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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.

Cells secrete numerous bioactive molecules essential for the function of healthy organisms. However, there are no scalable methods to link individual cell secretions to their transcriptional state. By developing and using secretion encoded single-cell sequencing (SEC-seq), which exploits hydrogel 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 (MSCs). We found that VEGF-A secretion is heterogeneous across the cell population and lowly correlated with theVEGFAtranscript level. While there is a modest population-wide increase in VEGF-A secretion by hypoxic induction, highest VEGF-A secretion across normoxic and hypoxic culture conditions occurs in a subpopulation of MSCs characterized by a unique gene expression signature. Taken together, SEC-seq enables the identification of specific genes involved in the control of secretory states, which may be exploited for developing means to modulate cellular secretion for disease treatment.

Summary The specification of inhibitory neurons has been described for the mouse and human brain, and many studies have shown that pluripotent stem cells (PSCs) can be used to create interneurons in vitro. It is unclear whether in vitro methods to produce human interneurons generate all the subtypes found in brain, and how similar in vitro and in vivo interneurons are. We applied single-nuclei and single-cell transcriptomics to model interneuron development from human cortex and interneurons derived from PSCs. We provide a direct comparison of various in vitro interneuron derivation methods to determine the homogeneity achieved. We find that PSC-derived interneurons capture stages of development prior to mid-gestation, and represent a minority of potential subtypes found in brain. Comparison with those found in fetal or adult brain highlighted decreased expression of synapse-related genes. These analyses highlight the potential to tailor the method of generation to drive formation of particular subtypes., Highlights • Comparison of interneurons derived from human pluripotent cells by various methods • Single-cell analyses define heterogeneity of in vitro-derived interneurons • Direct comparison of in vitro- and in vivo-derived interneurons • Identification of transcriptional modules that developmentally define interneurons, Plath, Lowry and colleagues profile interneurons generated from human pluripotent stem cells by various methods to understand the heterogeneity and cellular state of interneuron cultures in vitro. Using single-cell analyses, the authors define the homogeneity and maturity achieved with each in vitro method. By directly comparing these interneurons with those born in the human brain, the authors highlight distinctions particularly in synaptic genes and transcription factor modules that distinguish in vitro- and in vivo-derived neurons.

High-grade complex karyotype sarcomas are a heterogeneous group of more than seventy tumors that vary in histology, clinical course, and patient demographics. Despite these differences, sarcomas are treated similarly with varying efficacy. This is in part due to the rarity of the disease and the lack of preclinical models for the study of individual sarcoma subtypes. Here, we describe the development of a model of high-grade sarcoma formation from human mesenchymal stem cells by introducing genetic changes that are seen in complex karyotype sarcomas. RB1 and P53 are tumor suppressors that are often mutated or functionally inactivated in sarcomas and these were targeted using CRISPR-Cas9 technology which, alone did not result in sarcoma formation. To identify drivers, a lentiviral library screen was performed and the results were validated in a secondary screen. From this, YAP1 and K-RAS were found to robustly drive the formation of undifferentiated pleomorphic sarcoma and myxofibrosarcoma which exist on a disease spectrum. Other drivers were identified that resulted in high grade sarcomas. The majority of these tumors were undifferentiated pleomorphic sarcoma although pleomorphic leiomyosarcoma and osteosarcoma were present in a subset of tumors driven by CDK4 and PIK3CA. This tool allows for the study of human sarcoma subtype development from mesenchymal stem cells and provides a platform for further mechanistic studies, investigations of metastasis, and drug development for this aggressive and heterogeneous disease.

Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease. Single-cell RNA profiling of human cystic fibrosis proximal airway tissue reveals an overabundance of epithelial cells transitioning to specialized ciliated and secretory cells coupled with a decrease in cycling basal cells.

Summary CpG islands often exhibit low DNA methylation, high histone H3 lysine 4 trimethylation, low nucleosome density, and high DNase I hypersensitivity, yet the rules by which CpG islands are sensed remain poorly understood. In this study, we first evaluated the relationships between the DNA and the chromatin properties of CpG islands in embryonic stem cells using modified bacterial artificial chromosomes. Then, using a bioinformatic approach, we identified strict CpG-island density and length thresholds in mouse embryonic stem and differentiated cells that consistently specify low DNA methylation levels. Surprisingly, the human genome exhibited a dramatically different relationship between DNA properties and DNA methylation levels of CpG islands. Further analysis allowed speculation that this difference is accommodated in part by evolutionary changes in the nucleotide composition of orthologous promoters. Thus, a change in the rules by which CpG-island properties are sensed may have co-evolved with compensatory genome adaptation events during mammalian evolution., Graphical abstract, Highlights • Strict DNA property rules can predict low DNA methylation at murine CpG islands • Mice and humans differ greatly in how DNA properties influence DNA methylation • Evolutionary adaptation of CpG islands compensates for the species differences, Langerman and colleagues examined relationships between DNA and chromatin properties of CpG islands. They identified rules by which the length of a CpG-rich region and CpG density influence DNA methylation. These rules differ between humans and mice, with the differences permitted during evolution by adaptations at the DNA and chromatin levels.

