scholarly journals Diversification of reprogramming trajectories revealed by parallel single-cell transcriptome and chromatin accessibility sequencing

2020 ◽  
Vol 6 (37) ◽  
pp. eaba1190
Author(s):  
Q. R. Xing ◽  
C. A. El Farran ◽  
P. Gautam ◽  
Y. S. Chuah ◽  
T. Warrier ◽  
...  
Keyword(s):  
Single Cell ◽  
Chromatin Accessibility ◽  
Human Cells ◽  
Cellular Reprogramming ◽  
Surface Markers ◽  
Low Efficiency ◽  
Cell Transcriptome ◽  
Intermediate Cells ◽  
Single Cell Transcriptome ◽  
Accessible Chromatin

Cellular reprogramming suffers from low efficiency especially for the human cells. To deconstruct the heterogeneity and unravel the mechanisms for successful reprogramming, we adopted single-cell RNA sequencing (scRNA-Seq) and single-cell assay for transposase-accessible chromatin (scATAC-Seq) to profile reprogramming cells across various time points. Our analysis revealed that reprogramming cells proceed in an asynchronous trajectory and diversify into heterogeneous subpopulations. We identified fluorescent probes and surface markers to enrich for the early reprogrammed human cells. Furthermore, combinatory usage of the surface markers enabled the fine segregation of the early-intermediate cells with diverse reprogramming propensities. scATAC-Seq analysis further uncovered the genomic partitions and transcription factors responsible for the regulatory phasing of reprogramming process. Binary choice between a FOSL1 and a TEAD4-centric regulatory network determines the outcome of a successful reprogramming. Together, our study illuminates the multitude of diverse routes transversed by individual reprogramming cells and presents an integrative roadmap for identifying the mechanistic part list of the reprogramming machinery.

scholarly journals Diversification of Reprogramming Trajectories Revealed by Parallel Single-cell Transcriptome and Chromatin Accessibility Sequencing

2019 ◽  
Author(s):  
Qiaorui Xing ◽  
Chadi El Farran ◽  
Pradeep Gautam ◽  
Yu Song Chuah ◽  
Tushar Warrier ◽  
...  
Keyword(s):  
Single Cell ◽  
Regulatory Network ◽  
Chromatin Accessibility ◽  
Cellular Reprogramming ◽  
Binary Choice ◽  
Surface Markers ◽  
Part List ◽  
Cell Transcriptome ◽  
Intermediate Cells ◽  
Single Cell Transcriptome

To unravel the mechanism of human cellular reprogramming process at single-cell resolution, we performed parallel scRNA-Seq and scATAC-Seq analysis. Our analysis reveals that the cells undergoing reprogramming proceed in an asynchronous trajectory and diversify into heterogeneous sub-populations. BDD2-C8 fluorescent probe staining and negative staining for CD13, CD44 and CD201 markers, could enrich for the GDF3+ early reprogrammed cells. Combinatory usage of the surface markers enables the fine segregation of the early-intermediate cells with diverse reprogramming propensities. scATAC-Seq analysis further uncovered the genomic partitions and transcription factors responsible for the regulatory phasing of reprogramming process. Binary choice between a FOSL1 or a TEAD4-centric regulatory network determines the outcome of a successful reprogramming. Altogether, our study illuminates the multitude of diverse routes transversed by individual reprogramming cells and presents an integrative roadmap for identifying the mechanistic part-list of the reprogramming machinery.

scholarly journals Single cell transcriptome atlas of mouse mammary epithelial cells across development

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Bhupinder Pal ◽  
Yunshun Chen ◽  
Michael J. G. Milevskiy ◽  
François Vaillant ◽  
Lexie Prokopuk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Developmental Stages ◽  
Mammary Epithelial Cells ◽  
Expression Profiles ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Mammary Epithelial ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

scholarly journals Single-cell transcriptome analysis of embryonic and adult endothelial cells allows to rank the hemogenic potential of post-natal endothelium

