January 2023 Top 10 Publications - Advances in Single Cell Sequencing
Our monthly overview of single cell literature is back again. Past couple of months have brought us a diverse collection of exciting new studies that advanced our understanding of single cell methods and disease treatments. Here is our selection of top publications featuring new advances and applications of single cell sequencing in rheumatoid arthritis, SARS-CoV-2 infection, tuberculosis, hepatitis B liver failure and colorectal cancer.
Live slow-frozen human tumor tissues for scRNAseq
Restivo, G., Tastanova, A., Balázs, Z. et al. Live slow-frozen human tumor tissues viable for 2D, 3D, ex vivo cultures and single-cell RNAseq. Commun Biol 5, 1144 (2022). [PubMed]
Formalin-fixed and paraffin-embedded (FFPE), Tissue-Tek OCT embedded or snap-frozen tissues are staples in many biobanks worldwide and have been the basis of translational studies. Their usage is limited to assays that do not require viable cells and access to intact and viable human material is a prerequisite for translational validation of basic research, for novel therapeutic target discovery, and functional testing.
Restivo et al. showed that surplus tissues from multiple solid human cancers directly slow-frozen after resection can subsequently be used for different types of methods including the establishment of 2D, 3D, and ex vivo cultures as well as single-cell RNA sequencing with similar results when compared to freshly analyzed material.
Clonally expanded CD4+ T-cells identified in rheumatoid arthritis
Argyriou, A., Wadsworth, M.H., Lendvai, A. et al. Single cell sequencing identifies clonally expanded synovial CD4+ TPH cells expressing GPR56 in rheumatoid arthritis. Nat Commun 13, 4046 (2022). [PubMed]
Rheumatoid arthritis (RA) is an autoimmune disease affecting synovial joints where different CD4+ T cell subsets may contribute to pathology. Single-cell sequencing was performed in combination with TCRab sequencing on peripheral blood (PB) and synovial fluid (SF) from ACPA+ and ACPA- RA patients to investigate CD4 T-cell subsets.
Argyriou et al. showed that the adhesion G-protein coupled receptor 56 (GPR56) delineates synovial CXCL13high peripheral helper T cell (TPH) CD4+ T cells expressing LAG-3 and the tissue-resident memory receptors CXCR6 and CD69. In ACPA- SF, TPH cells display lower levels of GPR56 and LAG-3. Further, most expanded T cell clones in the joint are within CXCL13high TPH CD4+ T cells. RNA-velocity analysis suggests a common differentiation pathway between the two TPH clusters and effector CD4+ T cells. This study provides comprehensive immunoprofiling of the synovial CD4+ T cell subsets in ACPA+ and ACPA- RA.
Single cell roadmap of human gonadal development
Garcia-Alonso, L., Lorenzi, V., Mazzeo, C.I. et al. Single-cell roadmap of human gonadal development. Nature 607, 540–547 (2022). [PubMed]
Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries. The understanding of this process is hampered by the lack of good in vitro models and differences between animal models (mice) and humans.
Garcia-Alonso et al. generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.
Bronchoalveolar epithelial cells characterization in SARS-CoV-2 patients
Deng, Z., Li, Q., Li, Y. et al. Single-cell RNA-seq data analysis characterizing bronchoalveolar epithelial cells in patients with SARS-CoV-2 infection. J Inflamm 19, 13 (2022). [PubMed]
This study uses a NCBI GEO expression profile (GSE145926) to compare scRNA-seq data generated with BaLF from bronchoalveolar epithelial cells of SARS-CoV-2 patients with different disease severities and healthy individuals. Patients with a severe phenotype showed increased ACE2 expression levels. 11 different epithelial cells clusters were identified in SARS-CoV-2 patients, which had two major differentiation directions in upregulating pathways either related to inflammation or apoptosis.
Hahaut, V., Pavlinic, D., Carbone, W. et al. Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq. Nat Biotechnol 40, 1447–1451 (2022). [PubMed]
FLASH-seq (FS), a full-length single-cell RNA sequencing (scRNA-seq) method with increased sensitivity and reduced hands-on time compared to Smart-seq3. The entire FS protocol can be performed in ~4.5 hours, is simple to automate and can be easily miniaturized to decrease resource consumption.
Two epithelial tumor cell states are identified and refined the consensus molecular classification of colorectal cancer
Joanito, I., Wirapati, P., Zhao, N. et al. Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer. Nat Genet 54, 963–975 (2022). [PubMed]
The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear.
Joanito et al. identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. It is defined in this study that the intrinsic epithelial axis of colorectal cancer and propose a refined ‘IMF’ classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F).
