Single-Cell RNA Sequencing Resolves Spatiotemporal Development of Pre-thymic Lymphoid Progenitors and Thymus Organogenesis in Human Embryos.

Generation of the first T lymphocytes in the human embryo involves the emergence, migration, and thymus seeding of lymphoid progenitors together with concomitant thymus organogenesis, which is the initial step to establish the entire adaptive immune system. However, the cellular and molecular programs regulating this process remain unclear. We constructed a single-cell transcriptional landscape of human early T lymphopoiesis by using cells from multiple hemogenic and hematopoietic sites spanning embryonic and fetal stages. Among heterogenous early thymic progenitors, one subtype shared common features with a subset of lymphoid progenitors in fetal liver that are known as thymus-seeding progenitors. Unbiased bioinformatics analysis identified a distinct type of pre-thymic lymphoid progenitors in the aorta-gonad-mesonephros (AGM) region. In parallel, we investigated thymic epithelial cell development and potential cell-cell interactions during thymus organogenesis. Together, our data provide insights into human early T lymphopoiesis that prospectively direct T lymphocyte regeneration, which might lead to development of clinical applications.

Deciphering human macrophage development at single-cell resolution.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)1. However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45+ haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45+CD34+CD44+ yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs.

Advanced Composite Solid Electrolytes for Lithium Batteries: Filler Dimensional Design and Ion Path Optimization.

Composite solid electrolytes are considered to be the crucial components of all-solid-state lithium batteries, which are viewed as the next-generation energy storage devices for high energy density and long working life. Numerous studies have shown that fillers in composite solid electrolytes can effectively improve the ion-transport behavior, the essence of which lies in the optimization of the ion-transport path in the electrolyte. The performance is closely related to the structure of the fillers and the interaction between fillers and other electrolyte components including polymer matrices and lithium salts. In this review, the dimensional design of fillers in advanced composite solid electrolytes involving 0D-2D nanofillers, and 3D continuous frameworks are focused on. The ion-transport mechanism and the interaction between fillers and other electrolyte components are highlighted. In addition, sandwich-structured composite solid electrolytes with fillers are also discussed. Strategies for the design of composite solid electrolytes with high room temperature ionic conductivity are summarized, aiming to assist target-oriented research for high-performance composite solid electrolytes.

Transcriptomic landscape of circulating mononuclear phagocytes in Langerhans cell histiocytosis at the single-cell level.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm caused by aberrant activation of the mitogen-activated protein kinase (MAPK) pathway. Circulating myeloid cells from patients often carry disease-associated mutations and can be differentiated into langerinhigh LCH-like cells in vitro, but their detailed immune-phenotypic and molecular profiles are lacking and could shed key insights into disease biology. Here we recruited 217 pediatric LCH patients and took blood and tissue samples for BRAFV600E analysis. Immune-phenotyping of the circulating Lin-HLA-DR+ immune population in 49 of these patients revealed that decreased frequency of plasmacytoid dendritic cells was significantly linked to disease severity. By single-cell RNA sequencing of samples from 14 patients, we identified key changes in expression of RAS-MAPK-extracellular signal-regulated kinase (ERK) signaling-related genes and transcription factors in distinct members of the mononuclear phagocyte system in the presence of BRAFV600E. Moreover, treatment of patients with the BRAF inhibitor dabrafenib resulted in MAPK cascade inhibition, inflammation prevention, and regulation of cellular metabolism within mononuclear phagocytes. Finally, we also observed elevated expression of RAS-MAPK-ERK signaling-related genes in a CD207+CD1a+ cell subcluster in skin. Taken together, our data extend the molecular understanding of LCH biology at single-cell resolution, which might contribute to improvement of clinical diagnostics and therapeutics, and aid in the development of personalized medicine approaches.

Integrative transcriptomic analysis of developing hematopoietic stem cells in human and mouse at single-cell resolution.

