scholarly journals Integrated Single-Cell RNA-Sequencing Analysis of Aquaporin 5-Expressing Mouse Lung Epithelial Cells Identifies GPRC5A as a Novel Validated Type I Cell Surface Marker

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2460 ◽  
Author(s):  
Masafumi Horie ◽  
Alessandra Castaldi ◽  
Mitsuhiro Sunohara ◽  
Hongjun Wang ◽  
Yanbin Ji ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Cell ◽  
Rna Sequencing ◽  
Surface Marker ◽  
Mouse Lung ◽  
Type I ◽  
Cell Markers ◽  
Lung Epithelial ◽  
Single Cell Rna Sequencing

Molecular and functional characterization of alveolar epithelial type I (AT1) cells has been challenging due to difficulty in isolating sufficient numbers of viable cells. Here we performed single-cell RNA-sequencing (scRNA-seq) of tdTomato+ cells from lungs of AT1 cell-specific Aqp5-Cre-IRES-DsRed (ACID);R26tdTomato reporter mice. Following enzymatic digestion, CD31-CD45-E-cadherin+tdTomato+ cells were subjected to fluorescence-activated cell sorting (FACS) followed by scRNA-seq. Cell identity was confirmed by immunofluorescence using cell type-specific antibodies. After quality control, 92 cells were analyzed. Most cells expressed ‘conventional’ AT1 cell markers (Aqp5, Pdpn, Hopx, Ager), with heterogeneous expression within this population. The remaining cells expressed AT2, club, basal or ciliated cell markers. Integration with public datasets identified three robust AT1 cell- and lung-enriched genes, Ager, Rtkn2 and Gprc5a, that were conserved across species. GPRC5A co-localized with HOPX and was not expressed in AT2 or airway cells in mouse, rat and human lung. GPRC5A co-localized with AQP5 but not pro-SPC or CC10 in mouse lung epithelial cell cytospins. We enriched mouse AT1 cells to perform molecular phenotyping using scRNA-seq. Further characterization of putative AT1 cell-enriched genes revealed GPRC5A as a conserved AT1 cell surface marker that may be useful for AT1 cell isolation.

scholarly journals MBRS-46. CHARTING NEOPLASTIC AND IMMUNE CELL HETEROGENEITY IN HUMAN AND GEM MODELS OF MEDULLOBLASTOMA USING scRNAseq

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Andrew Donson ◽  
Kent Riemondy ◽  
Sujatha Venkataraman ◽  
Ahmed Gilani ◽  
Bridget Sanford ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Genetically Engineered ◽  
Cellular Heterogeneity ◽  
Cell Heterogeneity ◽  
Transcriptomic Data ◽  
Single Cell Rna Sequencing ◽  
Transcript Profiles

Abstract We explored cellular heterogeneity in medulloblastoma using single-cell RNA sequencing (scRNAseq), immunohistochemistry and deconvolution of bulk transcriptomic data. Over 45,000 cells from 31 patients from all main subgroups of medulloblastoma (2 WNT, 10 SHH, 9 GP3, 11 GP4 and 1 GP3/4) were clustered using Harmony alignment to identify conserved subpopulations. Each subgroup contained subpopulations exhibiting mitotic, undifferentiated and neuronal differentiated transcript profiles, corroborating other recent medulloblastoma scRNAseq studies. The magnitude of our present study builds on the findings of existing studies, providing further characterization of conserved neoplastic subpopulations, including identification of a photoreceptor-differentiated subpopulation that was predominantly, but not exclusively, found in GP3 medulloblastoma. Deconvolution of MAGIC transcriptomic cohort data showed that neoplastic subpopulations are associated with major and minor subgroup subdivisions, for example, photoreceptor subpopulation cells are more abundant in GP3-alpha. In both GP3 and GP4, higher proportions of undifferentiated subpopulations is associated with shorter survival and conversely, differentiated subpopulation is associated with longer survival. This scRNAseq dataset also afforded unique insights into the immune landscape of medulloblastoma, and revealed an M2-polarized myeloid subpopulation that was restricted to SHH medulloblastoma. Additionally, we performed scRNAseq on 16,000 cells from genetically engineered mouse (GEM) models of GP3 and SHH medulloblastoma. These models showed a level of fidelity with corresponding human subgroup-specific neoplastic and immune subpopulations. Collectively, our findings advance our understanding of the neoplastic and immune landscape of the main medulloblastoma subgroups in both humans and GEM models.

scholarly journals Unveiling the heterogeneity of NKT cells in the liver through single cell RNA sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hao Shen ◽  
Chan Gu ◽  
Tao Liang ◽  
Haifeng Liu ◽  
Fan Guo ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Subset ◽  
Nkt Cells ◽  
Organ Distribution ◽  
Marker Genes ◽  
Type I ◽  
Nkt Cell ◽  
Single Cell Rna Sequencing

