scholarly journals Quantitative comparison of in vitro and in vivo embryogenesis at a single cell resolution

2021 ◽  
Author(s):  
Yuqi Tan ◽  
Abby Spangler ◽  
Michael Farid ◽  
Da Peng ◽  
Patrick Cahan
Keyword(s):  
Single Cell ◽  
Web Application ◽  
Primitive Streak ◽  
Cell Types ◽  
Analysis Tool ◽  
Primary Analysis ◽  
Data Set ◽  
Correct Path

Stem cell engineering is a powerful platform to address numerous challenges in regenerative medicine and developmental biology. Typically, engineered populations are derived by exposing pluripotent stem cells to a series of signaling cues meant to recapitulate developmental milestones, such as the induction of the primitive streak. Morphologic, genetic, transcriptomic, and functional differences between fully differentiated in vivo and engineered populations have long been recognized. However, the correspondence between engineered and native embryonic progenitors has been relatively less well characterized, largely due to their transient and highly heterogenous nature, and therefore many studies have relied on expression of a few canonical markers to ensure that their cells are on the correct path. To address this challenge, we first generated an in vivo gastrulation mouse single cell RNA sequencing (scRNA-seq) reference data set and used it to train a collection of computational tools for comparing cell types, states, regulators, and trajectories across studies. Then we used these tools to evaluate a commonly used protocol designed to promote mesoderm derivation, as well as four previously published biomimetic protocols. Finally, we diversified our toolkits to reach a broader scientific community by implementing our primary analysis tool in Python and as an interactive web application.

scholarly journals Cell type resolved co-expression networks of core clock genes in brain development

2021 ◽  
Author(s):  
Surbhi Sharma ◽  
Asgar Hussain Ansari ◽  
Soundhar Ramasamy
Keyword(s):  
Circadian Clock ◽  
Single Cell ◽  
Radial Glia ◽  
Clock Genes ◽  
Neuronal Cell ◽  
Cell Types ◽  
Developing Brain ◽  
Knock Out

AbstractThe circadian clock regulates vital cellular processes by adjusting the physiology of the organism to daily changes in the environment. Rhythmic transcription of core Clock Genes (CGs) and their targets regulate these processes at the cellular level. Circadian clock disruption has been observed in people with neurodegenerative disorders like Alzheimer’s and Parkinson’s. Also, ablation of CGs during development has been shown to affect neurogenesis in both in vivo and in vitro models. Previous studies on the function of CGs in the brain have used knock-out models of a few CGs. However, a complete catalog of CGs in different cell types of the developing brain is not available and it is also tedious to obtain. Recent advancements in single-cell RNA sequencing (scRNA-seq) has revealed novel cell types and elusive dynamic cell states of the developing brain. In this study by using publicly available single-cell transcriptome datasets we systematically explored CGs-coexpressing networks (CGs-CNs) during embryonic and adult neurogenesis. Our meta-analysis reveals CGs-CNs in human embryonic radial glia, neurons and also in lesser studied non-neuronal cell types of the developing brain.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

scholarly journals Single-Cell Techniques Using Chromosomally Tagged Fluorescent Bacteria To Study Listeria monocytogenes Infection Processes

2010 ◽  
Vol 76 (11) ◽  
pp. 3625-3636 ◽  
Author(s):  
Damien Balestrino ◽  
M�lanie Anne Hamon ◽  
Laurent Dortet ◽  
Marie-Anne Nahori ◽  
Javier Pizarro-Cerda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Listeria Monocytogenes ◽  
Single Cell ◽  
Fluorescent Proteins ◽  
Cell Types ◽  
Intracellular Replication ◽  
Infectious Process ◽  
Infection Processes

ABSTRACT Listeria monocytogenes is a Gram-positive facultative intracellular pathogen which invades different cell types, including nonphagocytic cells, where it is able to replicate and survive. The different steps of the cellular infectious process have been well described and consist of bacterial entry, lysis of the endocytic vacuole, intracellular replication, and spreading to neighboring cells. To study the listerial infectious process, gentamicin survival assays, plaque formation, and direct microscopy observations are typically used; however, there are some caveats with each of these techniques. In this study we describe new single-cell techniques based on use of an array of integrative fluorescent plasmids (green, cyan, and yellow fluorescent proteins) to easily, rapidly, and quantitatively detect L. monocytogenes in vitro and in vivo. We describe construction of 13 integrative and multicopy plasmids which can be used for detecting intracellular bacteria, for measuring invasion, cell-to-cell spreading, and intracellular replication, for monitoring in vivo infections, and for generating transcriptional or translational reporters. Furthermore, we tested these plasmids in a variety of epifluorescence- and flow cytometry-based assays. We showed that we could (i) determine the expression of a particular promoter during the cell cycle, (ii) establish in one rapid experiment at which step in the cell cycle a particular mutant is defective, and (iii) easily measure the number of infected cells in vitro and in mouse organs. The plasmids that are described and the methods to detect them are new powerful tools to study host-Listeria interactions in a fast, robust, and high-throughput manner.

scholarly journals A simple, scalable approach to building a cross-platform transcriptome atlas

