Cells of the human intestinal tract mapped across space and time

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Background:Polymyalgia Rheumatica (PMR) is a common inflammatory disease in elderly persons whose pathogenesis is unclear [1-4].Objectives:We aimed to explore the pathogenetic features of PMR.Methods:We analyzed the cell subsets and their...

BackgroundAdipose-derived stem cells (ASCs) are obtained from a variety of sources in vivo where they present in large quantities. These cells are suitable for use in autologous transplantation and the construction of tissue-engineered adipose tissue. Studies have shown that ASCs differentiation is in a high degree of heterogeneity, yet the molecular basis including key regulators of differentiation remains to clarify.MethodsWe performed single-cell RNA sequencing and bioinformatics analysis on both undifferentiated (ASC-GM group) and adipogenically differentiated human ASCs (ASC-AD group, ASCs were cultured in adipogenic inducing medium for 1 week). And then, we verified the results of serum amyloid A1 (SAA1) with western blotting, immunofluorescence staining, oil red O staining. After these experiments, we down-regulated the expression of serum amyloid A1 (SAA1) gene to verify the adipogenic differentiation ability of ASCs.ResultsIn single-cell RNA sequence analyzing, we obtained 4415 cells in the ASC-GM group and 4634 cells in the ASC-AD group. The integrated sample cells could be divided into 11 subgroups (0–10 cluster). The cells in cluster 0, 2, 5 were came from ASC-GM group and the cells in cluster 1, 3, 7 came from ASC-AD group. The cells of cluster 4 and 6 came from both ASC-GM and ASC-AD groups. Fatty acid binding protein 4, fatty acid binding protein 5, complement factor D, fatty acid desaturase 1, and insulin like growth factor binding protein 5 were high expressed in category 1 and 7. Regulation of inflammatory response is the rank 1 biological processes. And cellular responses to external stimuli, negative regulation of defense response and acute inflammatory response are included in top 20 biological processes. Based on the MCODE results, we found that SAA1, C-C Motif Chemokine Ligand 5 (CCL5), and Annexin A1 (ANXA1) significantly highly expressed during adipogenic differentiation. Western blot and immunofluorescent staining results showed that SAA1 increased during adipogenesis. And the area of ORO positive staining in siSAA1 cells was significantly lower than in the siControl (negative control) cells.ConclusionsOur results also indicated that our adipogenic induction was successful, and there was great heterogeneity in the adipogenic differentiation of ASCs. SAA1 with the regulation of inflammatory response were involved in adipogenesis of ASCs based on single-cell RNA sequencing analysis. The data obtained will help to elucidate the intrinsic mechanism of heterogeneity in the differentiation process of stem cells, thus, guiding the regulation of self-renewal and differentiation of adult stem cells.

Heart and Brain Pericytes Exhibit a Pro-Fibrotic Response After Vascular Injury.

Background and aims: Nonalcoholic steatohepatitis (NASH) has become one of the major causes of cirrhosis and liver failure. However, there are currently no approved medications for managing NASH. Our study was designed to assess the effects of ginkgetin on NASH and the involved mechanisms. Methods: We constructed a mouse model of NASH by high-fat diet for 24weeks. The effects of ginkgetin on NASH were evaluated by histological study, Western blot, and biochemical analysis. RNA Sequencing (RNA-Seq) analysis was used to investigate the alteration in gene expression and signaling pathways at bulk and single-cell levels. Results: Administration of ginkgetin resulted in a marked improvement in hepatic lipid accumulation, inflammation, and fibrosis in the NASH model. And these results were supported by bulk RNA-Seq analysis, in which the related signaling pathways and gene expression were markedly downregulated. Furthermore, single-cell RNA-Seq (scRNA-Seq) analysis revealed that the effects of ginkgetin on NASH were associated with the reprogramming of macrophages, hepatic stellate cells, and endothelial cells. Especially, ginkgetin induced a marked decrease in macrophages and a shift from pro-inflammatory to anti-inflammatory phenotype in NASH mice. And the NASH-associated macrophages (NAMs), which emerge during NASH, were also significantly downregulated by ginkgetin. Conclusion: Ginkgetin exhibits beneficial effects on improving NASH, supported by bulk and single-cell RNA-Seq. Our study may promote pharmacological therapy for NASH and raise the existent understanding of NASH.

