Live birth of chimeric monkey with high contribution from embryonic stem cells.

A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologous cells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditions for establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our results have major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.

Multimodal cell atlas of the ageing human skeletal muscle.

Muscle atrophy and functional decline (sarcopenia) are common manifestations of frailty and are critical contributors to morbidity and mortality in older people1. Deciphering the molecular mechanisms underlying sarcopenia has major implications for understanding human ageing2. Yet, progress has been slow, partly due to the difficulties of characterizing skeletal muscle niche heterogeneity (whereby myofibres are the most abundant) and obtaining well-characterized human samples3,4. Here we generate a single-cell/single-nucleus transcriptomic and chromatin accessibility map of human limb skeletal muscles encompassing over 387,000 cells/nuclei from individuals aged 15 to 99 years with distinct fitness and frailty levels. We describe how cell populations change during ageing, including the emergence of new populations in older people, and the cell-specific and multicellular network features (at the transcriptomic and epigenetic levels) associated with these changes. On the basis of cross-comparison with genetic data, we also identify key elements of chromatin architecture that mark susceptibility to sarcopenia. Our study provides a basis for identifying targets in the skeletal muscle that are amenable to medical, pharmacological and lifestyle interventions in late life.

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.

STOmicsDB: a comprehensive database for spatial transcriptomics data sharing, analysis and visualization.

Recent technological developments in spatial transcriptomics allow researchers to measure gene expression of cells and their spatial locations at the single-cell level, generating detailed biological insight into biological processes. A comprehensive database could facilitate the sharing of spatial transcriptomic data and streamline the data acquisition process for researchers. Here, we present the Spatial TranscriptOmics DataBase (STOmicsDB), a database that serves as a one-stop hub for spatial transcriptomics. STOmicsDB integrates 218 manually curated datasets representing 17 species. We annotated cell types, identified spatial regions and genes, and performed cell-cell interaction analysis for these datasets. STOmicsDB features a user-friendly interface for the rapid visualization of millions of cells. To further facilitate the reusability and interoperability of spatial transcriptomic data, we developed standards for spatial transcriptomic data archiving and constructed a spatial transcriptomic data archiving system. Additionally, we offer a distinctive capability of customizing dedicated sub-databases in STOmicsDB for researchers, assisting them in visualizing their spatial transcriptomic analyses. We believe that STOmicsDB could contribute to research insights in the spatial transcriptomics field, including data archiving, sharing, visualization and analysis. STOmicsDB is freely accessible at https://db.cngb.org/stomics/.

Uncovering the Bronchoalveolar Single-Cell Landscape of Patients With Pulmonary Tuberculosis With Human Immunodeficiency Virus Type 1 Coinfection.

BACKGROUND: Coinfection of human immunodeficiency virus type 1 (HIV-1) is the most significant risk factor for tuberculosis (TB). The immune responses of the lung are essential to restrict the growth of Mycobacterium tuberculosis and avoid the emergence of the disease. Nevertheless, there is still limited knowledge about the local immune response in people with HIV-1-TB coinfection. METHODS: We employed single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid from 9 individuals with HIV-1-TB coinfection and 10 with pulmonary TB. RESULTS: A total of 19 058 cells were grouped into 4 major cell types: myeloid cells, T/natural killer (NK) cells, B cells, and epithelial cells. The myeloid cells and T/NK cells were further divided into 10 and 11 subsets, respectively. The proportions of dendritic cell subsets, CD4+ T cells, and NK cells were lower in the HIV-1-TB coinfection group compared to the TB group, while the frequency of CD8+ T cells was higher. Additionally, we identified numerous differentially expressed genes between the CD4+ and CD8+ T-cell subsets between the 2 groups. CONCLUSIONS: HIV-1 infection not only affects the abundance of immune cells in the lungs but also alters their functions in patients with pulmonary TB.

High-Throughput Screening of Functional Neo-Antigens and Their Specific T-Cell Receptors via the Jurkat Reporter System Combined with Droplet Microfluidics.

