In silico, we identified myogenic as well as other cell types and constructed a “roadmap” of human skeletal muscle ontogeny across development. In a similar fashion, we also profiled the heterogeneous cell cultures generated from multiple human pluripotent stem cell (hPSC) myogenic differentiation protocols and mapped hPSC-derived myogenic progenitors to an embryonic-to-fetal transition period. We found differentially enriched biological processes and discovered co-regulated gene networks and transcription factors present at distinct myogenic stages. This work serves as a resource for advancing our knowledge of human myogenesis. It also provides a tool for a better understanding of hPSC-derived myogenic progenitors for translational applications in skeletal muscle-based regenerative medicine.
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•Human atlas of limb skeletal muscle in embryonic, fetal, and adult tissues•Human limb skeletal muscle populations and supportive cells vary across development•PAX7 muscle progenitor and stem cells are not identical across developmental states•hPSC-PAX7 cells align to the embryonic-to-fetal transition in human development
Xi et al. developed a comprehensive view of skeletal muscle and supportive cells across human development. This atlas revealed transcriptional differences among myogenic progenitors and stem cells at distinct developmental stages. This enabled identification of the developmental status of hPSC-derived muscle cells to the embryonic-to-fetal transition period in human development.
Current smoking is associated with increased risk of severe COVID-19, but it is not clear how cigarette smoke (CS) exposure affects SARS-CoV-2 airway cell infection. We directly exposed air-liquid ...interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term CS and then infected them with SARS-CoV-2. We found an increase in the number of infected airway cells after CS exposure with a lack of ABSC proliferation. Single-cell profiling of the cultures showed that the normal interferon response was reduced after CS exposure with infection. Treatment of CS-exposed ALI cultures with interferon β-1 abrogated the viral infection, suggesting one potential mechanism for more severe viral infection. Our data show that acute CS exposure allows for more severe airway epithelial disease from SARS-CoV-2 by reducing the innate immune response and ABSC proliferation and has implications for disease spread and severity in people exposed to CS.
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•Direct cigarette smoke exposure increases the number of SARS-CoV-2 infected cells•SARS-CoV-2 infection inhibits the airway basal stem cell repair response•Cigarette smoke reduces innate immune responses with worse SARS-CoV-2 infection
Purkayastha and colleagues modeled the direct effects of cigarette smoke on SARS-CoV-2 infection of the airway epithelium. Acute cigarette smoke exposure increased the number of infected and apoptotic cells, prevented the normal airway basal stem cell repair response, and blunted innate immune responses. Improving innate immunity impaired SARS-CoV-2 infection.
Much has been learned about the mechanisms of action of pluripotency factors Oct4 and Sox2. However, as with other regulators of cell identity, little is known about the impact of disrupting their ...binding motifs in a native environment or the characteristics of genes they regulate. By quantitatively examining dynamic ranges of gene expression instead of focusing on conventional measures of differential expression, we found that Oct4 and Sox2 enhancer binding is strongly enriched near genes subject to large dynamic ranges of expression among cell types, with binding sites near these genes usually within superenhancers. Mutagenesis of representative Oct4:Sox2 motifs near such active, dynamically regulated genes revealed critical roles in transcriptional activation during reprogramming, with more limited roles in transcriptional maintenance in the pluripotent state. Furthermore, representative motifs near silent genes were critical for establishing but not maintaining the fully silent state, while genes whose transcript levels varied by smaller magnitudes among cell types were unaffected by nearby Oct4:Sox2 motifs. These results suggest that Oct4 and Sox2 directly establish both active and silent transcriptional states in pluripotent cells at a large number of genes subject to dynamic regulation during mammalian development, but are less important than expected for maintaining transcriptional states.
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