Molecular profiling of adult C. elegans glia across sexes by single-nuclear RNA-seq.

A comprehensive understanding of nervous system function requires molecular insight into the diversity and sex dimorphism of both its component cell types, glia and neurons. Here, we present a single-nuclear RNA sequencing (RNA-seq) census of all neuroectoderm-derived glia in the adult C. elegans nervous system, across sexes.

Comparing phenotypic manifolds with Kompot: Detecting differential abundance and gene expression at single-cell resolution.

Kompot is a statistical framework for holistic comparison of multi-condition single-cell datasets, supporting both differential abundance and differential expression. Differential abundance captures changes in how cells populate the phenotypic manifold across conditions, while differential expression identifies condition-specific changes in gene regulation that may be localized to particular regions of that manifold. Kompot models the distribution of cells and gene expression as continuous functions over a low-dimensional representation of cell states, enabling single-cell resolution inference with calibrated uncertainty estimates.

Patterns of intra- and intertumor phenotypic heterogeneity in lethal prostate cancer.

Metastatic prostate cancer (mPC) is a clinically and molecularly heterogeneous disease. While there is increasing recognition of diverse tumor phenotypes across patients, less is known about the molecular and phenotypic heterogeneity present within an individual. In this study, we aimed to define the patterns, extent, and consequences of inter- and intratumoral heterogeneity in lethal prostate cancer.

Single-cell analysis of bidirectional reprogramming between early embryonic states identify mechanisms of differential lineage plasticities in mice.

Two distinct lineages, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common inner cell mass (ICM) progenitors in mammalian embryos. To study how these sister identities are forged, we leveraged mouse embryonic stem (ES) cells and extra-embryonic endoderm (XEN) stem cells-in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses showed distinct rates, efficiencies, and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions.

Age-related epithelial defects limit thymic function and regeneration.

The thymus is essential for establishing adaptive immunity yet undergoes age-related involution that leads to compromised immune responsiveness. The thymus is also extremely sensitive to acute insult and although capable of regeneration, this capacity declines with age for unknown reasons. We applied single-cell and spatial transcriptomics, lineage-tracing and advanced imaging to define age-related changes in nonhematopoietic stromal cells and discovered the emergence of two atypical thymic epithelial cell (TEC) states.

Quantifying cell-state densities in single-cell phenotypic landscapes using Mellon.

Cell-state density characterizes the distribution of cells along phenotypic landscapes and is crucial for unraveling the mechanisms that drive diverse biological processes. Here, we present Mellon, an algorithm for estimation of cell-state densities from high-dimensional representations of single-cell data. We demonstrate Mellon's efficacy by dissecting the density landscape of differentiating systems, revealing a consistent pattern of high-density regions corresponding to major cell types intertwined with low-density, rare transitory states.

Signals that control MAIT cell function in healthy and inflamed human tissues.

Mucosal-associated invariant T (MAIT) cells have a semi-invariant T-cell receptor that allows recognition of antigen in the context of the MHC class I-related (MR1) protein. Metabolic intermediates of the riboflavin synthesis pathway have been identified as MR1-restricted antigens with agonist properties. As riboflavin synthesis occurs in many bacterial species, but not human cells, it has been proposed that the main purpose of MAIT cells is antibacterial surveillance and protection.

DUX4 expression in cancer induces a metastable early embryonic totipotent program.

The transcription factor DUX4 regulates a portion of the zygotic gene activation (ZGA) program in the early embryo. Many cancers express DUX4 but it is unknown whether this generates cells similar to early embryonic stem cells. Here we identified cancer cell lines that express DUX4 and showed that DUX4 is transiently expressed in a small subset of the cells.

Quantifying Cell-State Densities in Single-Cell Phenotypic Landscapes using Mellon.

Cell-state density characterizes the distribution of cells along phenotypic landscapes and is crucial for unraveling the mechanisms that drive cellular differentiation, regeneration, and disease. Here, we present Mellon, a novel computational algorithm for high-resolution estimation of cell-state densities from single-cell data. We demonstrate Mellon's efficacy by dissecting the density landscape of various differentiating systems, revealing a consistent pattern of high-density regions corresponding to major cell types intertwined with low-density, rare transitory states.

Single-cell analysis of bidirectional reprogramming between early embryonic states reveals mechanisms of differential lineage plasticities.

Two distinct fates, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common progenitor cells, the inner cell mass (ICM), in mammalian embryos. To study how these sister identities are forged, we leveraged embryonic (ES) and etraembryonic doderm (XEN) stem cells - counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses uncovered distinct rates, efficiencies and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions.