T-cell receptor (TCR)-engineered T cells can precisely recognize a broad repertoire of targets derived from both intracellular and surface proteins of tumor cells. TCR-T adoptive cell therapy has shown safety and promising efficacy in solid tumor immunotherapy. However, antigen-specific functional TCR screening is time-consuming and expensive, which limits its application clinically. Here, we developed a novel integrated antigen-TCR screening platform based on droplet microfluidic technology, enabling high-throughput peptide-major histocompatibility complex (pMHC)-to-TCR paired screening with a high sensitivity and low background signal. We introduced DNA barcoding technology to label peptide antigen candidate-loaded antigen-presenting cells and Jurkat reporter cells to check the specificity of pMHC-TCR candidates. Coupled with the next-generation sequencing pipeline, interpretation of the DNA barcodes and the gene expression level of the Jurkat T-cell activation pathway provided a clear peptide-MHC-TCR recognition relationship. Our proof-of-principle study demonstrates that the platform could achieve pMHC-TCR paired high-throughput screening, which is expected to be used in the cross-reactivity and off-target high-throughput paired testing of candidate pMHC-TCRs in clinical applications.

The FASTQ+ format and PISA.

SUMMARY: The FASTQ+ format is designed for single-cell experiments. It extends various optional tags, including cell barcodes and unique molecular identifiers, to the sequence identifier and is fully compatible with the FASTQ format. In addition, PISA implements various utilities for processing sequences in the FASTQ format and alignments in the SAM/BAM/CRAM format from single-cell experiments, such as converting FASTQ format to FASTQ+, annotating alignments, PCR deduplication, feature counting and barcodes correction. The software is open-source and written in C language. AVAILABILITY AND IMPLEMENTATION: https://doi.org/10.5281/zenodo.7007056 or https://github.com/shiquan/PISA. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High-Throughput Functional Screening of Antigen-Specific T Cells Based on Droplet Microfluidics at a Single-Cell Level.

The lack of an efficient method for the identification of tumor antigen-specific T cell receptors (TCRs) impedes the development of T cell-based cancer immunotherapies. Here, we introduce a droplet-based microfluidic platform for function-based screening and sorting of tumor antigen-specific T cells with high throughput. We built a reporter cell line by co-transducing the TCR library and reporter genes at the downstream of TCR signaling, and reporter cells fluoresced upon functionally binding with antigens. We co-encapsulated reporter cells and antigen-presenting cells in droplets to allow for stimulation on a single-cell level. Functioning reporter cells specific against the antigen were identified in the microfluidic channel based on the fluorescent signals of the droplets, which were immediately sorted out using dielectrophoresis. We validated the reporter system and sorting results using flow cytometry. We then performed single-cell RNA sequencing on the sorted cells to further validate this platform and demonstrate the compatibility with genetic characterizations. Our platform provides a means for precise and efficient T cell immunotherapy, and the droplet-based high-throughput TCR screening method could potentially facilitate immunotherapeutic screening and promote T cell-based anti-tumor therapies.

Generation and Screening of Antigen-Specific Nanobodies from Mammalian Cells Expressing the BCR Repertoire Library Using Droplet-Based Microfluidics.

Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.

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.

Neuroimaging characteristics and long-term prognosis of myxoma-related intracranial diseases.

PURPOSE: Myxoma-related intracranial diseases were rarely documented in history. The main purpose of our study is to provide a more comprehensive and detailed understanding of the pathogenesis, imaging features, surgical procedures and pathology of such patients through long-term follow-up. METHODS: From March 2012 to July 2018, baseline information that included neuroimaging and neuropathology data from 12 cardiac myxoma patients with neurological symptoms were retrospectively analysed, and the treatment options were discussed. Nine patients underwent long-term postoperative follow-up. RESULTS: Twelve cardiac myxoma patients with neurological symptoms were identified, and among them, 10 patients were postoperative patients who had undergone excision of cardiac myxoma, 5 patients had received craniotomy, and the others had received conservative treatment. Positive neuroimaging findings were found in all patients, including cerebral infarction (12/12, 100%), multiple intracranial aneurysms (8/12, 67%), and extravascular metastasis (6/12, 50%). After a long-term average follow-up of 27 months, an increased number of metastatic lesions or an enlargement of the intracranial aneurysms was found in 4 patients. CONCLUSIONS: Neuroimaging findings of myxoma-related intracranial lesions were diversed and usually presented as multiple cerebral infarction, aneurysm formation, focal intracranial haemorrhage and space-occupying lesions. Progress is over a long period of time after primary tumour resection. It is necessary for patients to be regularly examined within 2 years after cardiac myxoma resection using MRI+CTA/MRA/DSA in order to be ruled out. Stable and effective chemotherapy drugs are urgently needed.