2020 ◽  
Author(s):  
Paul W Angel ◽  
Nadia Rajab ◽  
Yidi Deng ◽  
Chris M Pacheco ◽  
Tyrone Chen ◽  
...  
Keyword(s):  
Blood Cell ◽  
Single Cell ◽  
New Technologies ◽  
New Technology ◽  
Cell Types ◽  
Variance Partitioning ◽  
Primary Data ◽  
Batch Correction

ABSTRACTGene expression atlases have transformed our understanding of the development, composition and function of human tissues. New technologies promise improved cellular or molecular resolution, and have led to the identification of new cell types, or better defined cell states. But as new technologies emerge, information derived on old platforms becomes obsolete. We demonstrate that it is possible to combine a large number of different profiling experiments summarised from dozens of laboratories and representing hundreds of donors, to create an integrated molecular map of human tissue. As an example, we combine 850 samples from 38 platforms to build an integrated atlas of human blood cells. We achieve robust and unbiased cell type clustering using a variance partitioning method, selecting genes with low platform bias relative to biological variation. Other than an initial rescaling, no other transformation to the primary data is applied through batch correction or renormalisation. Additional data, including single-cell datasets, can be projected for comparison, classification and annotation. The resulting atlas provides a multi-scaled approach to visualise and analyse the relationships between sets of genes and blood cell lineages, including the maturation and activation of leukocytes in vivo and in vitro.In allowing for data integration across hundreds of studies, we address a key reproduciblity challenge which is faced by any new technology. This allows us to draw on the deep phenotypes and functional annotations that accompany traditional profiling methods, and provide important context to the high cellular resolution of single cell profiling. Here, we have implemented the blood atlas in the open access Stemformatics.org platform, drawing on its extensive collection of curated transcriptome data. The method is simple, scalable and amenable for rapid deployment in other biological systems or computational workflows.Graphical abstractRecursive approach to generating a multi-scaled atlas. Top panel: The method integrates data from all cell types in the Stemformatics database, and shows clear division of samples into global categories of stromal, pluripotent or blood (inset) cell types. Bottom panel: Integration of only the blood cell subsets provides a blood atlas. Projection of external samples (green) onto the blood atlas. Samples are coloured by curated annotations derived from the original studies, and can be viewed at Stemformatics.org

scholarly journals Single-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids

2019 ◽  
Author(s):  
Lisa M. Smits ◽  
Stefano Magni ◽  
Kamil Grzyb ◽  
Paul MA. Antony ◽  
Rejko Krüger ◽  
...  
Keyword(s):  
Single Cell ◽  
Dopaminergic Neurons ◽  
Neuronal Cell ◽  
Cell Types ◽  
Human Stem Cell ◽  
Glutamatergic Neurons ◽  
And Function ◽  
Excellent Tool

AbstractHuman stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulatein vitrothe organisation and function of a respective organ or part of an organ. Human midbrain organoids (hMOs) have been described to contain midbrain-specific dopaminergic neurons that release the neurotransmitter dopamine. However, the human midbrain contains also additional neuronal cell types, which are functionally interacting with each other. Here, we analysed hMOs at high-resolution by means of single-cell RNA-sequencing (scRNA-seq), imaging and electrophysiology to unravel cell heterogeneity. Our findings demonstrate that hMOs show essential neuronal functional properties as spontaneous electrophysiological activity of different neuronal subtypes, including dopaminergic, GABAergic, and glutamatergic neurons. Recapitulating thesein vivofeatures makes hMOs an excellent tool forin vitrodisease phenotyping and drug discovery.

scholarly journals Geometric Sketching Compactly Summarizes the Single-Cell Transcriptomic Landscape

2019 ◽  
Author(s):  
Brian Hie ◽  
Hyunghoon Cho ◽  
Benjamin DeMeo ◽  
Bryan Bryson ◽  
Bonnie Berger
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
High Sensitivity ◽  
Cell Types ◽  
Small Subset ◽  
Data Set ◽  
Cord Blood Cells ◽  
Umbilical Cord Blood Cells ◽  
Inflammatory Macrophages

SUMMARYLarge-scale single-cell RNA-sequencing (scRNA-seq) studies that profile hundreds of thousands of cells are becoming increasingly common, overwhelming existing analysis pipelines. Here, we describe how to enhance and accelerate single-cell data analysis by summarizing the transcriptomic heterogeneity within a data set using a small subset of cells, which we refer to as a geometric sketch. Our sketches provide more comprehensive visualization of transcriptional diversity, capture rare cell types with high sensitivity, and accurately reveal biological cell types via clustering. Our sketch of umbilical cord blood cells uncovers a rare subpopulation of inflammatory macrophages, which we experimentally validatedin vitro. The construction of our sketches is extremely fast, which enabled us to accelerate other crucial resource-intensive tasks such as scRNA-seq data integration. We anticipate that our algorithm will become an increasingly essential step when sharing and analyzing the rapidly-growing volume of scRNA-seq data and help enable the democratization of single-cell omics.

scholarly journals Human cerebral organoids recapitulate gene expression programs of fetal neocortex development