RATIONALE Pulmonaryfibrosis is a life-threatening incurable disorder associated with tissue dysfunctioncaused by the excessive deposition of extracellular matrix. Fibrosis isirreversible and there are currently no effective remedies that can reverse orcure for it. The phosphodiesterase 4B (PDE4B) inhibitor, Nerandomilast, is anovel and promising drug that has demonstrated innovative efficacy in clinicaltrials. PDE4 inhibition leads to the elevation of intracellular cAMP levels andactivation of PKA and EPAC1/2. However, the detailed cellular and molecularmechanisms by which it ameliorates fibrosis remain largely unknown.METHODS Weinvestigated the mechanism of action of Nerandomilast to inhibit theprogression of pulmonary fibrosis using both human fibrotic samples and mousefibrosis models. Bleomycin (BLM)-challenged mice were treated with Nerandomilastor placebo twice daily, starting 8 days after BLM administration. Sampling wasconducted at days 0, 8, 10, 14, and 21 for time-course analysis. We assessedthe progression of lung fibrosis and the degree of lung injury/inflammation. Additionally, we examined the mechanism of action at the cellular and molecular level incombination with time-course single cell RNA sequence analysis (scRNA-seq) and spatialtranscriptomic analysis. RESULTS Spatial transcriptomic analysis showedan accumulation of PDE4B expression in active fibrotic lesions. The expression of PDE4B increased with theprogression of fibrosis. PDE4B was preferentially expressed in non-immune cellssuch as capillary endothelial cells, fibroblasts and alveolar Type2 cells. Conversely, in immune cell population, expression levels were typically higher inmacrophages.Nerandomilastimproved body weight loss and reduced activity in BLM model. Nerandomilastsignificantly reduced fibrotic lesions and suppressed infiltration ofinflammatory cells. Epithelial cell apoptosis was reduced, and regeneratedepithelial cells were found in Nerandomilast-treated mice. In addition, migrationof a group of peripheral blood monocytes to the lungs and differentiation intofibrosis-associated macrophages was suppressed. Time-course scRNA-seq analysisrevealed distinct cellular and transcriptomic changes following Nerandomilasttreatments. CONCLUSIONS Nerandomilast reducedlung fibrosis and inflammatory response. The abundance of fibrosis specimensand detailed analysis of mouse models, coupled with scRNA-seq and spatialtranscriptomic analysis, revealed the details of Nerandomilast's mechanism ofaction on fibrosis and inflammation.

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and severe cardiopulmonary disease with a challenging prognosis, and its underlying pathogenesis remains elusive. A comprehensive understanding of IPAH is crucial to unveil potential diagnostic markers and therapeutic targets. In this study, we investigated cellular heterogeneity and molecular pathology in IPAH using single-cell RNA sequencing (scRNA-seq) analysis. Our scRNA-seq results revealed significant alterations in three crucial signaling pathways in IPAH: the hypoxia pathway, TGF β pathway, and ROS pathway, primarily attributed to changes in gene expression within arterial endothelial cells. Moreover, through bulk RNA sequencing analysis, we identified differentially expressed genes (DEGs) enriched in GO and KEGG pathways, implicated in regulating cell adhesion and oxidative phosphorylation in IPAH lungs. Similarly, DEGs-enriched pathways in IPAH arterial endothelial cells were also identified. By integrating DEGs from three IPAH datasets and applying protein-protein interaction (PPI) analysis, we identified 12 candidate biomarkers. Subsequent validation in two additional PAH datasets led us to highlight five potential biomarkers (CTNNB1, MAPK3, ITGB1, HSP90AA1, and DDX5) with promising diagnostic significance for IPAH. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) confirmed significant differences in the expression of these five genes in pulmonary arterial endothelial cells from PAH mice. In conclusion, our findings shed light on the pivotal role of arterial endothelial cells in the development of IPAH. Furthermore, the integration of single-cell and bulk RNA sequencing datasets allowed us to pinpoint novel candidate biomarkers for the diagnosis of IPAH. This work opens up new avenues for research and potential therapeutic interventions in IPAH management.