[Microsurgical treatment for giant invasive spinal schwannoma assisted by three-dimensional navigation].

OBJECTIVE: To summarize the clinical experience of microsurgical treatment for giant invasive spinal schwannoma assisted by three-dimensional navigation. METHODS: A total of 15 cases of giant invasive spinal schwannoma were retrospectively analyzed from 2013 to 2014 in Beijing Jishuitan Hospital.All patients were performed microsurgery assisted by three-dimensional navigation and were followed up for at least 12 months.A modified McCormick Scale was used to assess the patients' neurologic status and change. RESULTS: Four lesions were in the cervical region, 3 in the sacral, 2 each in the cervicothoracic, lumbar and thoracic regions, 1 each in the thoracolumbar and lumbosacral regions.A total of 28 pedicle screws were placed satisfactorily in the 5 patients with spinal instability.No severe complications were encountered.Gross total resection was performed in 13 of the 15 patients, and subtotal resection performed in 2 patients.Satisfactory decompression was achieved in all patients for neural compression.Postoperative clinical symptoms were improved in all patients, and none of the patients showed loosening or displacement of the implants. CONCLUSIONS: Three-dimensional navigation provides great help for neurosurgeons in surgical treatment of giant invasive spinal schwannoma, and it has great potential in raising the intraoperative localization accuracy, reducing operational damage and surgical complications.Total resection is suggested for giant invasive spinal schwannoma; if not, total resection of the intraspinal portion is recommended.

The hypoxia-inducible factor renders cancer cells more sensitive to vitamin C-induced toxicity.

Megadose vitamin C (Vc) is one of the most enduring alternative treatments for diverse human diseases and is deeply engrafted in popular culture. Preliminary studies in the 1970s described potent effects of Vc on prolonging the survival of patients with terminal cancer, but these claims were later criticized. An improved knowledge of the pharmacokinetics of Vc and recent reports using cancer cell lines have renewed the interest in this subject. Despite these findings, using Vc as an adjuvant for anticancer therapy remains questionable, among other things because there is no proper mechanistic understanding. Here, we show that a Warburg effect triggered by activation of the hypoxia-inducible factor (HIF) pathway greatly enhances Vc-induced toxicity in multiple cancer cell lines, including von Hippel-Lindau (VHL)-defective renal cancer cells. HIF increases the intracellular uptake of oxidized Vc through its transcriptional target glucose transporter 1 (GLUT1), synergizing with the uptake of its reduced form through sodium-dependent Vc transporters. The resulting high levels of intracellular Vc induce oxidative stress and massive DNA damage, which then causes metabolic exhaustion by depleting cellular ATP reserves. HIF-positive cells are particularly sensitive to Vc-induced ATP reduction because they mostly rely on the rather inefficient glycolytic pathway for energy production. Thus, our experiments link Vc-induced toxicity and cancer metabolism, providing a new explanation for the preferential effect of Vc on cancer cells.

Class IIa histone deacetylases and myocyte enhancer factor 2 proteins regulate the mesenchymal-to-epithelial transition of somatic cell reprogramming.

Class IIa histone deacetylases (HDACs) and myocyte enhancer factor 2 (MEF2) proteins compose a signaling module that orchestrates lineage specification during embryogenesis. We show here that this module also regulates the generation of mouse induced pluripotent stem cells by defined transcription factors. Class IIa HDACs and MEF2 proteins rise steadily during fibroblast reprogramming to induced pluripotent stem cells. MEF2 proteins tend to block the process by inducing the expression of Tgfß cytokines, which impairs the necessary phase of mesenchymal-to-epithelial transition (MET). Conversely, class IIa HDACs endeavor to suppress the activity of MEF2 proteins, thus enhancing the MET and colony formation efficiency. Our work highlights an unexpected role for a developmental axis in somatic cell reprogramming and provides new insight into how the MET is regulated in this context.

Expression characteristics and potential function of non-coding RNA in mouse cortical cells.

