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.

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The pluripotent state, which is first established in the primitive ectoderm cells (PE) of blastocysts, is lost progressively and irreversibly during subsequent development 1 . For example, development of postimplantation epiblast from PE involves significant transcriptional and epigenetic changes, including DNA methylation and X inactivation 2 , which creates a robust epigenetic barrier and prevents their reversion to a PE-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signaling, but they respond to Activin/bFGF to form self-renewing epiblast stem cells (EpiSC), which exhibit essential properties of epiblast cells 3,4 , that differ from embryonic stem cells (ESC) derived from PE 5 . Here we show reprogramming of advanced epiblast cells from E5.5 -E7.5 embryos with uniform expression of N-cadherin and inactive X chromosome, to ES-like cells (rESC) in response to LIF-STAT3 signaling. Cultured epiblast cells (cEpi) overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, involving DNA demethylation, X reactivation and expression of Ecadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Notably, using this new approach, we report reversion of established EpiSC to rESC. Furthermore, unlike epiblast and EpiSC, rESC contribute to somatic tissues and germ cells in chimeras. This is a tractable model to investigate signaling molecule induced epigenetic reprogramming that can promote reacquisition of the fundamental pluripotent state.Previous studies showed that epiblast cells, unlike PE, are refractory to LIF-STAT3 signaling 3,4 ; instead they respond to Activin/bFGF to generate self-renewing EpiSC. EpiSC differ epigenetically from ESC, as they have an inactive X-chromosome and they cannot form chimeras when introduced into blastocysts. However, we set out to re-examine if postimplantation epiblast cells could undergo reprogramming to ESC-like cells in response to LIF-STAT3 signaling. We isolated epiblast tissue on embryonic day (E) E5.5 -E7.5 from transgenic embryos with an Oct4-ΔPE-green fluorescent protein (GFP) reporter 6 . This reporter, with the distal enhancer and lacking the proximal enhancer for Oct4, shows Next, for the culture of epiblast, we used LIF and fetal calf serum (FCS) on mouse embryonic fibroblasts feeder cells (MEFs), which is the standard condition used for the derivation of ESC from PE, and for reprogramming of somatic cells to induced pluripotent stem cells (iPS) 5,[8][9][10][11] . The epiblast tissue was dissected to remove the most proximal region (the site of PGC and PGC precursors 2 ), and the outer visceral endoderm (Fig. 1a). All the epiblast cells uniformly showed an inactive X-chromosome, and were positive for Ncadherin (see below). Notably, we then trypsinised the epiblast tissue and used single cell suspension from individual epiblasts for culture, unlike previous studies where the epiblast tissue was left inta...