Creating Lineage Trajectory Maps Via Integration of Single-Cell RNA-Sequencing and Lineage Tracing

A subset of adult cardiac resident cells defined by the stem cell factor tyrosine kinase receptor termed c-kit, are believed to have myogenic potential and are now being delivered via intracoronary infusion to presumably promote cardiac regeneration and improve ventricular function after ischemic cardiac injury. However, recent studies have shown that despite these benefits, c-kit+ progenitor cells in the adult murine heart are more inclined to take on an endothelial rather than cardiomyocyte lineage. To better define the factors involved in early differentiation of these resident cardiac progenitor cells and to distinguish distinct cell subpopulations, we performed single cell RNA sequencing on c-kit+ cells from Kit-Cre lineage traced GFP reporter mice versus total mesenchymal cells from the heart that were CD31- and CD45-. Cells were isolated by cardiac digestion and FACS was performed, positively sorting for the c-kit+ lineage while negatively sorting for CD31 and CD45 to eliminate endothelial and leukocyte progenitor contamination, respectively. Following this isolation, cells were examined to determine GFP reporter status and then submitted for single cell RNA sequencing using the Fluidigm A1 system. Clustering of 654 genes from this data identified 6 distinct subpopulations indicating various stages of early differentiation among CD31- and CD45-negative cardiac interstitial cells. Furthermore, comparison of GFP+ c-kit cells to the total non-GFP mesenchymal cells identified 75 differentially expressed transcripts. These unique gene signatures may help parse the genes that underlie cellular plasticity in the heart and define the best molecular lineages for transdifferentiation into cardiac myocytes.

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A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages

European Respiratory Journal ◽

10.1183/13993003.00646-2019 ◽

2019 ◽

Vol 55 (1) ◽

pp. 1900646 ◽

Cited By ~ 30

Author(s):

Nikita Joshi ◽

Satoshi Watanabe ◽

Rohan Verma ◽

Renea P. Jablonski ◽

Ching-I Chen ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Single Cell ◽

Rna Sequencing ◽

Single Molecule ◽

Alveolar Macrophages ◽

Lineage Tracing ◽

Single Cell Rna Sequencing ◽

Pharmacological Blockade ◽

Macrophage Colony ◽

Druggable Targets

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

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Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis

Circulation ◽

10.1161/circulationaha.120.046672 ◽

2020 ◽

Vol 142 (21) ◽

pp. 2045-2059 ◽

Cited By ~ 8

Author(s):

Gabriel F. Alencar ◽

Katherine M. Owsiany ◽

Santosh Karnewar ◽

Katyayani Sukhavasi ◽

Giuseppe Mocci ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Late Stage ◽

Lineage Tracing ◽

Stem Cell Marker ◽

Cell Phenotype ◽

Phenotypic Changes ◽

Atherosclerotic Lesions ◽

Single Cell Rna Sequencing

Background: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. Methods: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. Results: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11 - , Lgals3 + population with a chondrocyte-like gene signature that was markedly reduced with SMC- Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene–positive, extracellular matrix-remodeling, “pioneer” cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Conclusions: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3 + osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

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Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells

10.1101/2020.06.19.160424 ◽

2020 ◽

Author(s):

Kristen L. Wells ◽

Corey N. Miller ◽

Andreas R. Gschwind ◽

Wu Wei ◽

Jonah D. Phipps ◽

...

Keyword(s):

Epithelial Cells ◽

Single Cell ◽

Rna Sequencing ◽

Expression Patterns ◽

Critical Role ◽

Lineage Tracing ◽

Thymic Epithelial Cells ◽

Single Cell Rna Sequencing ◽

Medullary Thymic Epithelial Cells

AbstractMedullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene expression patterns within the mTEC compartment are remarkably heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single cell RNA-sequencing. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. Based on our findings, we propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a higher order roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

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Cellular basis of omentum activation and expansion revealed by single-cell RNA sequencing using a parabiosis model

Scientific Reports ◽

10.1038/s41598-021-93330-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kazuhiko Ishigaki ◽