Lineage-specific dynamic genetic regulation of gene expression during cell differentiation
Elorbany R, Popp JM, Rhodes K, Strober BJ, Barr K, et al. (2022) Single-cell sequencing reveals lineage-specific dynamic genetic regulation of gene expression during human cardiomyocyte differentiation. PLOS Genetics 18(1): e1009666. [PubMed]
Many of the gene variant and gene expression associations relevant to differentiation are unknown and cannot fully be explained by coded gene variation.
iPSCs were differentiated to cardiomyocytes and they sampled single cell data from 19 human cell lines at 7 different time points. They identified genetic regulatory effects that are “dynamic” over time in a complex environment containing diverse and transient cell states.
Two distinct trajectories for differentiation were identified. Hundreds of dynamic associations between regulatory variants and gene expression levels were also identified, including many specific to a single trajectory.
Alveolar macrophages in bronchioalveolar lavage fluids characterized from tuberculosis patients
Qianqian Chen, Chunmei Hu, Wei Lu, Tianxing Hang, Yan Shao, Cheng Chen, Yanli Wang, Nan Li, Linling Jin, Wei Wu, Hong Wang, Xiaoning Zeng, Weiping Xie. Characteristics of alveolar macrophages in bronchioalveolar lavage fluids from active tuberculosis patients identified by single-cell RNA sequencing[J]. The Journal of Biomedical Research, 2022, 36(3): 167-180. [PubMed]
Tuberculosis (TB), is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), and presents with high morbidity and mortality. Alveolar macrophages play an important role in TB pathogenesis although there is heterogeneity and functional plasticity.
Chen et al. showed the characteristics of alveolar macrophages from bronchioalveolar lavage fluid (BALF) in active TB patients: Alveolar macrophage subclusters with increased percentages were involved in inflammatory signaling pathways as well as the basic macrophage functions. The TB-increased alveolar macrophage subclusters were shown to be derived from M1-like polarization state, before switching to an M2-like polarization state with the development of M. tuberculosis infection. This study demonstrated the characteristics of alveolar macrophages from BALF in active TB patients.
State of Hepatic Lymphatic Endothelial Cells in Hepatitis B Liver Failure
Zhang, P.; Li, H.; Zhou, C.; Liu, K.; Peng, B.; She, X.; Cheng, K.; Liu, H.; Ming, Y. Single-Cell RNA Transcriptomics Reveals the State of Hepatic Lymphatic Endothelial Cells in Hepatitis B Virus-Related Acute-on-Chronic Liver Failure. J. Clin. Med. 2022, 11, 2910. [PubMed]
Acute-on-chronic liver failure (ACLF) is an acutely decompensated cirrhosis syndrome with high short-term mortality. The relationship between the lymphatic system and ACLF is less known. Zhang et al. explored the role of hepatic lymphatic vessels (LVs) and lymphatic endothelial cells (LyECs) in ACLF using human liver samples. ACLF exhibited more severe liver injury and inflammation than cirrhosis, as indicated by significant increases in plasma levels of alanine/aspartate aminotransferases and total bilirubin. It is revealed that many monocyte/macrophages infiltrated into the liver of ACLF cases. Meanwhile, a group of apoptotic and dysfunctional LyECs, which were the result of secreted phosphoprotein 1 (SPP1) released from infiltrating monocyte/macrophages. In conclusion, ACLF is associated with less LV and LyEC dysfunction, at least in part mediated by SPP1 released from infiltrating monocyte/macrophages. Hepatic LVs and LyECs can be a novel therapeutic strategy for ACLF.
Comparative analysis of testes in wild-type and knockout mice
Hong, S.; Shen, X.; Luo, C.; Sun, F. Comparative analysis of the testes from wild-type and Alkbh5-knockout mice using single-cell RNA sequencing. Oxford G3. 2022, 12(8), jka130. [PubMed]
ALKBH5 deficiency causes male infertility in mice; however, the mechanisms that confer disruption of spermatogenesis are not completely clear. In this study, testis samples from wild-type and Alkbh5-knockout mice were profiled using single-cell RNA sequencing. Differences were analyzed between the transcriptional profiles of the groups at various germ cell developmental stages. Genes related to spermatogenesis in germ cells and somatic cells (Sertoli cells and Leydig cells) were detected and evaluated for their functions and associated pathways, such as chromatin-related functional pathways, through gene ontology enrichment analysis. This study highlights ALKBH5 as an important gene for germ cell development and spermatogenesis and offers new molecular mechanistic insights and provide the basis for further research into the causes and treatment of male infertility.
a) Western blots showing the efficiency of ALKBH5 protein KO. b-Actin served as a loading control. b) Immunofluorescence images of testis from 12-week-old WT and Alkbh5 KO mice stained for ALKBH5 (green) and DAPI (blue) (scale bars: 50 lm). c) Left panel: representative testes gross morphology of 12-week-old WT control and age-matched Alkbh5 KO mice. Right panel: testis weights from WT and Alkbh5 KO mice (n.8 per group; ****P <0.0001, Student’s t-test, error bars are 6 SEM).
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