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Trajectory inference approaches analyze genome-wide omics data from thousands of single cells and computationally infer the order of these cells along developmental trajectories. Although more than 70 trajectory inference tools have already been developed, it is challenging to compare their performance because the input they require and output models they produce vary substantially. Here, we benchmark 45 of these methods on 110 real and 229 synthetic datasets for cellular ordering, topology, scalability and usability. Our results highlight the complementarity of existing tools, and that the choice of method should depend mostly on the dataset dimensions and trajectory topology. Based on these results, we develop a set of guidelines to help users select the best method for their dataset. Our freely available data and evaluation pipeline (https://benchmark.dynverse.org) will aid in the development of improved tools designed to analyze increasingly large and complex single-cell datasets.The authors comprehensively benchmark the accuracy, scalability, stability and usability of 45 single-cell trajectory inference methods.

Key Points Alternative splicing explains how a single gene can generate more than one mRNA transcript, thus expanding the complexity of the proteome. During normal development, a large number of alternative splicing changes occur, and it is now apparent that these transitions between alternatively spliced isoforms contribute to the acquisition of adult tissue functions and identity. Individual splicing changes are coordinated during development, establishing splicing networks. As a result of recent progress, we now better understand the mechanisms that coordinate alternative splicing networks and the roles of these networks in cell differentiation, organ development and tissue homeostasis. Alternative splicing expands the complexity of the proteome by generating multiple transcript isoforms from a single gene. Numerous alternative splicing events occur during cell differentiation and tissue maturation, suggesting that alternative splicing supports proper development. Recent studies shed light on how alternative splicing and its coordination contribute to organ development and tissue homeostasis. Alternative splicing of eukaryotic transcripts is a mechanism that enables cells to generate vast protein diversity from a limited number of genes. The mechanisms and outcomes of alternative splicing of individual transcripts are relatively well understood, and recent efforts have been directed towards studying splicing networks. It has become apparent that coordinated splicing networks regulate tissue and organ development, and that alternative splicing has important physiological functions in different developmental processes in humans.

T helper 2 (TH 2) cells orchestrate protective type 2 immune responses, such as those that target helminths and facilitate tissue repair, but also contribute to chronic inflammatory diseases, such as asthma and allergy. Here, we review recent insights into how diverse molecular signals from cellular sources, including dendritic cells, innate lymphoid cells and the epithelium, are integrated by T cells to guide the transcriptional and epigenetic changes necessary for TH 2 cell differentiation. Our improved understanding of these pathways has opened new avenues for therapeutically targeting TH 2 cells in asthma and allergy. The advent of comprehensive single-cell transcriptomics along with improvements in single-cell proteomics and the generation of novel in vivo cell fate mapping techniques promise to expand our understanding of T cell diversity and offer new insight into disease-related heterogeneity and plasticity of TH cell responses.

Computational trajectory inference enables the reconstruction of cell state dynamics from single-cell RNA sequencing experiments. However, trajectory inference requires that the direction of a biological process is known, largely limiting its application to differentiating systems in normal development. Here, we present CellRank ( https://cellrank.org ) for single-cell fate mapping in diverse scenarios, including regeneration, reprogramming and disease, for which direction is unknown. Our approach combines the robustness of trajectory inference with directional information from RNA velocity, taking into account the gradual and stochastic nature of cellular fate decisions, as well as uncertainty in velocity vectors. On pancreas development data, CellRank automatically detects initial, intermediate and terminal populations, predicts fate potentials and visualizes continuous gene expression trends along individual lineages. Applied to lineage-traced cellular reprogramming data, predicted fate probabilities correctly recover reprogramming outcomes. CellRank also predicts a new dedifferentiation trajectory during postinjury lung regeneration, including previously unknown intermediate cell states, which we confirm experimentally. CellRank infers directed cell state transitions and cell fates incorporating RNA velocity information into a graph based Markov process.