Keiki Kumano ◽

Kyohei Fujita ◽

Hiroo Ueno

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Immune Cells ◽

Physiological Function ◽

Cell Types ◽

Lineage Tracing ◽

Mesenchymal Progenitors ◽

Fluorescent Markers ◽

Circulating Cells ◽

Single Cell Rna Sequencing

AbstractAlthough the physiological function of the omentum remains elusive, it has been proposed that it plays an important role in fat storage, immune regulation, and regeneration of injured tissues and organs. Although the omentum undergoes expansion upon activation, reports on the accurate assessment of increased cell types and the origin of the increased cells remain limited. To investigate this aspect, the omenta of parabiotic mice were subjected to activation using distinct fluorescent markers and single-cell RNA sequencing (scRNA-seq) was performed to identify circulation-derived omental cells. We found that a considerable number of circulating cells contributed to the activation of the omentum. The omental cells derived from circulating cells exhibited morphological features similar to those of fibroblasts. scRNA-seq revealed the existence of a novel cell population that co-expressed macrophage and fibroblast markers in the activated omentum, suggesting that it corresponded to circulating macrophage-derived fibroblast-like cells. Lineage tracing experiments revealed that most fibroblasts in the activated omentum were not derived from WT1-positive mesenchymal progenitors. The cell cluster also expressed various chemokine genes, indicating its role in the activation and recruitment of immune cells. These results provide important information regarding the activation of the omentum.

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Overcoming Genetic Drop-outs in Variants-based Lineage Tracing from Single-cell RNA Sequencing Data

10.1101/2020.01.01.892562 ◽

2020 ◽

Author(s):

Tianshi Lu ◽

Seongoh Park ◽

James Zhu ◽

Xiaowei Zhan ◽

Xinlei Wang ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Developmental Process ◽

Variant Calling ◽

Lineage Tracing ◽

Whole Genome ◽

Sequencing Data ◽

Bayesian Hierarchical ◽

Single Cell Rna Sequencing ◽

Drop Outs

ABSTRACTLineage tracing provides key insights into the fates of individual cells in complex tissues. Recent works on lineage reconstruction based on the single-cell expression data are suitable for short time frames while tracing lineage based on more stable genetic markers is needed for studies that span time scales over months or years. However, variant calling from the single-cell RNA sequencing (scRNA-Seq) data suffers from “genetic drop-outs”, including low coverage and allelic bias, which presents significant obstacles for lineage reconstruction. Prior studies focused only on mitochondrial (chrM) variants and need to be expanded to the whole genome to capture more variants with clearer physiological meaning. However, non-chrM variants suffer even more severe drop-outs than chrM variants, although drop-outs affect all variants. We developed strategies to overcome genetic drop-outs in scRNA-Seq-derived whole genomic variants for accurate lineage tracing, and we developed SClineger, a Bayesian Hierarchical model, to implement our approach. Our validation analyses on a series of sequencing protocols demonstrated the necessity of correction for genetic drop-outs and consideration of variants in the whole genome, and also showed the improvement that our approach provided. We showed that genetic-based lineage tracing is applicable for single-cell studies of both tumors and non-tumor tissues using our approach, and can reveal novel biological insights not afforded by expressional analyses. Interestingly, we showed that cells of various lineages grew under the spatial constraints of their respective organs during the developmental process. Overall, our work provides a powerful tool that can be applied to the large amounts of already existing scRNA-Seq data to construct the lineage histories of cells and derive new knowledge.

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Rainbow-Seq: Combining Cell Lineage Tracing with Single-Cell RNA Sequencing in Preimplantation Embryos

iScience ◽

10.1016/j.isci.2018.08.009 ◽

2018 ◽

Vol 7 ◽

pp. 16-29 ◽

Cited By ~ 3

Author(s):

Fernando H. Biase ◽

Qiuyang Wu ◽

Riccardo Calandrelli ◽

Marcelo Rivas-Astroza ◽

Shuigeng Zhou ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Cell Lineage ◽

Lineage Tracing ◽

Preimplantation Embryos ◽

Single Cell Rna Sequencing

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Sex-Stratified Gene Regulatory Networks Reveal Female Key Driver Genes of Atherosclerosis Involved in Smooth Muscle Cell Phenotype Switching

Circulation ◽

10.1161/circulationaha.120.051231 ◽

2021 ◽

Vol 143 (7) ◽

pp. 713-726 ◽

Cited By ~ 1

Author(s):

Robin J.G. Hartman ◽

Katie Owsiany ◽

Lijiang Ma ◽

Simon Koplev ◽

Ke Hao ◽

...

