scholarly journals Characterization of normal and cancer stem-like cell populations in murine lingual epithelial organoids using single-cell RNA sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Erik Johansson ◽  
Hiroo Ueno
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Targeted Therapies ◽  
Tongue Cancer ◽  
Cell Populations ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Molecular Targeted Therapies ◽  
Single Cell Rna Sequencing

AbstractThe advances in oral cancer research and therapies have not improved the prognosis of patients with tongue cancer. The poor treatment response of tongue cancer may be attributed to the presence of heterogeneous tumor cells exhibiting stem cell characteristics. Therefore, there is a need to develop effective molecular-targeted therapies based on the specific gene expression profiles of these cancer stem-like cell populations. In this study, the characteristics of normal and cancerous organoids, which are convenient tools for screening anti-cancer drugs, were analyzed comparatively. As organoids are generally generated by single progenitors, they enable the exclusion of normal cell contamination from the analyses. Single-cell RNA sequencing analysis revealed that p53 signaling activation and negative regulation of cell cycle were enriched characteristics in normal stem-like cells whereas hypoxia-related pathways, such as HIF-1 signaling and glycolysis, were upregulated in cancer stem-like cells. The findings of this study improved our understanding of the common features of heterogeneous cell populations with stem cell properties in tongue cancers, that are different from those of normal stem cell populations; this will enable the development of novel molecular-targeted therapies for tongue cancer.

scholarly journals Single-Cell RNA Sequencing in Human Retinal Degeneration Reveals Distinct Glial Cell Populations

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 438 ◽  
Author(s):  
Andrew P. Voigt ◽  
Elaine Binkley ◽  
Miles J. Flamme-Wiese ◽  
Shemin Zeng ◽  
Adam P. DeLuca ◽  
...  
Keyword(s):  
Single Cell ◽  
Retinal Degeneration ◽  
Rna Sequencing ◽  
Glial Cell ◽  
Glial Cells ◽  
Peripheral Retina ◽  
Cell Populations ◽  
Specific Gene ◽  
Autoimmune Retinopathy ◽  
Single Cell Rna Sequencing

Degenerative diseases affecting retinal photoreceptor cells have numerous etiologies and clinical presentations. We clinically and molecularly studied the retina of a 70-year-old patient with retinal degeneration attributed to autoimmune retinopathy. The patient was followed for 19 years for progressive peripheral visual field loss and pigmentary changes. Single-cell RNA sequencing was performed on foveal and peripheral retina from this patient and four control patients, and cell-specific gene expression differences were identified between healthy and degenerating retina. Distinct populations of glial cells, including astrocytes and Müller cells, were identified in the tissue from the retinal degeneration patient. The glial cell populations demonstrated an expression profile consistent with reactive gliosis. This report provides evidence that glial cells have a distinct transcriptome in the setting of human retinal degeneration and represents a complementary clinical and molecular investigation of a case of progressive retinal disease.

scholarly journals 973. Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Cerebral Organoids

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S33-S34
Author(s):  
Karen Ocwieja ◽  
Alexandra Stanton ◽  
Alexsia Richards ◽  
Jenna Antonucci ◽  
Travis Hughes ◽  
...  
Keyword(s):  
Stem Cell ◽  
Human Brain ◽  
Single Cell ◽  
Rna Sequencing ◽  
Zika Virus ◽  
Model Systems ◽  
Sequencing Analysis ◽  
Human Stem Cell ◽  
Zikv Infection ◽  
Single Cell Rna Sequencing

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. One barrier has been the lack of relevant model systems for the developing human brain; however, thanks to advances in the stem cell field, we can now evaluate ZIKV central nervous system infections in human stem cell-derived cerebral organoids which recapitulate complex 3-dimensional neural architecture. Methods We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize cerebral organoids infected with ZIKV. Using this sequencing method, and published transcriptional profiles, we identify multiple cellular populations in our organoids, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, defining transcriptional features of uninfected cells and infected cells. Results In this model of the developing brain, we identify preferred tropisms of ZIKV infection and pronounced effects on cell division, differentiation, and death. Our data additionally reveal differences in cellular populations and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. This finding might help explain phenotypic differences attributed to the viruses, including variable propensity to cause microcephaly. Conclusion Overall, our work provides insight into normal and diseased human brain development, and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection. Disclosures All Authors: No reported Disclosures.

