Single-cell RNA sequencing (scRNA-seq) data are commonly affected by technical artifacts known as “doublets,” which limit cell throughput and lead to spurious biological conclusions. Here, we present ...a computational doublet detection tool—DoubletFinder—that identifies doublets using only gene expression data. DoubletFinder predicts doublets according to each real cell’s proximity in gene expression space to artificial doublets created by averaging the transcriptional profile of randomly chosen cell pairs. We first use scRNA-seq datasets where the identity of doublets is known to show that DoubletFinder identifies doublets formed from transcriptionally distinct cells. When these doublets are removed, the identification of differentially expressed genes is enhanced. Second, we provide a method for estimating DoubletFinder input parameters, allowing its application across scRNA-seq datasets with diverse distributions of cell types. Lastly, we present “best practices” for DoubletFinder applications and illustrate that DoubletFinder is insensitive to an experimentally validated kidney cell type with “hybrid” expression features.
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•DoubletFinder uses gene expression features to predict doublets in scRNA-seq data•DoubletFinder identifies doublets derived from transcriptionally distinct cells•Doublet removal improves differential gene expression analysis performance•DoubletFinder is insensitive to bona fide cells with “hybrid” expression profiles
scRNA-seq data interpretation is confounded by technical artifacts known as doublets—single-cell transcriptome data representing more than one cell. Moreover, scRNA-seq cellular throughput is purposefully limited to minimize doublet formation rates. By identifying cells sharing expression features with simulated doublets, DoubletFinder detects many real doublets and mitigates these two limitations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Multicellular organs comprise differentiated cell types with discrete yet interdependent functions. The cells' spatial arrangements and interconnectivities, both critical elements of higher-order ...function, derive from complex developmental programs in vivo and are often difficult or impossible to emulate in vitro. Here, we report the bottom-up synthesis of microtissues composed of multiple cell types with programmed connectivity. We functionalized cells with short oligonucleotides to impart specific adhesive properties. Hybridization of complementary DNA sequences enabled the assembly of multicellular structures with defined cell-cell contacts. We demonstrated that the kinetic parameters of the assembly process depend on DNA sequence complexity, density, and total cell concentration. Thus, cell assembly can be highly controlled, enabling the design of microtissues with defined cell composition and stoichiometry. We used this strategy to construct a paracrine signaling network in isolated 3-dimensional microtissues.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell ...specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.
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Sample multiplexing enables pooled analysis during single-cell RNA sequencing workflows, thereby increasing throughput and reducing batch effects. A challenge for all multiplexing techniques is to ...link sample-specific barcodes with cell-specific barcodes, then demultiplex sample identity post-sequencing. However, existing demultiplexing tools fail under many real-world conditions where barcode cross-contamination is an issue. We therefore developed deMULTIplex2, an algorithm inspired by a mechanistic model of barcode cross-contamination. deMULTIplex2 employs generalized linear models and expectation-maximization to probabilistically determine the sample identity of each cell. Benchmarking reveals superior performance across various experimental conditions, particularly on large or noisy datasets with unbalanced sample compositions.
Microglia are resident macrophages in the brain that emerge in early development and respond to the local environment by altering their molecular and phenotypic states. Fundamental questions about ...microglia diversity and function during development remain unanswered because we lack experimental strategies to interrogate their interactions with other cell types and responses to perturbations ex vivo. We compared human microglia states across culture models, including cultured primary and pluripotent stem cell-derived microglia. We developed a “report card” of gene expression signatures across these distinct models to facilitate characterization of their responses across experimental models, perturbations, and disease conditions. Xenotransplantation of human microglia into cerebral organoids allowed us to characterize key transcriptional programs of developing microglia in vitro and reveal that microglia induce transcriptional changes in neural stem cells and decrease interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.
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•Microglia culture models differentially attenuate and preserve gene signatures•Brain organoid microenvironment preserves microglial homeostatic state•Genes in the interferon response pathway are attenuated in the presence of microglia•Microglia decrease double-stranded DNA breaks and cell stress in radial glia
Our “microglia report card” reveals that human microglia selectively regulate different biological gene cluster modules in an environment-dependent fashion. The presence of microglia in brain organoids protects against double-stranded DNA breaks and attenuates the gene response on the interferon signaling pathway. Additionally, microglia accelerate neural network synchronization in organoids.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Embryonic tissue boundaries are critical to not only cement newly patterned structures during development, but also to serve as organizing centers for subsequent rounds of morphogenesis. Although ...this latter role is especially difficult to study in vivo, synthetic embryology offers a new vantage point and fresh opportunities. In this review, we cover recent progress towards understanding and controlling in vitro boundaries and how they impact synthetic model systems. A key point this survey highlights is that the outcome of self-organization is strongly dependent on the boundary imposed, and new insight into the complex functions of embryonic boundaries will be necessary to create better self-organizing tissues for basic science, drug development, and regenerative medicine.
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•Review of recent progress in synthetic embryology to reveal the role of embryonic boundaries during development.•Summary of current state-of-the-art tools for creating and shaping boundaries in vitro.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
A T cell mounts an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the ...extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling.
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•A synthetic T cell signaling system was created using receptor ligands made of DNA•T cell signaling can discriminate between DNA ligands differing by a single base pair•Higher-affinity ligands promote the clustering and phosphorylation of receptors•Receptor clustering provides a kinetic proofreading mechanism for ligand discrimination
The spatial reorganization of T cell receptors is critical for ligand discrimination in T cell signaling.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of ...phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.
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•KP-tracer mice enable continuous, high-resolution in vivo cancer lineage tracing•Rare subclones with distinct expression programs expand during tumor evolution•Lineage tracing reveals cellular plasticity and evolutionary paths•Metastases are derived from spatially localized, expanding subclones of the tumor
Yang et al. developed a genetically engineered mouse model of lung cancer capable of continuous lineage tracing with single-cell RNA-seq readout. They identified the subclonal dynamics of tumors, gene modules underlying expansion, transient increases in cellular plasticity, stereotypical evolutionary paths to aggressiveness across tumor genotypes, and the spatial and phylogenetic origins of metastases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Sample multiplexing facilitates scRNA-seq by reducing costs and identifying artifacts such as cell doublets. However, universal and scalable sample barcoding strategies have not been described. We ...therefore developed MULTI-seq: multiplexing using lipid-tagged indices for single-cell and single-nucleus RNA sequencing. MULTI-seq reagents can barcode any cell type or nucleus from any species with an accessible plasma membrane. The method involves minimal sample processing, thereby preserving cell viability and endogenous gene expression patterns. When cells are classified into sample groups using MULTI-seq barcode abundances, data quality is improved through doublet identification and recovery of cells with low RNA content that would otherwise be discarded by standard quality-control workflows. We use MULTI-seq to track the dynamics of T-cell activation, perform a 96-plex perturbation experiment with primary human mammary epithelial cells and multiplex cryopreserved tumors and metastatic sites isolated from a patient-derived xenograft mouse model of triple-negative breast cancer.
The complexity of the human body derives from numerous modular building blocks assembled hierarchically across multiple length scales. These building blocks, spanning sizes ranging from single cells ...to organs, interact to regulate development and normal organismal function but become disorganized during disease. Here, we review methods for the bottom-up and directed assembly of modular, multicellular, and tissue-like constructs in vitro . These engineered tissues will help refine our understanding of the relationship between form and function in the human body, provide new models for the breakdown in tissue architecture that accompanies disease, and serve as building blocks for the field of regenerative medicine.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
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