CellRegMap: a statistical framework for mapping context‐specific regulatory variants using scRNA‐seq

Single‐cell RNA sequencing (scRNA‐seq) enables characterizing the cellular heterogeneity in human tissues. Recent technological advances have enabled the first population‐scale scRNA‐seq studies in hundreds of individuals, allowing to assay genetic effects with single‐cell resolution. However, existing strategies to analyze these data remain based on principles established for the genetic analysis of bulk RNA‐seq. In particular, current methods depend on a priori definitions of discrete cell types, and hence cannot assess allelic effects across subtle cell types and cell states. To address this, we propose the Cell Regulatory Map (CellRegMap), a statistical framework to test for and quantify genetic effects on gene expression in individual cells. CellRegMap provides a principled approach to identify and characterize genotype–context interactions of known eQTL variants using scRNA‐seq data. This model‐based approach resolves allelic effects across cellular contexts of different granularity, including genetic effects specific to cell subtypes and continuous cell transitions. We validate CellRegMap using simulated data and apply it to previously identified eQTL from two recent studies of differentiating iPSCs, where we uncover hundreds of eQTL displaying heterogeneity of genetic effects across cellular contexts. Finally, we identify fine‐grained genetic regulation in neuronal subtypes for eQTL that are colocalized with human disease variants.     

Advances in single‐cell sequencing and its application to musculoskeletal system research

In recent years, single‐cell sequencing (SCS) technologies have continued to advance with improved operating procedures and reduced cost, leading to increasing practical adoption among researchers. These emerging technologies have superior abilities to analyse cell heterogeneity at a single‐cell level, which have elevated multi‐omics research to a higher level. In some fields of research, application of SCS has enabled many valuable discoveries, and musculoskeletal system offers typical examples. This article reviews some major scientific issues and recent advances in musculoskeletal system. In addition, combined with SCS technologies, the research of cell or tissue heterogeneity in limb development and various musculoskeletal system clinical diseases also provides new possibilities for treatment strategies. Finally, this article discusses the challenges and future development potential of SCS and recommends the direction of future applications of SCS to musculoskeletal medicine.     

Cellular and molecular atlas of the placenta from a COVID‐19 pregnant woman infected at midgestation highlights the defective impacts on foetal health

ObjectivesThe impacts of the current COVID‐19 pandemic on maternal and foetal health are enormous and of serious concern. However, the influence of SARS‐CoV‐2 infection at early‐to‐mid gestation on maternal and foetal health remains unclear.Materials and methodsHere, we report the follow‐up study of a pregnant woman of her whole infective course of SARS‐CoV‐2, from asymptomatic infection at gestational week 20 to mild and then severe illness state, and finally cured at Week 24. Following caesarean section due to incomplete uterine rupture at Week 28, histological examinations on the placenta and foetal tissues as well as single‐cell RNA sequencing (scRNA‐seq) for the placenta were performed.ResultsCompared with the gestational age‐matched control placentas, the placenta from this COVID‐19 case exhibited more syncytial knots and lowered expression of syncytiotrophoblast‐related genes. The scRNA‐seq analysis demonstrated impaired trophoblast differentiation, activation of antiviral and inflammatory CD8 T cells, as well as the tight association of increased inflammatory responses in the placenta with complement over‐activation in macrophages. In addition, levels of several inflammatory factors increased in the placenta and foetal blood.ConclusionThese findings illustrate a systematic cellular and molecular signature of placental insufficiency and immune activation at the maternal–foetal interface that may be attributed to SARS‐CoV‐2 infection at the midgestation stage, which highly suggests the extensive care for maternal and foetal outcomes in pregnant women suffering from COVID‐19.     

Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri‐implantation period

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1⁺ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single‐cell RNA sequencing (scRNA‐seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue‐resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function‐related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA‐seq and cytotoxicity assays confirmed that CYP26A1⁺ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal‐foetal interface by regulating the activity of NK cells.     

Neural G0: a quiescent‐like state found in neuroepithelial‐derived cells and glioma

Single‐cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA‐seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent‐like state in neuroepithelial‐derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non‐dividing neural progenitors. Putative glioblastoma stem‐like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down‐regulation of quiescence‐associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent‐like state found in neuroepithelial‐derived cells and gliomas.     

Single‐cell transcriptome analysis reveals defective decidua stromal niche attributes to recurrent spontaneous abortion

ObjectivesSuccessful pregnancy involves the homeostasis between maternal decidua and fetoplacental units, whose disruption contributes to compromised pregnancy outcomes, including recurrent spontaneous abortion (RSA). The role of cell heterogeneity of maternal decidua in RSA is yet to be illustrated.Materials and methodsA total of 66,078 single cells from decidua samples isolated from patients with RSA and healthy controls were analysed by unbiased single‐cell RNA sequencing (scRNA‐seq).ResultsOur scRNA‐seq results revealed that stromal cells are the most abundant cell type in decidua during early pregnancy. RSA samples are accompanied by aberrant decidualization and obviously obstructed communication between stromal cells and other cell types, such as abnormal activation of macrophages and NK cells. In addition, the over‐activated TNF superfamily member 12 (TNFSF12, TWEAK) and FASLG in RSA are closely related to stromal cell demise and pregnancy failure.ConclusionsOur research reveals that the cell composition and communications in normal and RSA decidua at early pregnancy and provides insightful information for the pathology of RSA and will pave the way for pregnancy loss prevention.