Non-coding RNAs (ncRNAs) play essential regulatory functions in various physiological and pathological processes in the brain. To systematically characterize the ncRNA profile in cortical cells, we downloaded single-cell SMART-Seq v4 data of mouse cerebral cortex. Our results revealed that the ncRNAs alone are sufficient to define the identity of most cortical cell types. We identified 1,600 ncRNAs that exhibited cell type specificity, even yielding to distinguish microglia from perivascular macrophages with ncRNA. Moreover, we characterized cortical layer and region specific ncRNAs, in line with the results by spatial transcriptome (ST) data. By constructing a co-expression network of ncRNAs and protein-coding genes, we predicted the function of ncRNAs. By integrating with genome-wide association studies data, we established associations between cell type-specific ncRNAs and traits related to neurological disorders. Collectively, our study identified differentially expressed ncRNAs at multiple levels and provided the valuable resource to explore the functions and dysfunctions of ncRNAs in cortical cells.

Unsupervised spatially embedded deep representation of spatial transcriptomics.

Optimal integration of transcriptomics data and associated spatial information is essential towards fully exploiting spatial transcriptomics to dissect tissue heterogeneity and map out inter-cellular communications. We present SEDR, which uses a deep autoencoder coupled with a masked self-supervised learning mechanism to construct a low-dimensional latent representation of gene expression, which is then simultaneously embedded with the corresponding spatial information through a variational graph autoencoder. SEDR achieved higher clustering performance on manually annotated 10 × Visium datasets and better scalability on high-resolution spatial transcriptomics datasets than existing methods. Additionally, we show SEDR's ability to impute and denoise gene expression (URL: https://github.com/JinmiaoChenLab/SEDR/ ).

A single-cell chromatin accessibility dataset of human primed and naïve pluripotent stem cell-derived teratoma.

Teratoma, due to its remarkable ability to differentiate into multiple cell lineages, is a valuable model for studying human embryonic development. The similarity of the gene expression and chromatin accessibility patterns in these cells to those observed in vivo further underscores its potential as a research tool. Notably, teratomas derived from human naïve (pre-implantation epiblast-like) pluripotent stem cells (PSCs) have larger embryonic cell diversity and contain extraembryonic lineages, making them more suitable to study developmental processes. However, the cell type-specific epigenetic profiles of naïve PSC teratomas have not been yet characterized. Using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), we analyzed 66,384 cell profiles from five teratomas derived from human naïve PSCs and their post-implantation epiblast-like (primed) counterparts. We observed 17 distinct cell types from both embryonic and extraembryonic lineages, resembling the corresponding cell types in human fetal tissues. Additionally, we identified key transcription factors specific to different cell types. Our dataset provides a resource for investigating gene regulatory programs in a relevant model of human embryonic development.

VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification.

In contrast to rodents, the mechanisms underlying human trophectoderm and early placenta specification are understudied due to ethical barriers and the scarcity of embryos. Recent reports have shown that human pluripotent stem cells (PSCs) can differentiate into trophectoderm (TE)-like cells (TELCs) and trophoblast stem cells (TSCs), offering a valuable in vitro model to study early placenta specification. Here, we demonstrate that the VGLL1 (vestigial-like family member 1), which is highly expressed during human and non-human primate TE specification in vivo but is negligibly expressed in mouse, is a critical regulator of cell fate determination and self-renewal in human TELCs and TSCs derived from naïve PSCs. Mechanistically, VGLL1 partners with the transcription factor TEAD4 (TEA domain transcription factor 4) to regulate chromatin accessibility at target gene loci through histone acetylation and acts in cooperation with GATA3 and TFAP2C. Our work is relevant to understand primate early embryogenesis and how it differs from other mammalian species.

Region-specific transcriptomic responses to obesity and diabetes in macaque hypothalamus.

The hypothalamus plays a crucial role in the progression of obesity and diabetes; however, its structural complexity and cellular heterogeneity impede targeted treatments. Here, we profiled the single-cell and spatial transcriptome of the hypothalamus in obese and sporadic type 2 diabetic macaques, revealing primate-specific distributions of clusters and genes as well as spatial region, cell-type-, and gene-feature-specific changes. The infundibular (INF) and paraventricular nuclei (PVN) are most susceptible to metabolic disruption, with the PVN being more sensitive to diabetes. In the INF, obesity results in reduced synaptic plasticity and energy sensing capability, whereas diabetes involves molecular reprogramming associated with impaired tanycytic barriers, activated microglia, and neuronal inflammatory response. In the PVN, cellular metabolism and neural activity are suppressed in diabetic macaques. Spatial transcriptomic data reveal microglia's preference for the parenchyma over the third ventricle in diabetes. Our findings provide a comprehensive view of molecular changes associated with obesity and diabetes.