Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients.

The coronavirus disease 2019 (COVID-19) pandemic poses a current world-wide public health threat. However, little is known about its hallmarks compared to other infectious diseases. Here, we report the single-cell transcriptional landscape of longitudinally collected peripheral blood mononuclear cells (PBMCs) in both COVID-19- and influenza A virus (IAV)-infected patients. We observed increase of plasma cells in both COVID-19 and IAV patients and XIAP associated factor 1 (XAF1)-, tumor necrosis factor (TNF)-, and FAS-induced T cell apoptosis in COVID-19 patients. Further analyses revealed distinct signaling pathways activated in COVID-19 (STAT1 and IRF3) versus IAV (STAT3 and NFκB) patients and substantial differences in the expression of key factors. These factors include relatively increase of interleukin (IL)6R and IL6ST expression in COVID-19 patients but similarly increased IL-6 concentrations compared to IAV patients, supporting the clinical observations of increased proinflammatory cytokines in COVID-19 patients. Thus, we provide the landscape of PBMCs and unveil distinct immune response pathways in COVID-19 and IAV patients.

Rolling back human pluripotent stem cells to an eight-cell embryo-like stage.

After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.

Cell transcriptomic atlas of the non-human primate Macaca fascicularis.

Studying tissue composition and function in non-human primates (NHPs) is crucial to understand the nature of our own species. Here we present a large-scale cell transcriptomic atlas that encompasses over 1 million cells from 45 tissues of the adult NHP Macaca fascicularis. This dataset provides a vast annotated resource to study a species phylogenetically close to humans. To demonstrate the utility of the atlas, we have reconstructed the cell-cell interaction networks that drive Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases, and intersected our data with human genetic disease orthologues to establish potential clinical associations. Our M. fascicularis cell atlas constitutes an essential reference for future studies in humans and NHPs.

Single-cell transcriptional diversity of neonatal umbilical cord blood immune cells reveals neonatal immune tolerance.

The characteristics of neonatal immune cells display intrinsic differences compared with adult immune cells. Therefore, a comprehensive analysis of key gene expression regulation is required to understand the response of the human fetal immune system to infections. Here, we applied single-cell RNA sequencing (scRNA-seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) to systematically profile umbilical cord blood (UCB) nucleated cells and peripheral blood mononuclear cells (PBMCs) to identify their composition and differentially expressed genes. The immune cells in neonatal UCB demonstrated the expression of key genes, such as HBG2, NFKBIA, JUN, FOS, and TNFAIP3. In contrast, natural killer and T cells, which are constituents of adult PBMCs, exhibited high cytotoxic gene expression. Furthermore, we obtained similar results from the data of scATAC-seq by identifying the status of chromatin accessibility of key genes. Therefore, scRNA-seq and scATAC-seq of neonatal UCB nucleated cells and adult PBMCs could serve as an invaluable resource for elucidating the regulatory mechanisms of responses of distinct immune cell types and further identifying the differences between neonatal and adult immune responses to predict the potential underlying mechanism for neonatal immune tolerance.

A single-cell transcriptome atlas of human euploid and aneuploid blastocysts.

Aneuploidy is frequently detected in early human embryos as a major cause of early pregnancy failure. However, how aneuploidy affects cellular function remains elusive. Here, we profiled the transcriptomes of 14,908 single cells from 203 human euploid and aneuploid blastocysts involving autosomal and sex chromosomes. Nearly all of the blastocysts contained four lineages. In aneuploid chromosomes, 19.5% ± 1.2% of the expressed genes showed a dosage effect, and 90 dosage-sensitive domains were identified. Aneuploidy leads to prevalent genome-wide transcriptome alterations. Common effects, including apoptosis, were identified, especially in monosomies, partially explaining the lower cell numbers in autosomal monosomies. We further identified lineage-specific effects causing unstable epiblast development in aneuploidies, which was accompanied by the downregulation of TGF-ß and FGF signaling, which resulted in insufficient trophectoderm maturation. Our work provides crucial insights into the molecular basis of human aneuploid blastocysts and may shed light on the cellular interaction during blastocyst development.

