A rapid co-culture stamping device for studying intercellular communication.

Regulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists. We report here development of a temporarily sealed microfluidic stamping device which utilizes a novel valve design for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell interactions. We demonstrate post-stamping cell viability of >95%, the stamping of multiple adherent cell types, and the ability to control the seeded cell density. We also show viability, proliferation and migration of cultured cells, enabling analysis of co-culture boundary conditions on cell fate. We also developed an in-vitro model of endothelial and cardiac stem cell interactions, which are thought to regulate coronary repair after myocardial injury. The stamp is fabricated using microfabrication techniques, is operated with a lab pipettor and uses very low reagent volumes of 20 μl with cell injection efficiency of >70%. This easy-to-use device provides a general strategy for micro-patterning of multiple cell types and will be important for studying cell-cell interactions in a multitude of applications

Benchmark and Parameter Sensitivity Analysis of Single-Cell RNA Sequencing Clustering Methods

Single-cell RNA-seq (scRNAseq) is a powerful tool to study heterogeneity of cells. Recently, several clustering based methods have been proposed to identify distinct cell populations. These methods are based on different statistical models and usually require to perform several additional steps, such as preprocessing or dimension reduction, before applying the clustering algorithm. Individual steps are often controlled by method-specific parameters, permitting the method to be used in different modes on the same datasets, depending on the user choices. The large number of possibilities that these methods provide can intimidate non-expert users, since the available choices are not always clearly documented. In addition, to date, no large studies have invistigated the role and the impact that these choices can have in different experimental contexts. This work aims to provide new insights into the advantages and drawbacks of scRNAseq clustering methods and describe the ranges of possibilities that are offered to users. In particular, we provide an extensive evaluation of several methods with respect to different modes of usage and parameter settings by applying them to real and simulated datasets that vary in terms of dimensionality, number of cell populations or levels of noise. Remarkably, the results presented here show that great variability in the performance of the models is strongly attributed to the choice of the user-specific parameter settings. We describe several tendencies in the performance attributed to their modes of usage and different types of datasets, and identify which methods are strongly affected by data dimensionality in terms of computational time. Finally, we highlight some open challenges in scRNAseq data clustering, such as those related to the identification of the number of clusters

Single-cell transcriptomics reveals involution mimicry during the specification of the basal breast cancer subtype

Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.</p

Single cell analysis of adult cardiac stromal cells

The mammalian adult heart is comprised of multiple cell types which interact with each other to maintain structure and function in homeostasis and disease. Extensive characterisation of such populations is therefore crucial to understanding how these cells can be manipulated for better injury resolution and repair. It is hypothesised that a subset of cardiac cells labelled as SCA1+PDGFR?+CD31- cells (S+P+) which reside in the cardiac interstitium are dedicated to cardiac homeostasis and regeneration. They may also support cardiomyocytes and vascular tissue through dedicated paracrine functions. To investigate the activation of these cells during initial stages of inflammation after myocardial infarction (MI), the gene expression (GE) profiles of more than 30,000 cells were analysed following induction of MI by coronary artery ligation. High throughput single cell RNA-seq (scRNAseq) was performed using GFP+CD31- and total interstitial cells (TIP) isolated from a mouse with a H2B-eGFP fusion gene knocked-in to the PDGFR? locus. In-depth bioinformatic analysis including clustering and diffusion mapping analysis revealed known and novel cell states and the presence of cell subsets with potential progenitor cell activity. While the major differentiation pathway directs S+P+ cells towards myofibroblasts differentiation, a novel minority progenitor population was characterised by expression of Wif1, an inhibitor of Wnt signalling. The growth factors PDGF and FGF may recruit S+P+ cells to form immature myofibroblast in vitro with similar GE signature to myofibroblasts isolated from mice following MI in vivo. Both Fluidigm and Chromium 10X scRNAseq technologies had complementary roles: While Fluidigm had much greater depth; there were too few cells for detailed cluster analysis, though cluster identity could be assigned to Fluidigm sequenced cells using a Random Forests classifier trained on the Chromium 10X data-set. High content, single cell GE analysis provides an extraordinary resolution of the cellular repair process. Our findings highlight the role of resident S+P+ as cardiac progenitor cells in the healthy and diseased heart. S+P+ cells are activated following MI, providing a source of differentiated cell types and secretory functions required for cardiac repair. These molecular findings may identify new therapeutic approaches for improving patient survival following MI

Droplet-based single cell RNAseq tools: A practical guide

© 2019 The Royal Society of Chemistry. Droplet based scRNA-seq systems such as Drop-seq, inDrop and Chromium 10X have been the catalyst for the wide adoption of high-throughput scRNA-seq technologies in the research laboratory. In order to understand the capabilities of these systems to deeply interrogate biology; here we provide a practical guide through all the steps involved in a typical scRNA-seq experiment. Through comparing and contrasting these three main droplet based systems (and their derivatives), we provide an overview of all critical considerations in obtaining high quality and biologically relevant data. We also discuss the limitations of these systems and how they fit into the emerging field of Genomic Cytometry

Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro- fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration

A rapid co-culture stamping device for studying intercellular communication

Regulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists. We report here development of a temporarily sealed microfluidic stamping device which utilizes a novel valve design for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell interactions. We demonstrate post-stamping cell viability of >95%, the stamping of multiple adherent cell types, and the ability to control the seeded cell density. We also show viability, proliferation and migration of cultured cells, enabling analysis of co-culture boundary conditions on cell fate. We also developed an in-vitro model of endothelial and cardiac stem cell interactions, which are thought to regulate coronary repair after myocardial injury. The stamp is fabricated using microfabrication techniques, is operated with a lab pipettor and uses very low reagent volumes of 20 μl with cell injection efficiency of >70%. This easy-to-use device provides a general strategy for micro-patterning of multiple cell types and will be important for studying cell-cell interactions in a multitude of applications

A Rapid microfluidic stamping device for studying cardiac stem cells and endothelial cells co-culture

Many biological processes in the body are regulated by synchronized activity between two cell types. Recent advances in cell μcontact printing have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. However, these techniques are still complicated to perform and are seldom used by biologists. We report here development of a novel microfluidic stamping device for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell spatial interactions. To demonstrate the stamp's capabilities, we developed an in-vitro model of endothelial and cardiac mesenchymal stem cell interactions, which are thought to regulate coronary repair after myocardial injury.2 page(s

A rapid co-culture stamping device for studying intercellular communication

Regulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists. We report here development of a temporarily sealed microfluidic stamping device which utilizes a novel valve design for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell interactions. We demonstrate post-stamping cell viability of \u3e95%, the stamping of multiple adherent cell types, and the ability to control the seeded cell density. We also show viability, proliferation and migration of cultured cells, enabling analysis of co-culture boundary conditions on cell fate. We also developed an in-vitro model of endothelial and cardiac stem cell interactions, which are thought to regulate coronary repair after myocardial injury. The stamp is fabricated using microfabrication techniques, is operated with a lab pipettor and uses very low reagent volumes of 20 μl with cell injection efficiency of \u3e70%. This easy-to-use device provides a general strategy for micro-patterning of multiple cell types and will be important for studying cell-cell interactions in a multitude of applications