Rationale: Recurrent injury to, and dysregulated repair of the alveolar epithelium is believed to play a central role in the development of interstitial lung diseases. Our recent work identified dynamic Yap/Taz activity in AT2 cells following alveolar injury, in which, Yap was by activated 7-days, Taz activated by 14-days, and both were down-regulated by 21-days after bleomycin induced injury and subsequent functional repair. Our previous work identified aberrant sustained Yap activation in AT2 cells of patients with idiopathic pulmonary fibrosis. Herein, we sought to determine whether sustained AT2 cell Yap/Taz activity prevents functional alveolar repair and promotes the pathogenesis of pulmonary fibrosis. Methods: Lineage labeled transgenic mice with AT2-cell specific conditional activation of Yap/Taz (SftpcCreert2 Stk3f/f Stk4f/f) (referred to as YTAT2 hereafter) were given single dose (0.08iU) bleomycin injury 3-weeks post tamoxifen induced activation. Two weeks post injury, the Yap/Taz inhibitor verteporfin (or vehicle control) was administered via single dose (60mg/kg) intraperitoneal injection. Lung fibrosis was quantified by Sircol collagen assays and Ashcroft scoring, and immunofluorescence analysis of epithelial cell type markers was used to assess alveolar epithelial cell differentiation. Single-Cell RNA and ATAC sequencing was used to define the transcriptional networks regulated by Yap/Taz activity. Isolated human epithelial cells were treated with Yap/Taz activator XMU-MP1 (3μM), plated as organoids in expansion or differentiation media, and collected at 14 days for single cell RNA sequencing. Results: Single cell RNA sequencing analysis of human epithelial cell organoids showed the addition of XMU-MP1 (Yap/Taz activation) led to the increased presence of aberrant epithelial cell populations. YTAT2 mice had increased fibrotic remodeling at 28 days compared to wild-type mice. The addition of verteporfin partially rescued these phenotypes. Immunofluorescence analysis of lineage labeled Yap/Taz active AT2 cells showed a subset of AT2 cells expressing both AT2 and AT1 cell markers suggesting persistence of transitional cells that normally resolve after regeneration. Analysis of single-cell ATAC sequencing showed bleomycin injured Yap/Taz active AT2 cells had open chromatin associated with promoters of genes normally restricted to AT1 (Ager) and proximal airways (Scgb1a1, Muc5b) consistent with abnormal alveolar epithelial cell differentiation. Conclusion: These studies find that sustained Yap/Taz activity in AT2 cells results in the abnormal differentiation of AT2 cells in human and mouse leading to failed alveolar repair. This failure of normal adaptive repair leads to increased fibrotic remodeling at 28-days post injury.

BackgroundTumor is a complex and dynamic ecosystem formed by the interaction of numerous diverse cells types and the microenvironments they inhabit. Determining how cellular states change and develop distinct cellular communities in response to the tumor microenvironment is critical to understanding cancer progression. Tumour-associated macrophages (TAMs) are an important component of the tumour microenvironment and play a crucial role in cancer progression. This study was designed to identify cell-state-specific M2 macrophage markers associated with gastric cancer (GC) prognosis through integrative analysis of single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data using a machine learning framework named EcoTyper.ResultsThe results showed that TAMs were classified into M1 macrophages, M2 macrophages, monocytes, undefined macrophages and dendritic cells, with M2 macrophages predominating. EcoTyper assigned macrophages to different cell states and ecotypes. A total of 168 cell-state-specific M2 macrophage markers were obtained by integrative analysis of scRNA-seq and bulk RNA-seq data. These markers could categorize GC patients into two clusters (clusters A and B) with different survival and M2 macrophages infiltration abundance. Cell adhesion molecules, cytokine-cytokine receptor interaction, JAK/STAT pathway, MAPK pathway were significantly enriched in cluster A, which had worse survival and higher M2 macrophages infiltration.ConclusionIn conclusion, this study profiles a single-cell atlas of intratumor heterogeneity and defines the cell states and ecotypes of TAMs in GC. Furthermore, we have identified prognostically relevant cell-state-specific M2 macrophage markers. These findings provide novel insights into the tumor ecosystem and cancer progression.