A spatiotemporal atlas of cholestatic injury and repair in mice.

Cholestatic liver injuries, characterized by regional damage around the bile ductular region, lack curative therapies and cause considerable mortality. Here we generated a high-definition spatiotemporal atlas of gene expression during cholestatic injury and repair in mice by integrating spatial enhanced resolution omics sequencing and single-cell transcriptomics. Spatiotemporal analyses revealed a key role of cholangiocyte-driven signaling correlating with the periportal damage-repair response. Cholangiocytes express genes related to recruitment and differentiation of lipid-associated macrophages, which generate feedback signals enhancing ductular reaction. Moreover, cholangiocytes express high TGFß in association with the conversion of liver progenitor-like cells into cholangiocytes during injury and the dampened proliferation of periportal hepatocytes during recovery. Notably, Atoh8 restricts hepatocyte proliferation during 3,5-diethoxycarbonyl-1,4-dihydro-collidin damage and is quickly downregulated after injury withdrawal, allowing hepatocytes to respond to growth signals. Our findings lay a keystone for in-depth studies of cellular dynamics and molecular mechanisms of cholestatic injuries, which may further develop into therapies for cholangiopathies.

A spatiotemporal atlas of mouse liver homeostasis and regeneration.

The mechanism by which mammalian liver cell responses are coordinated during tissue homeostasis and perturbation is poorly understood, representing a major obstacle in our understanding of many diseases. This knowledge gap is caused by the difficulty involved with studying multiple cell types in different states and locations, particularly when these are transient. We have combined Stereo-seq (spatiotemporal enhanced resolution omics-sequencing) with single-cell transcriptomic profiling of 473,290 cells to generate a high-definition spatiotemporal atlas of mouse liver homeostasis and regeneration at the whole-lobe scale. Our integrative study dissects in detail the molecular gradients controlling liver cell function, systematically defining how gene networks are dynamically modulated through intercellular communication to promote regeneration. Among other important regulators, we identified the transcriptional cofactor TBL1XR1 as a rheostat linking inflammation to Wnt/ß-catenin signaling for facilitating hepatocyte proliferation. Our data and analytical pipelines lay the foundation for future high-definition tissue-scale atlases of organ physiology and malfunction.

Comparative analysis of mesenchymal stem/stromal cells derived from human induced pluripotent stem cells and the cognate umbilical cord mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells (MSCs) show tremendous potential for regenerative medicine due to their self-renewal, multi-differentiation and immunomodulatory capabilities. Largely studies had indicated conventional tissue-derived MSCs have considerable limited expandability and donor variability which hinders further application. Induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) have created exciting source for standardized cellular therapy. However, the cellular and molecular differences between iMSCs and the cognate tissue-derived MSCs remains poorly explored. In this study, we first successfully reprogrammed human umbilical cords-derived mesenchymal stem/stromal cells (UMSCs) into iPSCs by using the cocktails of mRNA. Subsequently, iPSCs were further differentiated into iMSCs in xeno-free induction medium. Then, iMSCs were compared with the donor matched UMSCs by assessing proliferative state, differentiation capability, immunomodulatory potential through immunohistochemical analysis, flow cytometric analysis, transcriptome sequencing analysis, and combine with coculture with immune cell population. The results showed that iMSCs exhibited high expression of MSCs positive-makers CD73, CD90, CD105 and lack expression of negative-maker cocktails CD34, CD45, CD11b, CD19, HLA-DR; also successfully differentiated into osteocytes, chondrocytes and adipocytes. Further, the iMSCs were similar with their parental UMSCs in cell proliferative state detected by the CCK-8 assay, and in cell rejuvenation state assessed by ß-Galactosidase staining and telomerase activity related mRNA and protein analysis. However, iMSCs exhibited similarity to resident MSCs in Homeobox (Hox) genes expression profile and presented better neural differentiation potential by activation of NESTIN related pathway. Moreover, iMSCs owned enhanced immunosuppression capacity through downregulation pools of pro-inflammatory factors, including IL6, IL1B etc. and upregulation anti-inflammatory factors NOS1, TGFB etc. signals. In summary, our study provides an attractive cell source for basic research and offers fundamental biological insight of iMSCs-based therapy.