The concept of differentiation therapy emerged from the fact that hormones or cytokines may promote differentiation ex vivo, thereby irreversibly changing the phenotype of cancer cells. Its hallmark success has been the treatment of acute promyelocytic leukaemia (APL), a condition that is now highly curable by the combination of retinoic acid (RA) and arsenic. Recently, drugs that trigger differentiation in a variety of primary tumour cells have been identified, suggesting that they are clinically useful. This Opinion article analyses the basis for the clinical successes of RA or arsenic in APL by assessing the respective roles of terminal maturation and loss of self-renewal. By reviewing other successful examples of drug-induced tumour cell differentiation, novel approaches to transform differentiating drugs into more efficient therapies are proposed.

Human development requires intricate cell specification and communication pathways that allow an embryo to generate and appropriately connect more than 200 different cell types. Key to the successful completion of this differentiation programme is the quantitative and reversible regulation of core signalling networks, and post-translational modification with ubiquitin provides embryos with an essential tool to accomplish this task. Instigated by E3 ligases and reversed by deubiquitylases, ubiquitylation controls many processes that are fundamental for development, such as cell division, fate specification and migration. As aberrant function or regulation of ubiquitylation enzymes is at the roots of developmental disorders, cancer, and neurodegeneration, modulating the activity of ubiquitylation enzymes is likely to provide strategies for therapeutic intervention.

The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury–repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance 1 . Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the ‘pulmonary ionocyte’, which co-expresses FOXI1 , multiple subunits of the vacuolar-type H + -ATPase (V-ATPase) and CFTR , the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium. Single-cell RNA sequencing analysis is used to identify cell types in the tracheal epithelium, including previously unidentified ionocytes, which express high levels of the cystic fibrosis transmembrane conductance regulator, CFTR .

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation 1 . Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking 1 , 2 . Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs) 3 . Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions) 4 . Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13–14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior–posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development. The culture of genetically unmodified human naive embryonic stem cells in specific growth conditions gives rise to structures that recapitulate those of post-implantation human embryos up to 13–14 days after fertilization.

Vascular smooth muscle cells (VSMCs) are a major cell type present at all stages of an atherosclerotic plaque. According to the ‘response to injury’ and ‘vulnerable plaque’ hypotheses, contractile VSMCs recruited from the media undergo phenotypic conversion to proliferative synthetic cells that generate extracellular matrix to form the fibrous cap and hence stabilize plaques. However, lineage-tracing studies have highlighted flaws in the interpretation of former studies, revealing that these studies had underestimated both the content and functions of VSMCs in plaques and have thus challenged our view on the role of VSMCs in atherosclerosis. VSMCs are more plastic than previously recognized and can adopt alternative phenotypes, including phenotypes resembling foam cells, macrophages, mesenchymal stem cells and osteochondrogenic cells, which could contribute both positively and negatively to disease progression. In this Review, we present the evidence for VSMC plasticity and summarize the roles of VSMCs and VSMC-derived cells in atherosclerotic plaque development and progression. Correct attribution and spatiotemporal resolution of clinically beneficial and detrimental processes will underpin the success of any therapeutic intervention aimed at VSMCs and their derivatives.

In eukaryotes, the genome does not exist as a linear molecule but instead is hierarchically packaged inside the nucleus. This complex genome organization includes multiscale structural units of chromosome territories, compartments, topologically associating domains, which are often demarcated by architectural proteins such as CTCF and cohesin, and chromatin loops. The 3D organization of chromatin modulates biological processes such as transcription, DNA replication, cell division and meiosis, which are crucial for cell differentiation and animal development. In this Review, we discuss recent progress in our understanding of the general principles of chromatin folding, its regulation and its functions in mammalian development. Specifically, we discuss the dynamics of 3D chromatin and genome organization during gametogenesis, embryonic development, lineage commitment and stem cell differentiation, and focus on the functions of chromatin architecture in transcription regulation. Finally, we discuss the role of 3D genome alterations in the aetiology of developmental disorders and human diseases.