Abstract CD1d-dependent type I NKT cells, which are activated by lipid antigen, are known to play important roles in innate and adaptive immunity, as are a portion of type II NKT cells. However, the heterogeneity of NKT cells, especially NKT-like cells, remains largely unknown. Here, we report the profiling of NKT (NK1.1+CD3e+) cells in livers from wild type (WT), Jα18-deficient and CD1d-deficient mice by single-cell RNA sequencing. Unbiased transcriptional clustering revealed distinct cell subsets. The transcriptomic profiles identified the well-known CD1d-dependent NKT cells and defined two CD1d-independent NKT cell subsets. In addition, validation of marker genes revealed the differential organ distribution and landscape of NKT cell subsets during liver tumor progression. More importantly, we found that CD1d-independent Sca-1−CD62L+ NKT cells showed a strong ability to secrete IFN-γ after costimulation with IL-2, IL-12 and IL-18 in vitro. Collectively, our findings provide a comprehensive characterization of NKT cell heterogeneity and unveil a previously undefined functional NKT cell subset.

scholarly journals Single-cell RNA Sequencing Reveals That Injected ADSCs Change the Macrophage Profile in a Murine Model of Pulmonary Fibrosis

2021 ◽  
Author(s):  
Mamatali Rahman ◽  
Zhao-Yan Wang ◽  
Jun-Xiang Li ◽  
Hao-Wei Xu ◽  
Qiong Wu
Keyword(s):  
Pulmonary Fibrosis ◽  
Single Cell ◽  
Rna Sequencing ◽  
Treatment Options ◽  
Mouse Lung ◽  
Therapeutic Effects ◽  
Intratracheal Administration ◽  
Cell Based Therapy ◽  
Single Cell Rna Sequencing ◽  
Underlying Mechanisms

Abstract Background: Idiopathic pulmonary fibrosis (IPF) is a deadly chronic interstitial lung disease with no effective treatment options other than lung transplantation. Allogeneic adipose-derived mesenchymal stem cells (ADSCs) are considered ideal as seed cells for stem cell-based therapy, and some studies illustrated the therapeutic effect of ADSCs on IPF, but the underlying mechanisms remain unclear.Methods: A single intratracheal dose of bleomycin (BLM) was administered to induce pulmonary injury/fibrosis in C57BL/6 mice, after GFP-labeled mouse ADSCs (mADSCs) were implanted intratracheally to explore their potential therapeutic effects in the inflamed/fibrotic lung microenvironment. The mADSCs were then retrieved through fluorescence-activated cell sorting and subjected to single-cell RNA sequencing (scRNA-seq).Results: Our data indicate that the single-dose intratracheal administration of mADSCs could significantly increase the life span of IPF mice by remodeling the extracellular matrix and promoting the polarization of macrophages to an anti-inflammatory phenotype. Conclusions: A single intratracheal injection of mADSCs alleviated BLM-induced pulmonary fibrosis by readjustment of the mouse lung microenvironment, which was reflected in changes of the lung C1QB+, APOE+ and TREM2+ macrophages in the mouse model.

scholarly journals Re-investigation of classic T cell subsets and identification of novel cell subpopulations by single-cell RNA sequencing

2021 ◽  
Author(s):  
Xuefei Wang ◽  
Xiangru Shen ◽  
Shan Chen ◽  
Hongyi Liu ◽  
Ni Hong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Single Cell ◽  
Rna Sequencing ◽  
T Cell Activation ◽  
Expression Profiles ◽  
T Cell Subsets ◽  
Cell Subpopulation ◽  
Single Cell Rna Sequencing

AbstractClassic T cell subsets are defined by a small set of cell surface markers, while single cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq Clustered-Populations (scCPops) and cell surface marker-defined classic T cell subsets remain unclear. Here, we interrogated 6 bead-enriched T cell subsets with 62,235 single cell transcriptomes and re-grouped them into 9 scCPops. Bead-enriched CD4 Naïve and CD8 Naïve were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops including mucosal-associated invariant T cells and natural killer T cells. The multiple T cell subsets that form a single scCPop exhibited similar expression pattern, but not vice versa, indicating scCPops are much homogeneous cell populations with similar cell states. Interestingly, we discovered and named IFNhi T, a new T cell subpopulation that highly expressed Interferon Signaling Associated Genes (ISAGs). We further enriched IFNhi T by FACS sorting of BST2 for scRNA-seq analyses. IFNhi T cluster disappeared on tSNE plot after removing ISAGs, while IFNhi T cluster showed up by tSNE analyses of ISAGs alone, indicating ISAGs are the major contributor of IFNhi T cluster. BST2+ T cells and BST2− T cells showing different efficiencies of T cell activation indicates high level of ISAGs may contribute to quick immune responses.

scholarly journals Expressed barcodes enable clonal characterization of chemotherapeutic responses in chronic lymphocytic leukemia