2021 ◽  
Author(s):  
Artem Adamov ◽  
Yasmin Natalia Serina Sechanecia ◽  
Christophe Lancrin
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Rational Design ◽  
Continuous Production ◽  
Cellular Reprogramming ◽  
Hematopoietic Stem ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Hematopoietic stem cells are crucial for the continuous production of blood cells during life. The transplantation of these cells is one of the most common treatments to cure patient suffering of blood diseases. However, the lack of suitable donors is a major limitation. One option to get hematopoietic stem cells matching perfectly a patient is cellular reprogramming. Hematopoietic stem cells emerge from endothelial cells in blood vessels during embryogenesis through the endothelial to hematopoietic transition. Here, we used single-cell transcriptomics analysis to compare embryonic and post-natal endothelial cells to investigate the potential of adult vasculature to be reprogrammed in hematopoietic stem cells. Although transcriptional similarities have been found between embryonic and adult endothelial cells, we found some key differences in term of transcription factors expression. There is a deficit of expression of Runx1, Tal1, Lyl1 and Cbfb in adult endothelial cells compared to their embryonic counterparts. Using a combination of gene expression profiling and gene regulatory network analysis, we found that endothelial cells from the pancreas, brain, kidney and liver appear to be the most suitable targets for cellular reprogramming into hematopoietic stem cells. Overall, our work provides an important resource for the rational design of a reprogramming strategy for the generation of hematopoietic stem cells.

scholarly journals Lifelong single-cell profiling of cranial neural crest diversification

2021 ◽  
Author(s):  
Peter Fabian ◽  
Kuo-Chang Tseng ◽  
Mathi Thiruppathy ◽  
Claire Arata ◽  
Hung-Jhen Chen ◽  
...  
Keyword(s):  
Neural Crest ◽  
Single Cell ◽  
Cell Fate ◽  
Intrinsic Property ◽  
Chromatin Accessibility ◽  
Specific Cell ◽  
List Type ◽  
Cranial Neural Crest ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

AbstractThe cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being an intrinsic property of cranial neural crest, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse neural crest lineage potential.HighlightsSingle-cell transcriptome and chromatin atlas of cranial neural crestProgressive emergence of region-specific cell fate competencyChromatin accessibility mapping identifies candidate lineage regulatorsGata3 function linked to gill-specific respiratory programGraphical Abstract

scholarly journals Single-cell atlas of a non-human primate reveals new pathogenic mechanisms of COVID-19

2020 ◽  
Author(s):  
Lei Han ◽  
Xiaoyu Wei ◽  
Chuanyu Liu ◽  
Giacomo Volpe ◽  
Zhifeng Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Viral Entry ◽  
Immune Cell ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
List Type ◽  
Innate Immune Responses ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Substantial Heterogeneity

ABSTRACTStopping COVID-19 is a priority worldwide. Understanding which cell types are targeted by SARS-CoV-2 virus, whether interspecies differences exist, and how variations in cell state influence viral entry is fundamental for accelerating therapeutic and preventative approaches. In this endeavor, we profiled the transcriptome of nine tissues from a Macaca fascicularis monkey at single-cell resolution. The distribution of SARS-CoV-2 facilitators, ACE2 and TMRPSS2, in different cell subtypes showed substantial heterogeneity across lung, kidney, and liver. Through co-expression analysis, we identified immunomodulatory proteins such as IDO2 and ANPEP as potential SARS-CoV-2 targets responsible for immune cell exhaustion. Furthermore, single-cell chromatin accessibility analysis of the kidney unveiled a plausible link between IL6-mediated innate immune responses aiming to protect tissue and enhanced ACE2 expression that could promote viral entry. Our work constitutes a unique resource for understanding the physiology and pathophysiology of two phylogenetically close species, which might guide in the development of therapeutic approaches in humans.Bullet pointsWe generated a single-cell transcriptome atlas of 9 monkey tissues to study COVID-19.ACE2+TMPRSS2+ epithelial cells of lung, kidney and liver are targets for SARS-CoV-2.ACE2 correlation analysis shows IDO2 and ANPEP as potential therapeutic opportunities.We unveil a link between IL6, STAT transcription factors and boosted SARS-CoV-2 entry.

scholarly journals Parallel Bimodal Single-cell Sequencing of Transcriptome and Chromatin Accessibility

2019 ◽  
Author(s):  
Qiao Rui Xing ◽  
Chadi EL Farran ◽  
Yao Yi ◽  
Tushar Warrier ◽  
Pradeep Gautam ◽  
...  
Keyword(s):  
Single Cell ◽  
Target Genes ◽  
Chromatin Accessibility ◽  
Human Cell Lines ◽  
Microfluidic Chips ◽  
Primary Cells ◽  
Parallel Sequencing ◽  
Single Cell Sequencing ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

SUMMARYWe developed ASTAR-Seq (Assay for Single-cell Transcriptome and Accessibility Regions) integrated with automated microfluidic chips, which allows for parallel sequencing of transcriptome and chromatin accessibility within the same single-cell. Using ASTAR-Seq, we profiled 192 mESCs cultured in serum+LIF and 2i medium, 424 human cell lines including BJ, K562, JK1, and Jurkat, and 480 primary cells undergoing erythroblast differentiation. Integrative analysis using Coupled NMF identified the distinct sub-populations and uncovered sets of regulatory regions and the respective target genes determining their distinctions. Analysis of epigenetic regulomes further unravelled the key transcription factors responsible for the heterogeneity observed.