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., The pandemic of COVID-19, caused by SARS-CoV-2 infection, warrants immediate investigation for therapy options. Here the authors show, using epithelial and air-liquid interface cultures, that SARS-CoV-2 hijacks host cell metabolism to facilitate viral replication, and that inhibition of mTORC1, a master metabolic regulator, suppresses viral replication.

Introduction/AbstractCystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts in excess of 70,000 people globally. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most significant determinant of morbidity and mortality. In this study we report results from a multi-institute consortium in which single cell transcriptomics were applied to define disease-related changes to the proximal airway of CF donors (n=19) undergoing transplantation for end-stage lung disease compared to the proximal airway of previously healthy lung donors (n=19). We found that all major airway epithelial cell types were conserved between control and CF donors. Disease-dependent differences were observed, including an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subtypes coupled with an unexpected decrease in cycling basal cells. This study developed a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

Summary 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., Graphical Abstract, Highlights • 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.

Applications of embryonic stem cells (ESCs) require faithful chromatin changes during differentiation, but the fate of the X chromosome state in differentiating ESCs is unclear. Female human ESC lines either carry two active X chromosomes (XaXa), an Xa and inactive X chromosome with or without XIST RNA coating (XiXIST+Xa;XiXa), or an Xa and an eroded Xi (XeXa) where the Xi no longer expresses XIST RNA and has partially reactivated. Here, we established XiXa, XeXa, and XaXa ESC lines and followed their X chromosome state during differentiation. Surprisingly, we found that the X state pre-existing in primed ESCs is maintained in differentiated cells. Consequently, differentiated XeXa and XaXa cells lacked XIST, did not induce X inactivation, and displayed higher X-linked gene expression than XiXa cells. These results demonstrate that X chromosome dosage compensation is not required for ESC differentiation. Our data imply that XiXIST+Xa ESCs are most suited for downstream applications and show that all other X states are abnormal byproducts of our ESC derivation and propagation method.

The developmental trajectory of human skeletal myogenesis and the transition between progenitor and stem cell states are unclear. We employed 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.

Defining the number, proportion, or lineage of distinct cell types in the developing human brain is an important goal of modern brain research. We defined single cell transcriptomic profiles for 40,000 cells at mid-gestation to identify cell types in the developing human neocortex. We define expression profiles corresponding to all known major cell types at this developmental period and identify multiple transcription factors and co-factors expressed in specific cell types, providing an unprecedented resource for understanding human neocortical development including the first single-cell characterization of human subplate neurons. We characterize major developmental trajectories during early neurogenesis, showing that cell type differentiation occurs on a continuum that involves transitions that tie cell cycle progression with early cell fate decisions. We use these data to deconvolute regulatory networks and map neuropsychiatric disease genes to specific cell types, implicating dysregulation of specific cell types, as the mechanistic underpinnings of several neurodevelopmental disorders. Together these results provide an extensive catalog of cell types in human neocortex and extend our understanding of early cortical development, human brain evolution and the cellular basis of neuropsychiatric disease.One Sentence SummaryComprehensive single cell transcriptomes in developing human cortex inform models of cell diversity, differentiation and disease risk.

We performed RNA sequencing on 40,000 cells to create a high-resolution single-cell gene expres-sion atlas of developing human cortex, providing the first single-cell characterization of previously uncharacterized cell types, including human sub-plate 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.

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.

Summary Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single-cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm-biased stem cell state., Graphical Abstract, Highlights • Subsets of hESCs can co-express pluripotency-associated and lineage-specific genes • hESCs co-expressing GATA6 are capable of long-term self-renewal • Single GATA6-expressing hESCs regenerate GATA6-negative cells • GATA6-expressing hESCs are biased in their propensity for differentiation, Human embryonic stem cells have the capacity to turn into any cell type within the adult. Peter Andrews and colleagues have shown that subtle differences between individual cells can functionally bias the resulting cell type that is produced. Generating and purifying these biased cells may therefore improve the derivation of medically relevant cell types.