2019 ◽  
Author(s):  
Aziz Al’Khafaji ◽  
Catherine Gutierrez ◽  
Eric Brenner ◽  
Russell Durrett ◽  
Kaitlyn E. Johnson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Single Cell ◽  
Rna Sequencing ◽  
Genetic Alterations ◽  
Response To Therapy ◽  
Lymphocytic Leukemia ◽  
Single Cell Rna Sequencing ◽  
Cancer Cell Population

AbstractThe remarkable evolutionary capacity of cancer is a major challenge to current therapeutic efforts. Fueling this evolution is its vast clonal heterogeneity and ability to adapt to diverse selective pressures. Although the genetic and transcriptional mechanisms underlying these responses have been independently evaluated, the ability to couple genetic alterations present within individual clones to their respective transcriptional or functional outputs has been lacking in the field. To this end, we developed a high-complexity expressed barcode library that integrates DNA barcoding with single-cell RNA sequencing through use of the CROP-seq sgRNA expression/capture system, and which is compatible with the COLBERT clonal isolation workflow for subsequent genomic and epigenomic characterization of specific clones of interest. We applied this approach to study chronic lymphocytic leukemia (CLL), a mature B cell malignancy notable for its genetic and transcriptomic heterogeneity and variable disease course. Here, we demonstrate the clonal composition and gene expression states of HG3, a CLL cell line harboring the common alteration del(13q), in response to front-line cytotoxic therapy of fludarabine and mafosfamide (an analog of the clinically used cyclophosphamide). Analysis of clonal abundance and clonally-resolved single-cell RNA sequencing revealed that only a small fraction of clones consistently survived therapy. These rare highly drug tolerant clones comprise 94% of the post-treatment population and share a stable, pre-existing gene expression state characterized by upregulation of CXCR4 and WNT signaling and a number of DNA damage and cell survival genes. Taken together, these data demonstrate at unprecedented resolution the diverse clonal characteristics and therapeutic responses of a heterogeneous cancer cell population. Further, this approach provides a template for the high-resolution study of thousands of clones and the respective gene expression states underlying their response to therapy.

scholarly journals A survey of human brain transcriptome diversity at the single cell level

2015 ◽  
Vol 112 (23) ◽  
pp. 7285-7290 ◽  
Author(s):  
Spyros Darmanis ◽  
Steven A. Sloan ◽  
Ye Zhang ◽  
Martin Enge ◽  
Christine Caneda ◽  
...  
Keyword(s):  
Human Brain ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cortical Neurons ◽  
Cell Types ◽  
Type I ◽  
Neuronal Populations ◽  
Brain Transcriptome ◽  
Single Cell Rna Sequencing ◽  
Whole Transcriptome

The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

scholarly journals Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
David Brown ◽  
Michael Altermatt ◽  
Tatyana Dobreva ◽  
Sisi Chen ◽  
Alexander Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Type ◽  
Gene Therapies ◽  
P53 Signaling ◽  
Single Cell Rna Sequencing ◽  
Delivery Vehicles ◽  
Tissue Tropisms

Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at high resolution. Using this platform, we have both corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Transcriptomic analysis revealed that this neuronal bias is due mainly to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific transitory responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. The presented experimental and computational approaches for parallel characterization of AAV tropism will facilitate the advancement of safe and precise gene delivery vehicles, and showcase the power of understanding responses to gene therapies at the single-cell level.

scholarly journals Single-cell RNA-sequencing reveals the dynamic process and novel markers in porcine spermatogenesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lingkai Zhang ◽  
Fuyuan Li ◽  
Peipei Lei ◽  
Ming Guo ◽  
Ruifang Liu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Cell ◽  
Rna Sequencing ◽  
Germ Cells ◽  
Cell Types ◽  
Functional Enrichment ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Male Germ Cells ◽  
Single Cell Rna Sequencing

Abstract Background Spermatogenesis is the process by which male gametes are formed from spermatogonial stem cells and it is essential for the reliable transmission of genetic information between generations. To date, the dynamic transcriptional changes of defined populations of male germ cells in pigs have not been reported. Results To characterize the atlas of porcine spermatogenesis, we profiled the transcriptomes of ~ 16,966 testicular cells from a 150-day-old pig testis through single-cell RNA-sequencing (scRNA-seq). The scRNA-seq analysis identified spermatogonia, spermatocytes, spermatids and three somatic cell types in porcine testes. The functional enrichment analysis demonstrated that these cell types played diverse roles in porcine spermatogenesis. The accuracy of the defined porcine germ cell types was further validated by comparing the data from scRNA-seq with those from bulk RNA-seq. Since we delineated four distinct spermatogonial subsets, we further identified CD99 and PODXL2 as novel cell surface markers for undifferentiated and differentiating spermatogonia, respectively. Conclusions The present study has for the first time analyzed the transcriptome of male germ cells and somatic cells in porcine testes through scRNA-seq. Four subsets of spermatogonia were identified and two novel cell surface markers were discovered, which would be helpful for studies on spermatogonial differentiation in pigs. The datasets offer valuable information on porcine spermatogenesis, and pave the way for identification of key molecular markers involved in development of male germ cells.

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