scholarly journals Pancreatic cancer is marked by complement-high blood monocytes and tumor-associated macrophages

2021 ◽  
Vol 4 (6) ◽  
pp. e202000935
Author(s):  
Samantha B Kemp ◽  
Nina G Steele ◽  
Eileen S Carpenter ◽  
Katelyn L Donahue ◽  
Grace G Bushnell ◽  
...  
Keyword(s):  
Single Cell ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Rna Sequencing ◽  
Gene Expression Signature ◽  
Ductal Adenocarcinoma ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Tumor Associated Macrophages ◽  
Primary Tumors ◽  
Single Cell Rna Sequencing

Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3. Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8. In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.

#71: Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Neuroprogenitor Cells and Cerebral Organoids

2021 ◽  
Vol 10 (Supplement_1) ◽  
pp. S14-S14
Author(s):  
K E Ocwieja ◽  
T K Hughes ◽  
J M Antonucci ◽  
A L Richards ◽  
A C Stanton ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Zika Virus ◽  
Molecular Mechanisms ◽  
Sequencing Analysis ◽  
Human Stem Cell ◽  
Zikv Infection ◽  
Single Cell Sequencing ◽  
Single Cell Rna Sequencing

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. It is also unclear why congenital ZIKV disease was not observed prior to the recent epidemics in French Polynesia and the Americas, despite evidence that the Zika virus has actively circulated in parts of Africa and Asia since 1947 and 1966, respectively. Methods Due to advances in stem cell-based technologies, we can now model ZIKV infections of the central nervous system in human stem cell-derived neuroprogenitor cells and cerebral organoids, which recapitulate complex three-dimensional neural architecture. We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize these neural models infected with ZIKV. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, thereby defining transcriptional features of both uninfected and infected cells. Results In neuroprogenitor cells, single-cell sequencing reveals that while uninfected bystander cells strongly upregulate interferon pathway genes, these are largely suppressed in cells infected with ZIKV within the same culture dish. In our organoid model, single-cell sequencing allows us to identify multiple cellular populations, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. In this model of the developing brain, we identify preferred tropisms of ZIKV infection. Our data additionally reveal differences in cell-type frequencies and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. Conclusions These findings may help explain phenotypic differences attributed to the viruses, including variable propensities to cause microcephaly. Overall, our work provides insight into normal and diseased human brain development and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection.

scholarly journals The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

2019 ◽  
Author(s):  
Audrey C.A. Cleuren ◽  
Martijn A. van der Ent ◽  
Hui Jiang ◽  
Kristina L. Hunker ◽  
Andrew Yee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Single Cell ◽  
Blood Vessels ◽  
Rna Sequencing ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Vascular Beds

AbstractEndothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression following EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single cell suspensions for fluorescence-activated cell sorting (FACS) or single cell RNA sequencing analysis. Comparison of our EC-TRAP to published single cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.SignificanceEndothelial cells (ECs), which line all vertebrate blood vessels, are highly heterogeneous across different tissues. The present study uses a genetic approach to specifically tag mRNAs within ECs of the mouse, thereby allowing recovery and sequence analysis to evaluate the EC-specific gene expression program directly from intact organs. Our findings demonstrate marked heterogeneity in EC gene expression across different vascular beds under both normal and disease conditions, with a more accurate picture than can be achieved using other methods. The data generated in these studies advance our understanding of EC function in different blood vessels and provide a valuable resource for future studies.

scholarly journals Integrated single-cell RNA sequencing analysis reveals distinct cellular and transcriptional modules associated with survival in lung cancer

2022 ◽  
Vol 7 (1) ◽  
Author(s):  
Li Zhang ◽  
Yiming Zhang ◽  
Chengdi Wang ◽  
Ying Yang ◽  
Yinyun Ni ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Single Cell ◽  
Rna Sequencing ◽  
Squamous Carcinoma ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Basal Cells ◽  
Functional Roles ◽  
Pathologic Features ◽  
Single Cell Rna Sequencing