Current understanding of hematopoietic stem cell (HSC) development comes from mouse models is considered to be evolutionarily conserved in human. However, the cross-species comparison of the transcriptomic profiles of developmental HSCs at single-cell level is still lacking. Here, we performed integrative transcriptomic analysis of a series of key cell populations during HSC development in human and mouse, including HSC-primed hemogenic endothelial cells and pre-HSCs in mid-gestational aorta-gonad-mesonephros (AGM) region, and mature HSCs in fetal liver and adult bone marrow. We demonstrated the general similarity of transcriptomic characteristics between corresponding cell populations of the two species. Of note, one of the previously transcriptomically defined hematopoietic stem progenitor cell (HSPC) populations with certain arterial characteristics in AGM region of human embryos showed close transcriptomic similarity to pre-HSCs in mouse embryos. On the other hand, the other two HSPC populations in human AGM region displayed molecular similarity with fetal liver HSPCs, suggesting the maturation in AGM before HSCs colonizing the fetal liver in human, which was different to that in mouse. Finally, we re-clustered cells based on the integrated dataset and illustrated the evolutionarily conserved molecular signatures of major cell populations. Our results revealed transcriptomic conservation of critical cell populations and molecular characteristics during HSC development between human and mouse, providing a resource and theoretic basis for future studies on mammalian HSC development and regeneration by using mouse models.

CO2-Triggered ON/OFF Wettability Switching on Bioinspired Polylactic Acid Porous Films for Controllable Bioadhesion.

Bioinspired honeycomb-like porous films with switchable properties have drawn much attention recently owing to their potential application in scenarios in which the conversion between two opposite properties is required. Herein, the CO2-gas-triggered ON/OFF switching wettability of biocompatible polylactic acid (PLA) honeycomb porous films is fabricated. Highly ordered porous films with diameters between 2.0 and 2.8 µm are separately prepared from complexes of nonresponsive PLA and a CO2-sensitive melamine derivative [N2,N4,N6-tris(3-(dimethylamino)propyl)-1,3,5-triazine-2,4,6-triamine, MET] via the breath figure method. The hydrophilic CO2-sensitive groups can be precisely arranged in the pore's inner surface and/or top surface of the films by simply changing the PLA/MET ratio. The sensitive groups in the pore's inner surface act as a switch triggered by CO2 gas controlling water to enter the pores or not, thus resulting in ON/OFF switching wettability. The largest response of the water contact angle of honeycomb films reaches 35°, from 100 to 65°, leading to an obvious hydrophobic-hydrophilic conversion. The improved surface wettability enhances the interaction between the cell and honeycomb film surface, thus resulting in a better cell attachment. Such smart properties accompanying the biocompatible polymer and biological gas trigger facilitate possible biomedical and bioengineering applications in the future for these films.

Direct formation of hydrophilic honeycomb film by self-assembly in breath figure templating of hydrophobic polylacticacid/ionic surfactant complexes.

Honeycomb-patterned porous films with good surface wettability have great potential applications in various areas. However, hydrophilic honeycomb films are difficult to obtain using the direct self-assembly of pure (co)polymers. Thus, additional and special treatments are required to improve film wettability, which makes the procedure complicated and difficult to access. In this study, a facile way to prepare hydrophilic honeycomb-structured porous films is proposed that uses the direct self-assembly of complexes of biocompatible hydrophobic poly(l-lactic acid) and dodecyltrimethylammonium chloride by breath figure templating. The addition of ionic surfactant not only improves film quality but also confers good wettability. The obtained hydrophilic pore arrays were found to effectively promote cell attachment. Such a hydrophilic honeycomb-patterned porous film could find potential applications where pore wetting is required, including tissue engineering, lithography, and nanoparticle embedding.

Systematic study of cis-antisense miRNAs in animal species reveals miR-3661 to target PPP2CA in human cells.

MicroRNAs (miRNAs) suppress targeting gene expression through blocking translation or triggering mRNA degradation and, in general, act in trans, through a partially complementary interaction with the 3' untranslated region (3' UTR) or coding regions of a target gene. Although it has been reported previously that some miRNAs suppress their target genes on the opposite strand with a fully complementary sequence (i.e., natural antisense miRNAs that act in cis), there is no report to systematically study such cis-antisense miRNAs in different animal species. Here we report that cis-antisense miRNAs do exist in different animal species: 48 in Caenorhabditis elegans, 17 in Drosophila, 36 in Mus musculus, and 52 in Homo sapiens using a systematical bioinformatics approach. We show that most of these cis-antisense miRNAs can efficiently reduce the expression levels of their target genes in human cells. We further investigate hsa-miR-3661, one of the predicted cis-antisense miRNAs, in detail and demonstrate that this miRNA directly targets the coding sequence of PPP2CA located on the opposite DNA strand and inhibits the PPP2CA expression. Taken together, these results indicate that cis-antisense miRNAs are conservative and functional in animal species including humans.

A Tumor-Specific Prognostic Long Non-Coding RNA Signature in Gastric Cancer.

