Polyploidisation pleiotropically buffers ageing in hepatocytes.

BACKGROUND & AIMS: Polyploidy in hepatocytes has been proposed as a genetic mechanism to buffer against transcriptional dysregulation. Here, we aim to demonstrate the role of polyploidy in modulating gene regulatory networks in hepatocytes during ageing. METHODS: We performed single-nucleus RNA sequencing in hepatocyte nuclei of different ploidy levels isolated from young and old wild-type mice. Changes in the gene expression and regulatory network were compared to three independent strains that were haploinsufficient for HNF4A, CEBPA or CTCF, representing non-deleterious perturbations. Phenotypic characteristics of the liver section were additionally evaluated histologically, whereas the genomic allele composition of hepatocytes was analysed by BaseScope. RESULTS: We observed that ageing in wild-type mice results in nuclei polyploidy and a marked increase in steatosis. Haploinsufficiency of liver-specific master regulators (HFN4A or CEBPA) results in the enrichment of hepatocytes with tetraploid nuclei at a young age, affecting the genomic regulatory network, and dramatically suppressing ageing-related steatosis tissue wide. Notably, these phenotypes are not the result of subtle disruption to liver-specific transcriptional networks, since haploinsufficiency in the CTCF insulator protein resulted in the same phenotype. Further quantification of genotypes of tetraploid hepatocytes in young and old HFN4A-haploinsufficient mice revealed that during ageing, tetraploid hepatocytes lead to the selection of wild-type alleles, restoring non-deleterious genetic perturbations. CONCLUSIONS: Our results suggest a model whereby polyploidisation leads to fundamentally different cell states. Polyploid conversion enables pleiotropic buffering against age-related decline via non-random allelic segregation to restore a wild-type genome. IMPACT AND IMPLICATIONS: The functional role of hepatocyte polyploidisation during ageing is poorly understood. Using single-nucleus RNA sequencing and BaseScope approaches, we have studied ploidy dynamics during ageing in murine livers with non-deleterious genetic perturbations. We have identified that hepatocytes present different cellular states and the ability to buffer ageing-associated dysfunctions. Tetraploid nuclei exhibit robust transcriptional networks and are better adapted to genomically overcome perturbations. Novel therapeutic interventions aimed at attenuating age-related changes in tissue function could be exploited by manipulation of ploidy dynamics during chronic liver conditions.

Matching by OS Prognostic Score to Construct External Controls in Lung Cancer Clinical Trials.

External controls (eControls) leverage historical data to create non-randomized control arms. The lack of randomization can result in confounding between the experimental and eControl cohorts. To balance potentially confounding variables between the cohorts, one of the proposed methods is to match on prognostic scores. Still, the performance of prognostic scores to construct eControls in oncology has not been analyzed yet. Using an electronic health record-derived de-identified database, we constructed eControls using one of three methods: ROPRO, a state-of-the-art prognostic score, or either a propensity score composed of five (5Vars) or 27 covariates (ROPROvars). We compared the performance of these methods in estimating the overall survival (OS) hazard ratio (HR) of 11 recent advanced non-small cell lung cancer. The ROPRO eControls had a lower OS HR error (median absolute deviation (MAD), 0.072, confidence interval (CI): 0.036-0.185), than the 5Vars (MAD 0.081, CI: 0.025-0.283) and ROPROvars eControls (MAD 0.087, CI: 0.054-0.383). Notably, the OS HR errors for all methods were even lower in the phase III studies. Moreover, the ROPRO eControl cohorts included, on average, more patients than the 5Vars (6.54%) and ROPROvars cohorts (11.7%). The eControls matched with the prognostic score reproduced the controls more reliably than propensity scores composed of the underlying variables. Additionally, prognostic scores could allow eControls to be built on many prognostic variables without a significant increase in the variability of the propensity score, which would decrease the number of matched patients.

An integrated cell atlas of the lung in health and disease.

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

Cells of the adult human heart.

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.

A cellular census of human lungs identifies novel cell states in health and in asthma.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.

Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration.

Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates.