scholarly journals Massively parallel clonal analysis using CRISPR/Cas9 induced genetic scars

2016 ◽  
Author(s):  
Jan Philipp Junker ◽  
Bastiaan Spanjaard ◽  
Josi Peterson-Maduro ◽  
Anna Alemany ◽  
Bo Hu ◽  
...  
Keyword(s):  
Developmental Biology ◽  
Single Cell ◽  
Clonal Analysis ◽  
Lineage Tracing ◽  
Massively Parallel ◽  
Vertebrate Lineage ◽  
Lineage Trees ◽  
Made In

A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many cells. Although impressive progress has been made in lineage tracing using imaging approaches, analysis of vertebrate lineage trees has mostly been limited to relatively small subsets of cells. Here we present scartrace, a strategy for massively parallel clonal analysis based on Cas9 induced genetic scars in the zebrafish.

scholarly journals Massively parallel single cell lineage tracing using CRISPR/Cas9 induced genetic scars

2017 ◽  
Author(s):  
Bastiaan Spanjaard ◽  
Bo Hu ◽  
Nina Mitic ◽  
Jan Philipp Junker
Keyword(s):  
Single Cell ◽  
Computational Analysis ◽  
Systematic Approach ◽  
Single Cells ◽  
Cell Lineage ◽  
Transcriptome Profiling ◽  
Cell Types ◽  
Lineage Tracing ◽  
Lineage Trees ◽  
Different Cell Types

A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many different cell types. Single-cell RNA-sequencing (scRNA-seq) has become a widely-used method due to its ability to identify all cell types in a tissue or organ in a systematic manner 1–3. However, a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here, we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses, LINNAEUS (LINeage tracing by Nuclease-Activated Editing of Ubiquitous Sequences) can be used as a systematic approach for identifying the lineage origin of novel cell types, or of known cell types under different conditions.

scholarly journals Inference of Single-Cell Phylogenies from Lineage Tracing Data

2019 ◽  
Author(s):  
Matthew G. Jones ◽  
Alex Khodaverdian ◽  
Jeffrey J. Quinn ◽  
Michelle M. Chan ◽  
Jeffrey A. Hussmann ◽  
...  
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
Gene Editing ◽  
Lineage Tracing ◽  
Massively Parallel ◽  
Simulation Framework ◽  
Tree Reconstruction ◽  
Cassiopeia A ◽  
Mammalian Lineage ◽  
Insight Into

AbstractThe pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. To address this, we provide three resources. First, we introduce Cassiopeia - a suite of scalable and theoretically grounded maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date - consisting of 34,557 human cells continuously traced over 15 generations, 71% of which are uniquely marked - and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together these should broadly enable large-scale mammalian lineage tracing efforts. Cassiopeia and its benchmarking resources are publicly available at www.github.com/YosefLab/Cassiopeia.

scholarly journals Clonal analysis identifies hemogenic endothelium as the source of the blood-endothelial common lineage in the mouse embryo

Blood ◽  
2014 ◽  
Vol 124 (16) ◽  
pp. 2523-2532 ◽  
Author(s):  
Laura Padrón-Barthe ◽  
Susana Temiño ◽  
Cristina Villa del Campo ◽  
Laura Carramolino ◽  
Joan Isern ◽  
...  
Keyword(s):  
Single Cell ◽  
Mouse Embryo ◽  
Cell Lineage ◽  
Clonal Analysis ◽  
Lineage Tracing ◽  
Hemogenic Endothelium ◽  
Early Separation ◽  
Key Points ◽  
Endothelial Precursors

Key Points Single cell lineage tracing shows early separation of blood-endothelial precursors in the mouse embryo. Hemogenic endothelium in the YS generates the blood-endothelial common lineage and produces definitive precursors.

scholarly journals Clonal analysis and dynamic imaging identify multipotency of individual Gallus gallus caudal hindbrain neural crest cells toward cardiac and enteric fates

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weiyi Tang ◽  
Yuwei Li ◽  
Ang Li ◽  
Marianne E. Bronner
Keyword(s):  
Neural Crest ◽  
Single Cell ◽  
Cell Fate ◽  
Neural Crest Cells ◽  
Time Lapse ◽  
Cell Types ◽  
Clonal Analysis ◽  
Dynamic Imaging ◽  
Lineage Tracing ◽  
Enteric Ganglia

AbstractNeural crest stem cells arising from caudal hindbrain (often called cardiac and posterior vagal neural crest) migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects. Here, we explore whether individual caudal hindbrain neural crest precursors are multipotent or predetermined toward these particular fates and destinations. To this end, we perform lineage tracing of chick neural crest cells at single-cell resolution using two complementary approaches: retrovirally mediated multiplex clonal analysis and single-cell photoconversion. Both methods show that the majority of these neural crest precursors are multipotent with many clones producing mesenchymal as well as neuronal derivatives. Time-lapse imaging demonstrates that sister cells can migrate in distinct directions, suggesting stochasticity in choice of migration path. Perturbation experiments further identify guidance cues acting on cells in the pharyngeal junction that can influence this choice; loss ofCXCR4signaling results in failure to migrate to the heart but no influence on migration toward the foregut, whereas loss ofRETsignaling does the opposite. Taken together, the results suggest that environmental influences rather than intrinsic information govern cell fate choice of multipotent caudal hindbrain neural crest cells.

scholarly journals Sequential fate-switches in stem-like cells drive the tumorigenic trajectory from human neural stem cells to malignant glioma

Cell Research ◽  
2021 ◽  
Author(s):  
Xiaofei Wang ◽  
Ran Zhou ◽  
Yanzhen Xiong ◽  
Lingling Zhou ◽  
Xiang Yan ◽  
...  
Keyword(s):  
Malignant Glioma ◽  
Single Cell ◽  
De Novo ◽  
Early Stage ◽  
Lineage Tracing ◽  
Human Neural Stem Cells ◽  
Transcriptional Reprogramming ◽  
Neural Stem Progenitor Cells ◽  
Human Gbm ◽  
End Stage

AbstractGlioblastoma (GBM) is an incurable and highly heterogeneous brain tumor, originating from human neural stem/progenitor cells (hNSCs/hNPCs) years ahead of diagnosis. Despite extensive efforts to characterize hNSCs and end-stage GBM at bulk and single-cell levels, the de novo gliomagenic path from hNSCs is largely unknown due to technical difficulties in early-stage sampling and preclinical modeling. Here, we established two highly penetrant hNSC-derived malignant glioma models, which resemble the histopathology and transcriptional heterogeneity of human GBM. Integrating time-series analyses of whole-exome sequencing, bulk and single-cell RNA-seq, we reconstructed gliomagenic trajectories, and identified a persistent NSC-like population at all stages of tumorigenesis. Through trajectory analyses and lineage tracing, we showed that tumor progression is primarily driven by multi-step transcriptional reprogramming and fate-switches in the NSC-like cells, which sequentially generate malignant heterogeneity and induce tumor phenotype transitions. We further uncovered stage-specific oncogenic cascades, and among the candidate genes we functionally validated C1QL1 as a new glioma-promoting factor. Importantly, the neurogenic-to-gliogenic switch in NSC-like cells marks an early stage characterized by a burst of oncogenic alterations, during which transient AP-1 inhibition is sufficient to inhibit gliomagenesis. Together, our results reveal previously undercharacterized molecular dynamics and fate choices driving de novo gliomagenesis from hNSCs, and provide a blueprint for potential early-stage treatment/diagnosis for GBM.

A microfluidic device enabling deterministic single cell trapping and release

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Huichao Chai ◽  
Yongxiang Feng ◽  
Fei Liang ◽  
Wenhui Wang
Keyword(s):  
Chemical Analysis ◽  
Single Cell ◽  
Microfluidic Device ◽  
Single Cells ◽  
Cell Isolation ◽  
Cell Trapping ◽  
Analysis Techniques ◽  
Single Cell Trapping ◽  
Single Cell Isolation ◽  
Made In

Successful single-cell isolation is a pivotal technique for subsequent biological and chemical analysis of single cells. Although significant advances have been made in single-cell isolation and analysis techniques, most passive...

scholarly journals MEDALT: single-cell copy number lineage tracing enabling gene discovery

