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

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.

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

Science Advances ◽

10.1126/sciadv.aba1190 ◽

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.

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Single cell transcriptome atlas of mouse mammary epithelial cells across development

Breast Cancer Research ◽

10.1186/s13058-021-01445-4 ◽

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.

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

10.1101/2021.06.16.447688 ◽

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.

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Lifelong single-cell profiling of cranial neural crest diversification

10.1101/2021.08.19.456710 ◽

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

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Single-Cell Transcriptomics-Based Study of Transcriptional Regulatory Features in the Mouse Brain Vasculature

BioMed Research International ◽

10.1155/2021/7643209 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Wei-Wei Lin ◽

Lin-Tao Xu ◽

Yi-Sheng Chen ◽

Ken Go ◽

Chenyu Sun ◽

...

Keyword(s):

Single Cell ◽

Mouse Brain ◽

Regulatory Network ◽

Regulatory Networks ◽

Transcriptional Regulatory Network ◽

Rna Seq ◽

Brain Vasculature ◽

Transcriptional Regulatory ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Background. The critical role of vascular health on brain function has received much attention in recent years. At the single-cell level, studies on the developmental processes of cerebral vascular growth are still relatively few. Techniques for constructing gene regulatory networks (GRNs) based on single-cell transcriptome expression data have made significant progress in recent years. Herein, we constructed a single-cell transcriptional regulatory network of mouse cerebrovascular cells. Methods. The single-cell RNA-seq dataset of mouse brain vessels was downloaded from GEO (GSE98816). This cell clustering was annotated separately using singleR and CellMarker. We then used a modified version of the SCENIC method to construct GRNs. Next, we used a mouse version of SEEK to assess whether genes in the regulon were coexpressed. Finally, regulatory module analysis was performed to complete the cell type relationship quantification. Results. Single-cell RNA-seq data were used to analyze the heterogeneity of mouse cerebrovascular cells, whereby four cell types including endothelial cells, fibroblasts, microglia, and oligodendrocytes were defined. These subpopulations of cells and marker genes together characterize the molecular profile of mouse cerebrovascular cells. Through these signatures, key transcriptional regulators that maintain cell identity were identified. Our findings identified genes like Lmo2, which play an important role in endothelial cells. The same cell type, for instance, fibroblasts, was found to have different regulatory networks, which may influence the functional characteristics of local tissues. Conclusions. In this study, a transcriptional regulatory network based on single-cell analysis was constructed. Additionally, the study identified and profiled mouse cerebrovascular cells using single-cell transcriptome data as well as defined TFs that affect the regulatory network of the mouse brain vasculature.

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TRACING CO-REGULATORY NETWORK DYNAMICS IN NOISY, SINGLE-CELL TRANSCRIPTOME TRAJECTORIES

Biocomputing 2017 ◽

10.1142/9789813207813_0053 ◽

2016 ◽

Cited By ~ 5

Author(s):

PABLO CORDERO ◽

JOSHUA M. STUART

Keyword(s):

Single Cell ◽

Regulatory Network ◽

Network Dynamics ◽

Cell Transcriptome ◽

Single Cell Transcriptome

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

10.1101/2020.04.10.022103 ◽

2020 ◽

Cited By ~ 9

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.

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Parallel Bimodal Single-cell Sequencing of Transcriptome and Chromatin Accessibility

10.1101/829960 ◽

2019 ◽

Cited By ~ 2

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.

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