Cell state transitions are decoupled from cell division during early embryo development.

As tissues develop, cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. Here, to determine the role of cell division in differentiation, we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single-cell resolution.

Approximating mutual information of high-dimensional variables using learned representations.

Mutual information (MI) is a general measure of statistical dependence with widespread application across the sciences. However, estimating MI between multi-dimensional variables is challenging because the number of samples necessary to converge to an accurate estimate scales unfavorably with dimensionality. In practice, existing techniques can reliably estimate MI in up to tens of dimensions, but fail in higher dimensions, where sufficient sample sizes are infeasible.

A map of signaling responses in the human airway epithelium.

Receptor-mediated signaling plays a central role in tissue regeneration, and it is dysregulated in disease. Here, we build a signaling-response map for a model regenerative human tissue: the airway epithelium. We analyzed the effect of 17 receptor-mediated signaling pathways on organotypic cultures to determine changes in abundance and phenotype of epithelial cell types.

A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells.

Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites.

Global decoupling of cell differentiation from cell division in early embryo development.

As tissues develop, cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation, we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single-cell resolution.

The secreted neuronal signal Spock1 promotes blood-brain barrier development.

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restrict its permeability to proteins and small molecules. Classic chick-quail chimera studies have shown that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here, we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice.

A mannitol-based buffer improves single-cell RNA sequencing of high-salt marine cells.

Single-cell RNA sequencing (scRNA-seq) enables discovery of novel cell states by transcriptomic profiling with minimal prior knowledge, making it useful for studying non-model organisms. For most marine organisms, however, cells are viable at a higher salinity than is compatible with scRNA-seq, impacting data quality and cell representation. We show that a low-salinity phosphate buffer supplemented with D-mannitol (PBS-M) enables higher-quality scRNA-seq of blood cells from the tunicate .

A map of signaling responses in the human airway epithelium.

Receptor-mediated signaling plays a central role in tissue regeneration, and it is dysregulated in disease. Here, we build a signaling-response map for a model regenerative human tissue: the airway epithelium. We analyzed the effect of 17 receptor-mediated signaling pathways on organotypic cultures to determine changes in abundance and phenotype of all epithelial cell types.

CoSpar identifies early cell fate biases from single-cell transcriptomic and lineage information.

A goal of single-cell genome-wide profiling is to reconstruct dynamic transitions during cell differentiation, disease onset and drug response. Single-cell assays have recently been integrated with lineage tracing, a set of methods that identify cells of common ancestry to establish bona fide dynamic relationships between cell states. These integrated methods have revealed unappreciated cell dynamics, but their analysis faces recurrent challenges arising from noisy, dispersed lineage data.

Triple-Decker Sandwich Cultures of Intestinal Organoids for Long-Term Live Imaging, Uniform Perturbation, and Statistical Sampling.

Three-dimensional organoid cultures enable the study of stem cell and tissue biology ex vivo, providing improved access to cells for perturbation and live imaging. Typically, organoids are grown in hydrogel domes that are simple to prepare but that lead to non-uniform tissue growth and viability. We recently developed a simple alternative culture method to embed intestinal organoids in multilayered hydrogels, called "triple-decker sandwiches," that align organoids in a common z-plane with uniform access to medium.

Macrophage-Targeted Therapy Unlocks Antitumoral Cross-talk between IFNγ-Secreting Lymphocytes and IL12-Producing Dendritic Cells.

Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming.

Resident Kupffer cells and neutrophils drive liver toxicity in cancer immunotherapy.

Immunotherapy is revolutionizing cancer treatment but is often restricted by toxicities. What distinguishes adverse events from concomitant antitumor reactions is poorly understood. Here, using anti-CD40 treatment in mice as a model of T1-promoting immunotherapy, we showed that liver macrophages promoted local immune-related adverse events.

Inflation-collapse dynamics drive patterning and morphogenesis in intestinal organoids.

How stem cells self-organize to form structured tissues is an unsolved problem. Intestinal organoids offer a model of self-organization as they generate stem cell zones (SCZs) of typical size even without a spatially structured environment. Here we examine processes governing the size of SCZs.

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer.

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer.

Tumor-infiltrating dendritic cell states are conserved across solid human cancers.

Dendritic cells (DCs) contribute a small fraction of the tumor microenvironment but are emerging as an essential antitumor component based on their ability to foster T cell immunity and immunotherapy responses. Here, we discuss our expanding view of DC heterogeneity in human tumors, as revealed with meta-analysis of single-cell transcriptome profiling studies. We further examine tumor-infiltrating DC states that are conserved across patients, cancer types, and species and consider the fundamental and clinical relevance of these findings.

Tumor-Promoting Ly-6G SiglecF Cells Are Mature and Long-Lived Neutrophils.

Myeloid cells co-expressing the markers CD11b, Ly-6G, and SiglecF can be found in large numbers in murine lung adenocarcinomas and accelerate cancer growth by fostering tumor cell invasion, angiogenesis, and immunosuppression; however, some of these cells' fundamental features remain unexplored. Here, we show that tumor-infiltrating CD11b Ly-6G SiglecF cells are bona fide mature neutrophils and therefore differ from other myeloid cells, including SiglecF eosinophils, SiglecF macrophages, and CD11b Ly-6G myeloid-derived suppressor cells. We further show that SiglecF neutrophils gradually accumulate in growing tumors, where they can live for several days; this lifespan is in marked contrast to that of their SiglecF counterparts and neutrophils in general, which live for several hours only.

Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.

Bone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs). HSCs present a complex variety of regenerative behaviours at the clonal level, but the mechanisms underlying this diversity are still undetermined. Recent advances in single-cell RNA sequencing have revealed transcriptional differences among HSCs, providing a possible explanation for their functional heterogeneity.

Lineage commitment of hematopoietic stem cells and progenitors: insights from recent single cell and lineage tracing technologies.

Blood production is essential to maintain human health, and even small perturbations in hematopoiesis can cause disease. Hematopoiesis has therefore been the focus of much research for many years. Experiments determining the lineage potentials of hematopoietic stem and progenitor cells (HSPCs) in vitro and after transplantation revealed a hierarchy of progenitor cell states, where differentiating cells undergo lineage commitment-a series of irreversible changes that progressively restrict their potential.

Lineage reconstruction from clonal correlations.

A central task in developmental biology is to learn the sequence of fate decisions that leads to each mature cell type in a tissue or organism. Recently, clonal labeling of cells using DNA barcodes has emerged as a powerful approach for identifying cells that share a common ancestry of fate decisions. Here we explore the idea that stochasticity of cell fate choice during tissue development could be harnessed to read out lineage relationships after a single step of clonal barcoding.

Lineage tracing meets single-cell omics: opportunities and challenges.

A fundamental goal of developmental and stem cell biology is to map the developmental history (ontogeny) of differentiated cell types. Recent advances in high-throughput single-cell sequencing technologies have enabled the construction of comprehensive transcriptional atlases of adult tissues and of developing embryos from measurements of up to millions of individual cells. Parallel advances in sequencing-based lineage-tracing methods now facilitate the mapping of clonal relationships onto these landscapes and enable detailed comparisons between molecular and mitotic histories.

Lineage tracing on transcriptional landscapes links state to fate during differentiation.

A challenge in biology is to associate molecular differences among progenitor cells with their capacity to generate mature cell types. Here, we used expressed DNA barcodes to clonally trace transcriptomes over time and applied this to study fate determination in hematopoiesis. We identified states of primed fate potential and located them on a continuous transcriptional landscape.