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Fang Wang ◽  
Qihan Wang ◽  
Vakul Mohanty ◽  
Shaoheng Liang ◽  
Jinzhuang Dou ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Single Cell ◽  
Copy Number ◽  
Speciation Analysis ◽  
Population Based ◽  
Lineage Tracing ◽  
Breast Cancer Patients ◽  
Lineage Tree ◽  
History Of ◽  
A Cell

AbstractWe present a Minimal Event Distance Aneuploidy Lineage Tree (MEDALT) algorithm that infers the evolution history of a cell population based on single-cell copy number (SCCN) profiles, and a statistical routine named lineage speciation analysis (LSA), whichty facilitates discovery of fitness-associated alterations and genes from SCCN lineage trees. MEDALT appears more accurate than phylogenetics approaches in reconstructing copy number lineage. From data from 20 triple-negative breast cancer patients, our approaches effectively prioritize genes that are essential for breast cancer cell fitness and predict patient survival, including those implicating convergent evolution.The source code of our study is available at https://github.com/KChen-lab/MEDALT.

scholarly journals EPEN-22. SINGLE-CELL RNA SEQUENCING IDENTIFIES UPREGULATION OF IKZF1 IN PFA2 MYELOID SUBPOPULATION DRIVING AN ANTI-TUMOR PHENOTYPE

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii312-iii312
Author(s):  
Andrea Griesinger ◽  
Eric Prince ◽  
Andrew Donson ◽  
Kent Riemondy ◽  
Timothy Ritzman ◽  
...  
Keyword(s):  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
Expression Profiles ◽  
Myeloid Cells ◽  
Cell Subset ◽  
Lineage Tracing ◽  
Machine Learning Techniques ◽  
Cell Subpopulation ◽  
Immune Gene

Abstract We have previously shown immune gene phenotype variations between posterior fossa ependymoma subgroups. PFA1 tumors chronically secrete IL-6, which pushes the infiltrating myeloid cells to an immune suppressive function. In contrast, PFA2 tumors have a more immune activated phenotype and have a better prognosis. The objective of this study was to use single-cell(sc) RNAseq to descriptively characterize the infiltrating myeloid cells. We analyzed approximately 8500 cells from 21 PFA patient samples and used advanced machine learning techniques to identify distinct myeloid and lymphoid subpopulations. The myeloid compartment was difficult to interrupt as the data shows a continuum of gene expression profiles exist within PFA1 and PFA2. Through lineage tracing, we were able to tease out that PFA2 myeloid cells expressed more genes associated with an anti-viral response (MHC II, TNF-a, interferon-gamma signaling); while PFA1 myeloid cells had genes associated with an immune suppressive phenotype (angiogenesis, wound healing, IL-10). Specifically, we found expression of IKZF1 was upregulated in PFA2 myeloid cells. IKZF1 regulates differentiation of myeloid cells toward M1 or M2 phenotype through upregulation of either IRF5 or IRF4 respectively. IRF5 expression correlated with IKZF1, being predominately expressed in the PFA2 myeloid cell subset. IKZF1 is also involved in T-cell activation. While we have not completed our characterization of the T-cell subpopulation, we did find significantly more T-cell infiltration in PFA2 than PFA1. Moving forward these studies will provide us with valuable information regarding the molecular switches involved in the tumor-immune microenvironment and to better develop immunotherapy for PFA ependymoma.

scholarly journals Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lars Velten ◽  
Benjamin A. Story ◽  
Pablo Hernández-Malmierca ◽  
Simon Raffel ◽  
Daniel R. Leonce ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Single Cell ◽  
Lineage Tracing ◽  
Specific Gene ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Mutational Status

AbstractCancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

Abstract 305: Single Cell RNA Sequencing of Adult Cardiac c-kit+ Cells in a Murine Lineage Tracing Model

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Bryan D Maliken ◽  
Onur Kanisicak ◽  
Jeffery D Molkentin
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Rna Sequencing ◽  
Cardiac Injury ◽  
Mesenchymal Cells ◽  
Lineage Tracing ◽  
Tyrosine Kinase Receptor ◽  
Early Differentiation ◽  
Gfp Reporter ◽  
Single Cell Rna Sequencing

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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