Ischemic stroke (IS) is a fatal neurological disease that occurs when the blood flow to the brain is disrupted, leading to brain tissue damage and functional impairment. Cellular senescence, a vital characteristic of aging, is associated with a poor prognosis for IS. This study explores the potential role of cellular senescence in the pathological process following IS by analyzing transcriptome data from multiple datasets (GSE163654, GSE16561, GSE119121, and GSE174574). By using bioinformatics methods, we identified hub-senescence-related genes such as ANGPTL4, CCL3, CCL7, CXCL16, and TNF and verified them using quantitative reverse transcription polymerase chain reaction. Further analysis of single-cell RNA sequencing data suggests that MG4 microglial is highly correlated with cellular senescence in MCAO, and might play a crucial role in the pathological process after IS. Additionally, we identified retinoic acid as a potential drug for improving the prognosis of IS. This comprehensive investigation of cellular senescence in various brain tissues and peripheral blood cell types provides valuable insights into the underlying mechanisms of the pathology of IS and identifies potential therapeutic targets for improving patient outcomes.

Cancer immunotherapy is an increasingly successful strategy for treating patients with advanced or conventionally drug-resistant cancers. T cells have been proved to play important roles in anti-tumor and tumor microenvironment shaping, while these roles have not been explained in lung squamous cell carcinoma (LUSC). In this study, we first performed a comprehensive analysis of single-cell RNA sequencing (scRNA-seq) data from the gene expression omnibus (GEO) database to identify 72 T-cell marker genes. Subsequently, we constructed a 5-gene prognostic signature in the training cohort based on the T-cell marker genes from the cancer genome atlas (TCGA) database, which was further validated in the testing cohort and GEO cohort. The areas under the receiver operating characteristic curve at 1-, 3-, and 5-years were 0.614, 0.713 and 0.702 in the training cohort, 0.669, 0.603 and 0.645 in the testing cohort, 0.661, 0.628 and 0.590 in the GEO cohort, respectively. Furthermore, we created a highly reliable nomogram to facilitate clinical application. Gene set enrichment analysis showed that immune-related pathways were mainly enriched in the high-risk group. Tumor immune microenvironment indicated that high-risk group exhibited higher immune score, stromal score, and immune cell infiltration levels. Moreover, genes of the immune checkpoints and human leukocyte antigen family were all overexpressed in high-risk group. Drug sensitivity revealed that low-risk group was sensitive to 8 chemotherapeutic drugs and high-risk group to 4 chemotherapeutic drugs. In short, our study reveals a novel prognostic signature based on T-cell marker genes, which provides a new target and theoretical support for LUSC patients.

Recapitulating early human development with 8C-like cells.

Tumor microenvironment interacts with cancer cells, causing tumor progression. Tumor microenvironment consists of cellular and acelluar components. We investigated cellular components of tumor microenvironment and heterogeneity of ovarian cancer. We performed single cell RNA sequencing analysis of cellular components of malignant ascites from a patient with advanced ovarian cancer. Among the various cell types including cancer cells, B cells, macrophages and fibroblasts, macrophage was the most dominant in malignant ascites. To determine the heterogeneity of ovarian cancer, we obtained primary ovarian cancer tissue from ovarian cancer patient. We performed single cell RNA sequencing analysis of cellular components derived from ovarian cancer tissue. We determined intra-heterogeneity of ovarian cancer and cellular components of ovarian cancer tissue. Taken together, we determined cellular components of ovarian cancer tissue and ascites and intra-heterogeneity of ovarian cancer. This study can provide insight of tumor microvironment and help understanding of ovarian cancer heterogeneity. Citation Format: Untack Cho, Yong Sang Song, Woong-Yang Park, Hae-Ock Lee, Youngjin Han. Evaluating heterogeneity of tumor microenvironment using single cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 124.