BACKGROUND Aberrant expression of long non-coding RNAs (lncRNAs) is associated with prognosis of gastric cancer, some of which could be further evaluated as potential biomarkers. In this study, we attempted to identify a specific lncRNA signature to predict the prognosis of gastric cancer. MATERIAL AND METHODS The genome-wide lncRNA expression in the high-throughput RNA-sequencing data was retrieved from the Cancer Genome Atlas (TCGA). Differential expression of lncRNAs was identified using the Limma package. Survival analysis was conducted by use of univariate and multivariate Cox regression models. Functional enrichment analysis of lncRNAs was based on co-expressed mRNAs. DAVID was used to perform gene ontology and KEGG pathway analysis. RESULTS A total of 452 differentially expressed lncRNAs between gastric cancer and matched normal tissues were screened, of which 76 lncRNAs were identified to be gastric cancer-specific from a pan-cancer analysis of 12 types of human cancer. Among these 76 gastric cancer-specific lncRNAs, 5 lncRNAs (CTD-2616J11.14, RP1-90G24.10, RP11-150O12.3, RP11-1149O23.2, and MLK7-AS1) were significantly associated with the overall survival of patients with gastric cancer. A gastric cancer-specific 5-lncRNA signature was deduced to divide the patients into high- and low-risk groups with significantly different survival times (P<0.0001). Multivariate Cox regression analysis showed that this 5-lncRNA signature was an independent predictor of prognosis. Functional enrichment analysis of the 5 lncRNAs showed that they were mainly involved in DNA replication, mitotic cell cycle, programmed cell death, and RNA splicing. CONCLUSIONS Our results suggest that this tumor-specific lncRNA signature may be clinically useful in the prediction of gastric cancer prognosis.

Hematopoietic stem cell heterogeneity in non-human primates revealed by five-lineage output bias analysis.

Understanding hematopoietic stem cell (HSC) heterogeneity is crucial for treating malignant blood disorders. Compared with mice, we have limited knowledge of the heterogeneity of human HSCs. Fortunately, non-human primates (NHPs) have become the best animal models for studying human HSCs. Here, we employed a public dataset derived from NHP autologous bone marrow transplantation, and focused on a total of 820 HSC clones with reconstitution capacity of all available five lineages (granulocyte, monocyte, B cell, T cell, and natural killer cell) at two time points (11/12 and/or 42/43 months). Intriguingly, unsupervised clustering on these clones revealed six HSC subtypes, including a lymphoid/myeloid balanced (LM-balanced) subtype and five single-lineage-biased subtypes. We also observed that the subtypes of these HSC clones might change over time, and a given subtype could transition into any one of the other five subtypes, albeit with a certain degree of selectivity. Particularly, each of the six subtypes was more likely to turn into lymphoid-biased rather than myeloid-biased ones. Additionally, our five-lineage classification method exhibited strong correlation with traditional lymphoid/myeloid bias classification method. Specifically, our granulocyte- and monocyte-biased subtypes were predominantly attributed to α-HSCs, while LM-balanced, B cell-biased, and T cell-biased subtypes were primarily associated with ß-HSCs. The γ-HSCs were composed of a small subset of B cell-biased and T cell-biased subtypes. In summary, our five-lineage classification identifies more finely tuned HSC subtypes based on lineage output bias. These findings enrich our understanding of HSC heterogeneity in NHPs and provide important insights for human research.

Develop prediction model to help forecast advanced prostate cancer patients' prognosis after surgery using neural network.

Background: The effect of surgery on advanced prostate cancer (PC) is unclear and predictive model for postoperative survival is lacking yet. Methods: We investigate the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database, to collect clinical features of advanced PC patients. According to clinical experience, age, race, grade, pathology, T, N, M, stage, size, regional nodes positive, regional nodes examined, surgery, radiotherapy, chemotherapy, history of malignancy, clinical Gleason score (composed of needle core biopsy or transurethral resection of the prostate specimens), pathological Gleason score (composed of prostatectomy specimens) and prostate-specific antigen (PSA) are the potential predictive variables. All samples are divided into train cohort (70% of total, for model training) and test cohort (30% of total, for model validation) by random sampling. We then develop neural network to predict advanced PC patients' overall. Area under receiver operating characteristic curve (AUC) is used to evaluate model's performance. Results: 6380 patients, diagnosed with advanced (stage III-IV) prostate cancer and receiving surgery, have been included. The model using all collected clinical features as predictors and based on neural network algorithm performs best, which scores 0.7058 AUC (95% CIs, 0.7021-0.7068) in train cohort and 0.6925 AUC (95% CIs, 0.6906-0.6956) in test cohort. We then package it into a Windows 64-bit software. Conclusion: Patients with advanced prostate cancer may benefit from surgery. In order to forecast their overall survival, we first build a clinical features-based prognostic model. This model is accuracy and may offer some reference on clinical decision making.