Recurrent spontaneous abortion (RSA), characterized by pregnancy loss before 20 weeks of gestation more than twice, is an intricated pregnancy complication with enigmatic underlying mechanism ascribes to its complex pathogenesis. The homeostasis between the developing foetus and maternal decidua is critical for pregnancy maintenance. By exploring the cell heterogeneity in normal and RSA decidua utilizing scRNA‐Seq, we unravel the discrepancies in cell composition and communications in these two distinct deciduae. Our investigations uncover that stromal cells are the most abundant cell populations in the decidua, with three different subpopulations at various decidualization stages and two fibroblasts. There are two separated trajectories of stromal cell decidualization marked by PLA2G2A and WNT4. As the most abundant cell population in the decidua, the stromal cells dominate the communications with other cell types, including endothelial cells, macrophages, uNK cells and perivascular cells. Compared with normal decidua, decidualized stromal cells are overtly decreased in RSA decidua with augmented macrophages. In addition, we present some previously unappreciated signaling pathways among different cells types in decidua and also depict the remarkably changed communications between normal and RSA decidual. The aberrant activated TWEAK and FASLG in RSA are considered to be potential reasons for stromal cells demise and pregnancy failure. Our research reveals the cell composition and communications in normal and RSA decidua at early pregnancy and provides insightful information for the pathology of RSA and will pave the way for pregnancy loss prevention.     

Whole‐body senescent cell clearance alleviates age‐related brain inflammation and cognitive impairment in mice

Cellular senescence is characterized by an irreversible cell cycle arrest and a pro‐inflammatory senescence‐associated secretory phenotype (SASP), which is a major contributor to aging and age‐related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single‐nuclei and single‐cell RNA‐seq in the hippocampus from young and aged mice. We observed an age‐dependent increase in p16^Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK‐ATTAC mice, in which p16^Ink4a‐positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16^Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof‐of‐concept for senolytic interventions'' being a potential therapeutic avenue for alleviating age‐associated cognitive impairment.     

A genotyping‐in‐thousands by sequencing panel to inform invasive deer management using noninvasive fecal and hair samples

Studies in ecology, evolution, and conservation often rely on noninvasive samples, making it challenging to generate large amounts of high‐quality genetic data for many elusive and at‐risk species. We developed and optimized a Genotyping‐in‐Thousands by sequencing (GT‐seq) panel using noninvasive samples to inform the management of invasive Sitka black‐tailed deer (Odocoileus hemionus sitkensis) in Haida Gwaii (Canada). We validated our panel using paired high‐quality tissue and noninvasive fecal and hair samples to simultaneously distinguish individuals, identify sex, and reconstruct kinship among deer sampled across the archipelago, then provided a proof‐of‐concept application using field‐collected feces on SGang Gwaay, an island of high ecological and cultural value. Genotyping success across 244 loci was high (90.3%) and comparable to that of high‐quality tissue samples genotyped using restriction‐site associated DNA sequencing (92.4%), while genotyping discordance between paired high‐quality tissue and noninvasive samples was low (0.50%). The panel will be used to inform future invasive species operations in Haida Gwaii by providing individual and population information to inform management. More broadly, our GT‐seq workflow that includes quality control analyses for targeted SNP selection and a modified protocol may be of wider utility for other studies and systems where noninvasive genetic sampling is employed.     

TATA and paused promoters active in differentiated tissues have distinct expression characteristics

Core promoter types differ in the extent to which RNA polymerase II (Pol II) pauses after initiation, but how this affects their tissue‐specific gene expression characteristics is not well understood. While promoters with Pol II pausing elements are active throughout development, TATA promoters are highly active in differentiated tissues. We therefore used a genomics approach on late‐stage Drosophila embryos to analyze the properties of promoter types. Using tissue‐specific Pol II ChIP‐seq, we found that paused promoters have high levels of paused Pol II throughout the embryo, even in tissues where the gene is not expressed, while TATA promoters only show Pol II occupancy when the gene is active. The promoter types are associated with different chromatin accessibility in ATAC‐seq data and have different expression characteristics in single‐cell RNA‐seq data. The two promoter types may therefore be optimized for different properties: paused promoters show more consistent expression when active, while TATA promoters have lower background expression when inactive. We propose that tissue‐specific genes have evolved to use two different strategies for their differential expression across tissues.