No abstract

Although single cell RNA sequencing studies have begun providing compendia of cell expression profiles 1 – 9 , it has proven more difficult to systematically identify and localize all molecular types in individual organs to create a full molecular cell atlas. Here we describe droplet- and plate-based single cell RNA sequencing (scRNAseq) applied to ~75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, which have allowed us to define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 of 45 previously known cell types or subtypes and 14 new ones. This comprehensive molecular atlas elucidates the biochemical functions of lung cell types and the cell-selective transcription factors and optimal markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signaling interactions including sources and targets of chemokines in immune cell trafficking and expression changes on lung homing; and identifies the cell types directly affected by lung disease genes and respiratory viruses. Comparison to mouse identified 17 molecular types that appear to have been gained or lost during lung evolution and others whose expression profiles have been substantially altered, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions, and interactions are achieved in development and tissue engineering and altered in disease and evolution.

The mammalian lung is a highly branched network, in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types remain obscure, in part due to the limited number of lineage markers1-3 and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations1–5. We used microfluidic single cell RNA sequencing (RNA-seq) on 198 individual cells at 4 different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or prior purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell type diversity in the distal lung and led to the discovery of many novel cell type markers and transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full lifecycle of the alveolar type 2 cell lineage. This single cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.