Myeloid cells are physiologically related to innate immunity and inflammation. Tumor-associated myeloid cells gained increasing interest because of their critical roles in tumor progression and anticancer immune responses in human malignancies. However, the associations between tumor-associated myeloid cell-related genes and hepatocellular carcinoma have yet to be revealed. Here, through the integrating analysis of bulk and single-cell RNA (scRNA) sequencing of public HCC samples, we developed a gene signature to investigate the role of HCC-specific myeloid signature genes in HCC patients. We firstly defined 317 myeloid cell marker genes through analyzing scRNA data of HCC from the GEO dataset. After selecting the differentially expressed genes, eleven genes were also proved prognostic. Then we built a gene signature from the TCGA cohort and verified further with the ICGC dataset by applying the LASSO Cox method. An eight genes signature (FABP5, C15orf48, PABPC1, TUBA1B, AKR1C3, NQO1, AKR1B10, SPP1) was achieved finally. Patients in the high risk group correlated with higher tumor stages and poor survival than those in the low-risk group. The risk score was proved to be an independent risk factor for prognosis. The high risk group had higher infiltrations of dendritic cells, macrophages and Tregs. And the APC co-inhibition, T cell co-inhibition pathways were also activated. Besides, the risk score positively correlated with multidrug resistance proteins. In conclusion, our myeloid cell marker genes related signature can predict patients’ survival and may also indicate the levels of immune infiltration and drug resistance.

BackgroundOxidative stress plays a crucial role in the development of diabetic foot ulcers (DFU). However, its underlying mechanisms are not fully understood. The purpose of this study was to use bioinformatics and preliminary validation methods to preliminarily reveal the oxidative stress landscape in DFU.MethodsBased on the single-cell and bulk RNA sequencing data of DFU, we conducted differential genes screening, machine learning, PPI network construction, immune infiltration analysis, drug prediction, TF-mRNA-miRNA network, cell-cell interaction, pseudotime trajectory analysis, external cohort validation, and in vitro experiments to develop the oxidative stress landscape in DFU.ResultsBulk RNA-seq analysis identified 63 oxidative stress-related genes of DFU (DORGs), and the top 59 genes were screened out for key nodes with close functional associations. Functional enrichment analysis showed significant involvement in oxidative stress response. Drug prediction highlighted Thymoquinone and Erlotinib as potential therapeutic candidates. Machine learning algorithms (SVM-RFE, LASSO and RF) identified BCL2 and 和FOXP2 as candidate hub DORGs for DFU diagnosis. Immune cell infiltration analysis indicated a significant presence of naive B cells and CD8 T cells in DFU. The analysis of single-cell RNA sequencing identified a total of 31,787 cells across 10 distinct clusters, with a notably lower proportion of fibroblasts in DFU group than that in the control group. The expression patterns of BCL2 and 和FOXP2 across the different groups were consistent with findings from bulk RNA sequencing analysis. Notably, fibroblasts derived from DFU patients exhibited the highest oxidative stress scores. Intercellular signaling analysis indicated that fibroblasts serve as crucial communication cells, primarily engaged in COLLAGEN signaling network. Additionally, fibroblasts are categorized into five distinct clusters. Among these, COL6A5+ fibroblasts constitute the predominant cluster in DFU and exhibit low differentiation potential. Furthermore, in vitro experiments successfully established a DFU oxidative stress model of fibroblasts, revealing reduced migration ability in the absence of cell death. Both in vitro findings and external data corroborated the decreased expression levels of BCL2and和FOXP2in DFU.ConclusionThe oxidative stress-related genes BCL2 and FOXP2 could serve as diagnostic markers for DFU. Furthermore, we identified the novel pathogenic mechanism associated with oxidative stress in DFU fibroblasts. This study may offer new insights for the diagnosis and treatment of DFU.