Single-nucleus chromatin landscapes during zebrafish early embryogenesis.

Vertebrate embryogenesis is a remarkable process, during which numerous cell types of different lineages arise within a short time frame. An overwhelming challenge to understand this process is the lack of dynamic chromatin accessibility information to correlate cis-regulatory elements (CREs) and gene expression within the hierarchy of cell fate decisions. Here, we employed single-nucleus ATAC-seq to generate a chromatin accessibility dataset on the first day of zebrafish embryogenesis, including 3.3 hpf, 5.25 hpf, 6 hpf, 10 hpf, 12 hpf, 18 hpf and 24 hpf, obtained 51,620 high-quality nuclei and 23 clusters. Furthermore, by integrating snATAC-seq data with single-cell RNA-seq data, we described the dynamics of chromatin accessibility and gene expression across developmental time points, which validates the accuracy of the chromatin landscape data. Together, our data could serve as a fundamental resource for revealing the epigenetic regulatory mechanisms of zebrafish embryogenesis.

A scATAC-seq atlas of chromatin accessibility in axolotl brain regions.

Axolotl (Ambystoma mexicanum) is an excellent model for investigating regeneration, the interaction between regenerative and developmental processes, comparative genomics, and evolution. The brain, which serves as the material basis of consciousness, learning, memory, and behavior, is the most complex and advanced organ in axolotl. The modulation of transcription factors is a crucial aspect in determining the function of diverse regions within the brain. There is, however, no comprehensive understanding of the gene regulatory network of axolotl brain regions. Here, we utilized single-cell ATAC sequencing to generate the chromatin accessibility landscapes of 81,199 cells from the olfactory bulb, telencephalon, diencephalon and mesencephalon, hypothalamus and pituitary, and the rhombencephalon. Based on these data, we identified key transcription factors specific to distinct cell types and compared cell type functions across brain regions. Our results provide a foundation for comprehensive analysis of gene regulatory programs, which are valuable for future studies of axolotl brain development, regeneration, and evolution, as well as on the mechanisms underlying cell-type diversity in vertebrate brains.

Single-cell chromatin accessibility profiling of cell-state-specific gene regulatory programs during mouse organogenesis.

In mammals, early organogenesis begins soon after gastrulation, accompanied by specification of various type of progenitor/precusor cells. In order to reveal dynamic chromatin landscape of precursor cells and decipher the underlying molecular mechanism driving early mouse organogenesis, we performed single-cell ATAC-seq of E8.5-E10.5 mouse embryos. We profiled a total of 101,599 single cells and identified 41 specific cell types at these stages. Besides, by performing integrated analysis of scATAC-seq and public scRNA-seq data, we identified the critical cis-regulatory elements and key transcription factors which drving development of spinal cord and somitogenesis. Furthermore, we intersected accessible peaks with human diseases/traits-related loci and found potential clinical associated single nucleotide variants (SNPs). Overall, our work provides a fundamental source for understanding cell fate determination and revealing the underlying mechanism during postimplantation embryonic development, and expand our knowledge of pathology for human developmental malformations.

Molecular Identification of Commercial Fish Maws by DNA Sequencing of 16S rRNA and Cytochrome c Oxidase I Genes.

ABSTRACT: Fish maws (dried swim bladders) have long been used for medicinal tonics and as a valuable food resource in Southeast Asia. However, it is difficult to identify the original species of fish maws sold in markets due to a lack of taxonomic characteristics. In the present study, 37 kinds of commercial fish maws from various medicinal material markets were examined, and gene sequences were successfully obtained from ca. 95% of the samples. Partial sequences of the 16S rRNA gene and cytochrome c oxidase I (COI) gene were obtained and used to investigate the origin of these commercial fish maws. Thirty-five specimens belonged to nine species: five croakers and four noncroakers. All species identification was supported by both high homogeneity (98 to 100%) and clear clustering with low within-group Kimura two-parameter divergence scores (0 to 0.04 for 16S rRNA and 0 to 0.07 for COI) and high between-group divergence scores (0.07 to 0.15 for 16S rRNA and 0.11 to 0.24 for COI). Croakers were the predominant species, accounting for 74% of the total fish maw specimens. The large demand for croakers has put some species at the risk of extinction due to overfishing. As a valuable food, fish maw has progressively become more popular and has been used as a substitute for shark fin. The identification results allowed us to learn more about the fish species available on the fish maw market and provided an indicator for possible control of threatened or endangered fish species. A probable correlation between the molecular characteristics and morphological features of fish maws was also found and could provide both consumers and merchants with an important reference for identifying the origin of fish maws.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Mesenchymal Stem/Stromal Cells Derived from Human Placenta.