Chaos criteria and chaotification schemes on a class of first-order partial difference equations.

This article is involved in chaos criteria and chaotification schemes on one kind of first-order partial difference equations having non-periodic boundary conditions. Firstly, four chaos criteria are achieved by constructing heteroclinic cycles connecting repellers or snap-back repellers. Secondly, three chaotification schemes are obtained by using these two kinds of repellers. For illustrating the usefulness of these theoretical results, four simulation examples are presented.

Linear barycentric rational collocation method for solving generalized Poisson equations.

We consider the Poisson equation by collocation method with linear barycentric rational function. The discrete form of the Poisson equation was changed to matrix form. For the basis of barycentric rational function, we present the convergence rate of the linear barycentric rational collocation method for the Poisson equation. Domain decomposition method of the barycentric rational collocation method (BRCM) is also presented. Several numerical examples are provided to validate the algorithm.

Selenium-decorated biocompatible honeycomb films with redox-switchable surface for controlling cell adhesion/detachment.

HYPOTHESIS: Selenium (Se)-containing compound is sensitive to redox stimulation, showing hydrophobic-hydrophilic reversible transition. Introduction of such compound into honeycomb film could confer on it redox-switchable surface wettability, which is expected to control cell adhesion/detachment behavior. EXPERIMENTS: Didodecyl selenide was designed and mixed with polystyrene to prepare honeycomb films using "breath figure" method. The film microstructures were characterized by scanning electron microscope and atomic force microscopy, and the arrangement of Se atoms in honeycomb film was determined by X-ray photoelectron spectroscopy and energy dispersive spectrometry. The variation of film wettability upon the alternating stimulation of H2O2 and Vc was examined. Then the cell adhesion, proliferation, and controlled detachment on honeycomb films were conducted. FINDINGS: The introduction of didodecyl selenide helps to form ordered honeycomb film, and Se atoms were found to located on the bottom, pore walls, and top surface of the film. The presence of didodecyl selenide not only greatly improves film biocompatibility by enhancing cell thioredoxin reductase activity, but also imparts the film with H2O2-/vitamin C-regulated tunable wettability that controls cell adhesion and detachment. H2O2 treatment produces a hydrophilic surface for cell adhesion and proliferation, whereas the addition of vitamin C generates hydrophobic surfaces and allows cells to detach while remaining alive with high activity.

Divergent molecular events underlying initial T-cell commitment in human prenatal and postnatal thymus.

Introduction: Intrathymic T-cell development is a coordinated process accompanied by dynamic changes in gene expression. Although the transcriptome characteristics of developing T cells in both human fetal and postnatal thymus at single-cell resolution have been revealed recently, the differences between human prenatal and postnatal thymocytes regarding the ontogeny and early events of T-cell development still remain obscure. Moreover, the transcriptional heterogeneity and posttranscriptional gene expression regulation such as alternative polyadenylation at different stages are also unknown. Method: In this study, we performed integrative single-cell analyses of thymocytes at distinct developmental stages. Results: The subsets of prenatal CD4-CD8- double-negative (DN) cells, the most immature thymocytes responsible for T-cell lineage commitment, were characterized. By comprehensively comparing prenatal and postnatal DN cells, we revealed significant differences in some key gene expressions. Specifically, prenatal DN subpopulations exhibited distinct biological processes and markedly activated several metabolic programs that may be coordinated to meet the required bioenergetic demands. Although showing similar gene expression patterns along the developmental path, prenatal and postnatal thymocytes were remarkably varied regarding the expression dynamics of some pivotal genes for cell cycle, metabolism, signaling pathway, thymus homing, and T-cell commitment. Finally, we quantified the transcriptome-wide changes in alternative polyadenylation across T-cell development and found diverse preferences of polyadenylation site usage in divergent populations along the T-cell commitment trajectory. Discussion: In summary, our results revealed transcriptional heterogeneity and a dynamic landscape of alternative polyadenylation during T-cell development in both human prenatal and postnatal thymus, providing a comprehensive resource for understanding T lymphopoiesis in human thymus.

Nupr1 Negatively Regulates Endothelial to Hematopoietic Transition in the Aorta-Gonad-Mesonephros Region.