scholarly journals SINGLE CELL TRANSCRIPTOME ANALYSIS IDENTIFIES PUTATIVE CELL SURFACE MARKERS OF HUMAN SPERMATOGONIAL STEM CELLS

2020 ◽  
Vol 114 (3) ◽  
pp. e100-e101
Author(s):  
Sarah Munyoki ◽  
Adrienne N. Shami ◽  
Xianing Zheng ◽  
Qianyi Ma ◽  
Meena Sukhwani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Single Cell ◽  
Transcriptome Analysis ◽  
Spermatogonial Stem Cells ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

scholarly journals Expanding the CITE-seq tool-kit: Detection of proteins, transcriptomes, clonotypes and CRISPR perturbations with multiplexing, in a single assay

2018 ◽  
Author(s):  
Eleni Mimitou ◽  
Anthony Cheng ◽  
Antonino Montalbano ◽  
Stephanie Hao ◽  
Marlon Stoeckius ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Surface Markers ◽  
Protein Levels ◽  
Cellular Processes ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Lymphoma Sample

ABSTRACTRapid technological progress in the recent years has allowed the high-throughput interrogation of different types of biomolecules from single cells. Combining several of these readouts into integrated multi-omic assays is essential to comprehensively understand and model cellular processes. Here, we report the development of Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell: transcriptome, immune receptor clonotypes, surface markers, sample identity and sgRNAs. We demonstrate the use of ECCITE-seq to directly and efficiently capture sgRNA molecules and measure their effects on gene expression and protein levels, opening the possibility of performing high throughput single cell CRISPR screens with multimodal readout using existing libraries and commonly used vectors. Finally, by utilizing the combined phenotyping of clonotype and cell surface markers in immune cells, we apply ECCITE to study a lymphoma sample to discriminate cells and define molecular signatures of malignant cells within a heterogeneous population.

scholarly journals Combinatorial action of transcription factors in open chromatin contributes to early cellular heterogeneity and organizer mesendoderm specification

2020 ◽  
Author(s):  
Ann Rose Bright ◽  
Siebe van Genesen ◽  
Qingqing Li ◽  
Simon J. van Heeringen ◽  
Alexia Grasso ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Single Cell ◽  
Marginal Zone ◽  
Chromatin Accessibility ◽  
Cellular Heterogeneity ◽  
Open Chromatin ◽  
Cell Clusters ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

ABSTRACTDuring gastrulation, mesoderm is induced in pluripotent cells, concomitant with dorsal-ventral patterning and establishing of the dorsal axis. How transcription factors operate within the constraints of chromatin accessibility to mediate these processes is not well-understood. We applied chromatin accessibility and single cell transcriptome analyses to explore the emergence of heterogeneity and underlying gene-regulatory mechanisms during early gastrulation in Xenopus. ATAC-sequencing of pluripotent animal cap cells revealed a state of open chromatin of transcriptionally inactive lineage-restricted genes, whereas chromatin accessibility in dorsal marginal zone cells more closely reflected the transcriptional activity of genes. We characterized single cell trajectories in animal cap and dorsal marginal zone in early gastrula embryos, and inferred the activity of transcription factors in single cell clusters by integrating chromatin accessibility and single cell RNA-sequencing. We tested the activity of organizer-expressed transcription factors in mesoderm-competent animal cap cells and found combinatorial effects of these factors on organizer gene expression. In particular the combination of Foxb1 and Eomes induced a gene expression profile that mimicked those observed in head and trunk organizer single cell clusters. In addition, genes induced by Eomes, Otx2 or the Irx3-Otx2 combination, were enriched for promoters with maternally regulated H3K4me3 modifications, whereas promoters selectively induced by Lhx8 were marked more frequently by zygotically controlled H3K4me3. Our results show that combinatorial activity of zygotically expressed transcription factors acts on maternally-regulated accessible chromatin to induce organizer gene expression.

scholarly journals Lifelong single-cell profiling of cranial neural crest diversification in zebrafish

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Peter Fabian ◽  
Kuo-Chang Tseng ◽  
Mathi Thiruppathy ◽  
Claire Arata ◽  
Hung-Jhen Chen ◽  
...  
Keyword(s):  
Neural Crest ◽  
Single Cell ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Cranial Neural Crest ◽  
Cell Fates ◽  
Regulatory Circuit ◽  
Lineage Priming ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

AbstractThe cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe progressive and region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being established during cranial neural crest specification, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse potential.

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