Molecular heterogeneity of glia across sexes.

A comprehensive description of nervous system function, and sex dimorphism within, is incomplete without clear assessment of the diversity of its component cell types, neurons and glia. has an invariant nervous system with the first mapped connectome of a multicellular organism and single-cell atlas of component neurons. Here we present single nuclear RNA-seq evaluation of glia across the entire adult nervous system, including both sexes.

SEACells infers transcriptional and epigenomic cellular states from single-cell genomics data.

Metacells are cell groupings derived from single-cell sequencing data that represent highly granular, distinct cell states. Here we present single-cell aggregation of cell states (SEACells), an algorithm for identifying metacells that overcome the sparsity of single-cell data while retaining heterogeneity obscured by traditional cell clustering. SEACells outperforms existing algorithms in identifying comprehensive, compact and well-separated metacells in both RNA and assay for transposase-accessible chromatin (ATAC) modalities across datasets with discrete cell types and continuous trajectories.

Defining cellular population dynamics at single-cell resolution during prostate cancer progression.

Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigate prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in an in vivo murine model. We observe an expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients.

Lineage plasticity in prostate cancer depends on JAK/STAT inflammatory signaling.

Drug resistance in cancer is often linked to changes in tumor cell state or lineage, but the molecular mechanisms driving this plasticity remain unclear. Using murine organoid and genetically engineered mouse models, we investigated the causes of lineage plasticity in prostate cancer and its relationship to antiandrogen resistance. We found that plasticity initiates in an epithelial population defined by mixed luminal-basal phenotype and that it depends on increased Janus kinase (JAK) and fibroblast growth factor receptor (FGFR) activity.

CUT&Tag2for1: a modified method for simultaneous profiling of the accessible and silenced regulome in single cells.

Cleavage Under Targets and Tagmentation (CUT&Tag) is an antibody-directed transposase tethering strategy for in situ chromatin profiling in small samples and single cells. We describe a modified CUT&Tag protocol using a mixture of an antibody to the initiation form of RNA polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution to produce high-resolution maps of both the active and repressive regulomes in single cells. The ability to seamlessly map active promoters, enhancers, and repressive regulatory elements using a single workflow provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.

Tissue Schematics: A framework to decipher tissue architecture and assembly.

High-parameter spatial proteomics provide unprecedented opportunities to investigate how tissue architectures are assembled. In an article in this issue of Cell Systems, Bhate et al. present "Tissue Schematics," a conceptual framework and computational approach to decipher the rules of tissue assembly.

CellRank for directed single-cell fate mapping.

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.

Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in Alzheimer's disease.

Aberrant inflammation in the CNS has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a new approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular cross-talk along the neuroinflammatory axis in vitro. We used the tri-culture system to model neuroinflammation in Alzheimer's disease with hPSCs harboring the APP+/+ mutation and their isogenic control.

Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages.

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.

Regenerative lineages and immune-mediated pruning in lung cancer metastasis.

Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states.

Author Correction: Characterization of cell fate probabilities in single-cell data with Palantir.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The emergent landscape of the mouse gut endoderm at single-cell resolution.

Here we delineate the ontogeny of the mammalian endoderm by generating 112,217 single-cell transcriptomes, which represent all endoderm populations within the mouse embryo until midgestation. We use graph-based approaches to model differentiating cells, which provides a spatio-temporal characterization of developmental trajectories and defines the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncover a relationship between descendants of these two lineages, in which epiblast cells differentiate into endoderm at two distinct time points-before and during gastrulation.

Negative Co-stimulation Constrains T Cell Differentiation by Imposing Boundaries on Possible Cell States.

Co-stimulation regulates T cell activation, but it remains unclear whether co-stimulatory pathways also control T cell differentiation. We used mass cytometry to profile T cells generated in the genetic absence of the negative co-stimulatory molecules CTLA-4 and PD-1. Our data indicate that negative co-stimulation constrains the possible cell states that peripheral T cells can acquire.

Characterization of cell fate probabilities in single-cell data with Palantir.

Single-cell RNA sequencing studies of differentiating systems have raised fundamental questions regarding the discrete versus continuous nature of both differentiation and cell fate. Here we present Palantir, an algorithm that models trajectories of differentiating cells by treating cell fate as a probabilistic process and leverages entropy to measure cell plasticity along the trajectory. Palantir generates a high-resolution pseudo-time ordering of cells and, for each cell state, assigns a probability of differentiating into each terminal state.

Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment.

Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data.