In the aorta of mid-gestational mouse embryos, a specialized endothelial subpopulation termed hemogenic endothelial cells (HECs) develops into hematopoietic stem and progenitor cells (HSPCs), through a conserved process of endothelial-to-hematopoietic transition (EHT). EHT is tightly controlled by multiple intrinsic and extrinsic mechanisms. Nevertheless, the molecular regulators restraining this process remain poorly understood. Here, it is uncovered that, one of the previously identified HEC signature genes, Nupr1, negatively regulates the EHT process. Nupr1 deletion in endothelial cells results in increased HSPC generation in the aorta-gonad-mesonephros region. Furthermore, single-cell transcriptomics combined with serial functional assays reveals that loss of Nupr1 promotes the EHT process by promoting the specification of hematopoiesis-primed functional HECs and strengthening their subsequent hematopoietic differentiation potential toward HSPCs. This study further finds that the proinflammatory cytokine, tumor necrosis factor α (TNF-α), is significantly upregulated in Nupr1-deficient HECs, and the use of a specific TNF-α neutralizing antibody partially reduces excessive HSPC generation in the explant cultures from Nupr1-deficient embryos. This study identifies a novel negative regulator of EHT and the findings indicate that Nupr1 is a new potential target for future hematopoietic stem cell regeneration research.

Divergent expression of Neurl3 from hemogenic endothelial cells to hematopoietic stem progenitor cells during development.

Prior to the generation of hematopoietic stem cells (HSCs) from the hemogenic endothelial cells (HECs) mainly in the dorsal aorta in midgestational mouse embryos, multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs. These HSC-independent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth. However, little is known about yolk sac HECs. Here, combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays, we reveal that Neurl3-EGFP, in addition to marking the continuum throughout the ontogeny of HSCs from HECs, can also serve as a single enrichment marker for yolk sac HECs. Moreover, while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper, the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression. Interestingly, the B lymphoid potential of hematopoietic progenitors, but not for myeloid potentials, is exclusively detected in Neurl3-negative subpopulations in midgestational embryos. Taken together, these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.

Functional diversification and dynamics of CAR-T cells in patients with B-ALL.

Understanding of cellular evolution and molecular programs of chimeric antigen receptor-engineered (CAR)-T cells post-infusion is pivotal for developing better treatment strategies. Here, we construct a longitudinal high-precision single-cell transcriptomic landscape of 7,578 CAR-T cells from 26 patients with B cell acute lymphoblastic leukemia (B-ALL) post-infusion. We molecularly identify eight CAR-T cell subtypes, including three cytotoxic subtypes with distinct kinetics and three dual-identity subtypes with non-T cell characteristics. Remarkably, long-term remission is coincident with the dominance of cytotoxic subtypes, while leukemia progression is correlated with the emergence of subtypes with B cell transcriptional profiles, which have dysfunctional features and might predict relapse. We further validate in vitro that the generation of B-featured CAR-T cells is induced by excessive tumor antigen stimulation or suppressed TCR signaling, while it is relieved by exogenous IL-12. Moreover, we define transcriptional hallmarks of CAR-T cell subtypes and reveal their molecular changes along computationally inferred cellular evolution in vivo. Collectively, these results decipher functional diversification and dynamics of peripheral CAR-T cells post-infusion.

Pre-configuring chromatin architecture with histone modifications guides hematopoietic stem cell formation in mouse embryos.

The gene activity underlying cell differentiation is regulated by a diverse set of transcription factors (TFs), histone modifications, chromatin structures and more. Although definitive hematopoietic stem cells (HSCs) are known to emerge via endothelial-to-hematopoietic transition (EHT), how the multi-layered epigenome is sequentially unfolded in a small portion of endothelial cells (ECs) transitioning into the hematopoietic fate remains elusive. With optimized low-input itChIP-seq and Hi-C assays, we performed multi-omics dissection of the HSC ontogeny trajectory across early arterial ECs (eAECs), hemogenic endothelial cells (HECs), pre-HSCs and long-term HSCs (LT-HSCs) in mouse embryos. Interestingly, HSC regulatory regions are already pre-configurated with active histone modifications as early as eAECs, preceding chromatin looping dynamics within topologically associating domains. Chromatin looping structures between enhancers and promoters only become gradually strengthened over time. Notably, RUNX1, a master TF for hematopoiesis, enriched at half of these loops is observed early from eAECs through pre-HSCs but its enrichment further increases in HSCs. RUNX1 and co-TFs together constitute a central, progressively intensified enhancer-promoter interactions. Thus, our study provides a framework to decipher how temporal epigenomic configurations fulfill cell lineage specification during development.