Distinct Epithelial Cell Profiles in Normal Versus Induced-Congenital Diaphragmatic Hernia Fetal Lungs

Gas exchange in the lung occurs within alveoli, air-filled sacs composed of type 2 and type 1 epithelial cells (AEC2s and AEC1s), capillaries, and various resident mesenchymal cells. Here, we use a combination of in vivo clonal lineage analysis, different injury/repair systems, and in vitro culture of purified cell populations to obtain new information about the contribution of AEC2s to alveolar maintenance and repair. Genetic lineage-tracing experiments showed that surfactant protein C-positive (SFTPC-positive) AEC2s self renew and differentiate over about a year, consistent with the population containing long-term alveolar stem cells. Moreover, if many AEC2s were specifically ablated, high-resolution imaging of intact lungs showed that individual survivors undergo rapid clonal expansion and daughter cell dispersal. Individual lineage-labeled AEC2s placed into 3D culture gave rise to self-renewing "alveolospheres," which contained both AEC2s and cells expressing multiple AEC1 markers, including HOPX, a new marker for AEC1s. Growth and differentiation of the alveolospheres occurred most readily when cocultured with primary PDGFRα⁺ lung stromal cells. This population included lipofibroblasts that normally reside close to AEC2s and may therefore contribute to a stem cell niche in the murine lung. Results suggest that a similar dynamic exists between AEC2s and mesenchymal cells in the human lung.

Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested that AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that, during development, AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem-cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGFR (epidermal growth factor receptor) ligands in vitro and oncogenic Kras(G12D) in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair and cancer. We propose that local signals regulate AT2 stem-cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, whereas another signal controls reprogramming to AT1 fate.

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 markers 1 - 3 and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations 1 – 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.