Keyword(s):

Coronary Artery Disease ◽

Sex Differences ◽

Coronary Artery ◽

Single Cell ◽

Rna Sequencing ◽

Regulatory Networks ◽

Lineage Tracing ◽

Single Cell Rna Sequencing ◽

Key Drivers ◽

Artery Disease

Background: Although sex differences in coronary artery disease are widely accepted with women developing more stable atherosclerosis than men, the underlying pathobiology of such differences remains largely unknown. In coronary artery disease, recent integrative systems biological studies have inferred gene regulatory networks (GRNs). Within these GRNs, key driver genes have shown great promise but have thus far been unidentified in women. Methods: We generated sex-specific GRNs of the atherosclerotic arterial wall in 160 women and age-matched men in the STARNET study (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task). We integrated the female GRNs with single-cell RNA-sequencing data of the human atherosclerotic plaque and single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic smooth muscle cell (SMC) lineage-tracing mice. Results: By comparing sex-specific GRNs, we observed clear sex differences in network activity within the atherosclerotic tissues. Genes more active in women were associated with mesenchymal cells and endothelial cells, whereas genes more active in men were associated with the immune system. We determined that key drivers of GRNs active in female coronary artery disease were predominantly found in (SMCs by single-cell sequencing of the human atherosclerotic plaques, and higher expressed in female plaque SMCs, as well. To study the functions of these female SMC key drivers in atherosclerosis, we examined single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic SMC lineage-tracing mice. The female key drivers were found to be expressed by phenotypically modulated SMCs and affected by Klf4, suggesting that sex differences in atherosclerosis involve phenotypic switching of plaque SMCs. Conclusions: Our systems approach provides novel insights into molecular mechanisms that underlie sex differences in atherosclerosis. To discover sex-specific therapeutic targets for atherosclerosis, an increased emphasis on sex-stratified approaches in the analysis of multi-omics data sets is warranted.

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Lineage analysis reveals an endodermal contribution to the vertebrate pituitary

Science ◽

10.1126/science.aba4767 ◽

2020 ◽

Vol 370 (6515) ◽

pp. 463-467 ◽

Cited By ~ 1

Author(s):

Peter Fabian ◽

Kuo-Chang Tseng ◽

Joanna Smeeton ◽

Joseph J. Lancman ◽

P. Duc Si Dong ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Endocrine Cell ◽

Time Lapse ◽

Cell Types ◽

Lineage Tracing ◽

Single Cell Rna Sequencing ◽

Growth Metabolism ◽

Time Lapse Imaging ◽

Lineage Analysis

Vertebrate sensory organs arise from epithelial thickenings called placodes. Along with neural crest cells, cranial placodes are considered ectodermal novelties that drove evolution of the vertebrate head. The anterior-most placode generates the endocrine lobe [adenohypophysis (ADH)] of the pituitary, a master gland controlling growth, metabolism, and reproduction. In addition to known ectodermal contributions, we use lineage tracing and time-lapse imaging in zebrafish to identify an endodermal contribution to the ADH. Single-cell RNA sequencing of the adult pituitary reveals similar competency of endodermal and ectodermal epithelia to generate all endocrine cell types. Further, endoderm can generate a rudimentary ADH-like structure in the near absence of ectodermal contributions. The fish condition supports the vertebrate pituitary arising through interactions of an ancestral endoderm-derived proto-pituitary with newly evolved placodal ectoderm.

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Combined transient ablation and single-cell RNA-sequencing reveals the development of medullary thymic epithelial cells

eLife ◽

10.7554/elife.60188 ◽

2020 ◽

Vol 9 ◽

Author(s):

Kristen L Wells ◽

Corey N Miller ◽

Andreas R Gschwind ◽

Wu Wei ◽

Jonah D Phipps ◽

...

Keyword(s):

Epithelial Cells ◽

Single Cell ◽

Rna Sequencing ◽

Critical Role ◽

Lineage Tracing ◽

Thymic Epithelial Cells ◽

In Vivo Models ◽

Single Cell Rna Sequencing ◽

Medullary Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene-expression patterns within the mTEC compartment are heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single-cell RNA-sequencing in Mus musculus. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. We propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

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