Mesenchymal stem/stromal cells derived from placenta (PMSCs) are an attractive source for regenerative medicine because of their multidifferentiation potential and immunomodulatory capabilities. However, the cellular and molecular heterogeneity of PMSCs has not been fully characterized. Here, we applied single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin sequencing (scATAC-seq) techniques to cultured PMSCs from human full-term placenta. Based on the inferred characteristics of cell clusters, we identify several distinct subsets of PMSCs with specific characteristics, including immunomodulatory-potential and highly proliferative cell states. Furthermore, integrative analysis of gene expression and chromatin accessibility showed a clearer chromatin accessibility signature than those at the transcriptional level on immunomodulatory-related genes. Cell cycle gene-related heterogeneity can be more easily distinguished at the transcriptional than the chromatin accessibility level in PMSCs. We further reveal putative subset-specific cis-regulatory elements regulating the expression of immunomodulatory- and proliferation-related genes in the immunomodulatory-potential and proliferative subpopulations, respectively. Moreover, we infer a novel transcription factor PRDM1, which might play a crucial role in maintaining immunomodulatory capability by activating PRDM1-regulon loop. Collectively, our study first provides a comprehensive and integrative view of the transcriptomic and epigenomic features of PMSCs, which paves the way for a deeper understanding of cellular heterogeneity and offers fundamental biological insight of PMSC subset-based cell therapy.

A map of bat virus receptors derived from single-cell multiomics.

Bats are considered reservoirs of many lethal zoonotic viruses and have been implicated in several outbreaks of emerging infectious diseases, such as SARS-CoV, MERS-CoV, and SARS-CoV-2. It is necessary to systematically derive the expression patterns of bat virus receptors and their regulatory features for future research into bat-borne viruses and the prediction and prevention of pandemics. Here, we performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) of major organ samples collected from Chinese horseshoe bats (Rhinolophus affinis) and systematically checked the expression pattern of bat-related virus receptors and chromatin accessibility across organs and cell types, providing a valuable dataset for studying the nature of infection among bat-borne viruses.

Single-cell transcriptional profiling reveals cellular and molecular divergence in human maternal-fetal interface.

Placenta plays essential role in successful pregnancy, as the most important organ connecting and interplaying between mother and fetus. However, the cellular characteristics and molecular interaction of cell populations within the fetomaternal interface is still poorly understood. Here, we surveyed the single-cell transcriptomic landscape of human full-term placenta and revealed the heterogeneity of cytotrophoblast cell (CTB) and stromal cell (STR) with the fetal/maternal origin consecutively localized from fetal section (FS), middle section (Mid_S) to maternal section (Mat_S) of maternal-fetal interface. Then, we highlighted a subpopulation of CTB, named trophoblast progenitor-like cells (TPLCs) existed in the full-term placenta and mainly distributed in Mid_S, with high expression of a pool of putative cell surface markers. Further, we revealed the putative key transcription factor PRDM6 that might promote the differentiation of endovascular extravillous trophoblast cells (enEVT) by inhibiting cell proliferation, and down-regulation of PRDM6 might lead to an abnormal enEVT differentiation process in PE. Together, our study offers important resources for better understanding of human placenta and stem cell-based therapy, and provides new insights on the study of tissue heterogeneity, the clinical prevention and control of PE as well as the maternal-fetal interface.

Single-cell transcriptome profiling reveals molecular heterogeneity in human umbilical cord tissue and culture-expanded mesenchymal stem cells.