AbstractLung adenocarcinoma (LUAD) and squamous carcinoma (LUSC) are two major subtypes of non-small cell lung cancer with distinct pathologic features and treatment paradigms. The heterogeneity can be attributed to genetic, transcriptional, and epigenetic parameters. Here, we established a multi-omics atlas, integrating 52 single-cell RNA sequencing and 2342 public bulk RNA sequencing. We investigated their differences in genetic amplification, cellular compositions, and expression modules. We revealed that LUAD and LUSC contained amplifications occurring selectively in subclusters of AT2 and basal cells, and had distinct cellular composition modules associated with poor survival of lung cancer. Malignant and stage-specific gene analyses further uncovered critical transcription factors and genes in tumor progression. Moreover, we identified subclusters with proliferating and differentiating properties in AT2 and basal cells. Overexpression assays of ten genes, including sub-cluster markers AQP5 and KPNA2, further indicated their functional roles, providing potential targets for early diagnosis and treatment in lung cancer.

scholarly journals Characterization of Alzheimer’s Disease-Associated Excitatory Neurons via Single-Cell RNA Sequencing Analysis

2021 ◽  
Vol 13 ◽  
Author(s):  
Fanghong Shao ◽  
Meiting Wang ◽  
Qi Guo ◽  
Bowen Zhang ◽  
Xiangting Wang
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Single Cell ◽  
Rna Sequencing ◽  
Neuronal Cell ◽  
Cell Populations ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Excitatory Neurons

The detailed characteristics of neuronal cell populations in Alzheimer’s disease (AD) using single-cell RNA sequencing have not been fully elucidated. To explore the characterization of neuronal cell populations in AD, this study utilized the publicly available single-nucleus RNA-sequencing datasets in the transgenic model of 5X familial Alzheimer’s disease (5XFAD) and wild-type mice to reveal an AD-associated excitatory neuron population (C3:Ex.Neuron). The relative abundance of C3:Ex.Neuron increased at 1.5 months and peaked at 4.7 months in AD mice. Functional pathways analyses showed that the pathways positively related to neurodegenerative disease progression were downregulated in the C3:Ex.Neuron at 1.5 months in AD mice. Based on the differentially expressed genes among the C3:Ex.Neuron, four subtypes (C3.1–4) were identified, which exhibited distinct abundance regulatory patterns during the development of AD. Among these subtypes, the C3.1 neurons [marked by netrin G1 (Ntng1)] exhibited a similar regulatory pattern as the C3:Ex.Neuron in abundance during the development of AD. In addition, our gene set variation analysis (GSEA) showed that the C3.1 neurons, instead of other subtypes of the C3:Ex.Neuron, possessed downregulated AD pathways at an early stage (1.5 months) of AD mice. Collectively, our results identified a previously unidentified subset of excitatory neurons and provide a potential application of these neurons to modulate the disease susceptibility.

scholarly journals The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

2019 ◽  
Vol 116 (47) ◽  
pp. 23618-23624 ◽  
Author(s):  
Audrey C. A. Cleuren ◽  
Martijn A. van der Ent ◽  
Hui Jiang ◽  
Kristina L. Hunker ◽  
Andrew Yee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Expression Profiles ◽  
Affinity Purification ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Vascular Beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.

Molecular and Cellular Dynamics of Aortic Aneurysms Revealed by Single-Cell Transcriptomics

2021 ◽  
Author(s):  
Yanming Li ◽  
Scott A. LeMaire ◽  
Ying H. Shen
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Cellular Level ◽  
Aortic Aneurysms ◽  
Cellular Heterogeneity ◽  
Cell Populations ◽  
Sequencing Analysis ◽  
Molecular Features ◽  
Single Cell Rna Sequencing

The aorta is highly heterogeneous, containing many different types of cells that perform sophisticated functions to maintain aortic homeostasis. Recently, single-cell RNA sequencing studies have provided substantial new insight into the heterogeneity of vascular cell types, the comprehensive molecular features of each cell type, and the phenotypic interrelationship between these cell populations. This new information has significantly improved our understanding of aortic biology and aneurysms at the molecular and cellular level. Here, we summarize these findings, with a focus on what single-cell RNA sequencing analysis has revealed about cellular heterogeneity, cellular transitions, communications among cell populations, and critical transcription factors in the vascular wall. We also review the information learned from single-cell RNA sequencing that has contributed to our understanding of the pathogenesis of vascular disease, such as the identification of cell types in which aneurysm-related genes and genetic variants function. Finally, we discuss the challenges and future directions of single-cell RNA sequencing applications in studies of aortic biology and diseases.

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