2015 ◽  
Vol 112 (51) ◽  
pp. 15672-15677 ◽  
Author(s):  
J. Gray Camp ◽  
Farhath Badsha ◽  
Marta Florio ◽  
Sabina Kanton ◽  
Tobias Gerber ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Neuronal Differentiation ◽  
Cortical Development ◽  
Cell Types ◽  
Fetal Tissue ◽  
Neural Progenitor Cell ◽  
Neural Progenitor

Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

scholarly journals Assessing the Heterogeneity of Cardiac Non-myocytes and the Effect of Cell Culture with Integrative Single Cell Analysis

2020 ◽  
Author(s):  
Brian S. Iskra ◽  
Logan Davis ◽  
Henry E. Miller ◽  
Yu-Chiao Chiu ◽  
Alexander R. Bishop ◽  
...  
Keyword(s):  
Cell Culture ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Cell Types ◽  
Cell Analysis ◽  
Mass Cytometry ◽  
Single Cell Rna Sequencing ◽  
High Depth

AbstractCardiac non-myocytes comprise a diverse and crucial cell population in the heart that plays dynamic roles in cardiac wound healing and growth. Non-myocytes broadly fall into four cell types: endothelium, fibroblasts, leukocytes, and pericytes. Here we characterize the diversity of the non-myocytes in vivo and in vitro using mass cytometry. By leveraging single-cell RNA sequencing we inform the design of a mass cytometry panel. To aid in annotation of the mass cytometry datasets, we utilize data integration with a neural network. We introduce approximately 460,000∼ single cell proteomes of non-myocytes as well as 5,000∼ CD31 negative single cell transcriptomes. Using our data, as well as previously reported datasets, we characterize cardiac non-myocytes with high depth in six mice, characterizing novel surface markers (CD9, CD200, Notch3, and FolR2). Further, we find that extended cell culture promotes the proliferation of CD45+CD11b+FolR2+IAIE- myeloid cells in addition to fibroblasts.

scholarly journals A single-cell resolution developmental atlas of hematopoietic stem and progenitor cell expansion in zebrafish

2021 ◽  
Vol 118 (14) ◽  
pp. e2015748118
Author(s):  
Jun Xia ◽  
Zhixin Kang ◽  
Yuanyuan Xue ◽  
Yanyan Ding ◽  
Suwei Gao ◽  
...  
Keyword(s):  
Single Cell ◽  
Regulatory Mechanism ◽  
Fetal Liver ◽  
Cell Types ◽  
Hematopoietic Tissue ◽  
Hematopoietic Stem ◽  
Lineage Priming ◽  
Hematopoietic Niche

During vertebrate embryogenesis, fetal hematopoietic stem and progenitor cells (HSPCs) exhibit expansion and differentiation properties in a supportive hematopoietic niche. To profile the developmental landscape of fetal HSPCs and their local niche, here, using single-cell RNA-sequencing, we deciphered a dynamic atlas covering 28,777 cells and 9 major cell types (23 clusters) of zebrafish caudal hematopoietic tissue (CHT). We characterized four heterogeneous HSPCs with distinct lineage priming and metabolic gene signatures. Furthermore, we investigated the regulatory mechanism of CHT niche components for HSPC development, with a focus on the transcription factors and ligand–receptor networks involved in HSPC expansion. Importantly, we identified an endothelial cell-specific G protein–coupled receptor 182, followed by in vivo and in vitro functional validation of its evolutionally conserved role in supporting HSPC expansion in zebrafish and mice. Finally, comparison between zebrafish CHT and human fetal liver highlighted the conservation and divergence across evolution. These findings enhance our understanding of the regulatory mechanism underlying hematopoietic niche for HSPC expansion in vivo and provide insights into improving protocols for HSPC expansion in vitro.

scholarly journals Single-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids

2020 ◽  
Vol 382 (3) ◽  
pp. 463-476 ◽  
Author(s):  
Lisa M. Smits ◽  
Stefano Magni ◽  
Kaoru Kinugawa ◽  
Kamil Grzyb ◽  
Joachim Luginbühl ◽  
...  
Keyword(s):  
Single Cell ◽  
Dopaminergic Neurons ◽  
Neuronal Cell ◽  
Cell Types ◽  
Human Stem Cell ◽  
Electrophysiological Activity ◽  
And Function ◽  
Excellent Tool

AbstractHuman stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organization and function of a respective organ or part of an organ. Human midbrain organoids (hMOs) have been described to contain midbrain-specific dopaminergic neurons that release the neurotransmitter dopamine. However, the human midbrain contains also additional neuronal cell types, which are functionally interacting with each other. Here, we analysed hMOs at high-resolution by means of single-cell RNA sequencing (scRNA-seq), imaging and electrophysiology to unravel cell heterogeneity. Our findings demonstrate that hMOs show essential neuronal functional properties as spontaneous electrophysiological activity of different neuronal subtypes, including dopaminergic, GABAergic, glutamatergic and serotonergic neurons. Recapitulating these in vivo features makes hMOs an excellent tool for in vitro disease phenotyping and drug discovery.

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