Human umbilical cord-derived mesenchymal stem/stromal cells (UMSCs) demonstrate great therapeutic potential in regenerative medicine. The use of UMSCs for clinical applications requires high quantity and good quality of cells usually by in vitro expansion. However, the heterogeneity and the characteristics of cultured UMSCs and the cognate human umbilical cord tissue at single-cell resolution remain poorly defined. In this study, we created a single-cell transcriptome profile of human umbilical cord tissue and the cognate culture-expanded UMSCs. Based on the inferred characteristics of cell clusters and trajectory analysis, we identified three subgroups in culture-expanded UMSCs and putative novel transcription factors (TFs) in regulating UMSC state transition. Further, putative ligand-receptor interaction analysis demonstrated that cellular interactions most frequently occurred in epithelial-like cells with other cell groups in umbilical cord tissue. Moreover, we dissected the transcriptomic differences of in vitro and in vivo subgroups and inferred the telomere-related molecules and pathways that might be activated in UMSCs for cell expansion in vitro. Our study provides a comprehensive and integrative study of the transcriptomics of human umbilical cord tissue and their cognate-cultured counterparts, which paves the way for a deeper understanding of cellular heterogeneity and offers fundamental biological insight of UMSCs-based cell therapy.

Single-Nucleus Chromatin Accessibility Landscape Reveals Diversity in Regulatory Regions Across Distinct Adult Rat Cortex.

Rats have been widely used as an experimental organism in psychological, pharmacological, and behavioral studies by modeling human diseases such as neurological disorders. It is critical to identify and characterize cell fate determinants and their regulatory mechanisms in single-cell resolutions across rat brain regions. Here, we applied droplet-based single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) to systematically profile the single-cell chromatin accessibility across four dissected brain areas in adult Sprague-Dawley (SD) rats with a total of 59,023 single nuclei and identified 16 distinct cell types. Interestingly, we found that different cortex regions exhibit diversity in both cellular compositions and gene regulatory regions. Several cell-type-specific transcription factors (TFs), including SPI1, KLF4, KLF6, and NEUROD2, have been shown to play important roles during the pathogenesis of various neurological diseases, such as Alzheimer's disease (AD), astrocytic gliomas, autism spectrum disorder (ASD), and intellectual disabilities. Therefore, our single-nucleus atlas of rat cortex could serve as an invaluable resource for dissecting the regulatory mechanisms underlying diverse cortex cell fates and further revealing the regulatory networks of neuropathogenesis.

Serum-Free Culture System for Spontaneous Human Mesenchymal Stem Cell Spheroid Formation.

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

Single-cell RNA-seq reveals dynamic transcriptome profiling in human early neural differentiation.

Background: Investigating cell fate decision and subpopulation specification in the context of the neural lineage is fundamental to understanding neurogenesis and neurodegenerative diseases. The differentiation process of neural-tube-like rosettes in vitro is representative of neural tube structures, which are composed of radially organized, columnar epithelial cells and give rise to functional neural cells. However, the underlying regulatory network of cell fate commitment during early neural differentiation remains elusive. Results: In this study, we investigated the genome-wide transcriptome profile of single cells from six consecutive reprogramming and neural differentiation time points and identified cellular subpopulations present at each differentiation stage. Based on the inferred reconstructed trajectory and the characteristics of subpopulations contributing the most toward commitment to the central nervous system lineage at each stage during differentiation, we identified putative novel transcription factors in regulating neural differentiation. In addition, we dissected the dynamics of chromatin accessibility at the neural differentiation stages and revealed active cis-regulatory elements for transcription factors known to have a key role in neural differentiation as well as for those that we suggest are also involved. Further, communication network analysis demonstrated that cellular interactions most frequently occurred in the embryoid body stage and that each cell subpopulation possessed a distinctive spectrum of ligands and receptors associated with neural differentiation that could reflect the identity of each subpopulation. Conclusions: Our study provides a comprehensive and integrative study of the transcriptomics and epigenetics of human early neural differentiation, which paves the way for a deeper understanding of the regulatory mechanisms driving the differentiation of the neural lineage.