Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung

Abstract

The role of lung epithelial stem cells in maintenance and repair of the adult lung is ill-defined, and their identity remains contentious because of the lack of definitive markers for their prospective isolation and the absence of clonogenic assays able to measure their stem/progenitor cell potential. In this study, we show that replication of epithelial-mesenchymal interactions in a previously undescribed matrigel-based clonogenic assay enables the identification of lung epithelial stem/progenitor cells by their colony-forming potential in vitro. We describe a population of EpCAM(hi) CD49f(pos) CD104(pos) CD24(low) epithelial cfus that generate colonies comprising airway, alveolar, or mixed lung epithelial cell lineages when cocultured with EpCAM(neg) Sca-1(pos) lung mesenchymal cells. We show that soluble fibroblast growth factor-10 and hepatocyte growth factor partially replace the requirement for mesenchymal support of epithelial colony formation, allowing clonal passaging and demonstration of their capacity for self-renewal. These data support a model in which the adult mouse lung contains a minor population of multipotent epithelial stem/progenitor cells with the capacity for self-renewal and whose descendants give rise to airway and alveolar epithelial cell lineages in vitro.

Fig. 1.

Colony-forming potential of EpCAM^pos epithelial stem/progenitors is mediated by EpCAM^neg Sca-1^pos mesenchymal cells. (A) Gating strategy for subsetting EpCAM^pos epithelial cells and EpCAM^neg Sca-1^pos mesenchymal cells. (B) EpCAM^neg Sca-1^pos cell culture. (C) Coculture of EpCAM^pos cells with EpCAM^neg Sca-1^pos cells. (D–F) Red and green overlay of fluorescence from coculture of eGFP^pos EpCAM^pos and RFP^pos EpCAM^neg Sca-1^pos cells. (G) Effect of growth factor addition on colony growth of EpCAM^pos cells cocultured with EpCAM^neg Sca-1^pos mesenchymal cells. Data are presented relative to no factor control (% of control + SEM, n = 3 per group). (H) Incidence of cfus (% of cells seeded + SEM, n ≥ 3 per group) of EpCAM^pos cells supplemented with FGF-10 and/or HGF in stromal-free cultures. A statistically significant difference (**P < 0.01; ***P < 0.001) between growth factor-treated groups and controls was determined using one-way ANOVA with a Tukey posttest. (I) Image of colonies generated in stromal-free cultures with FGF-10 and HGF.

Fig. 2.

Lung epithelial cfus are enriched in the EpCAM^hi CD49f^pos CD104^pos CD24^low cell fraction. (A) Fractionation of CD45^neg CD31^neg lung cells based on EpCAM and CD49f expression. Sca-1 (B), proSP-C (C), CD104 (D), and CD24 (E) expression on EpCAM^hi (blue), EpCAM^med (green), and EpCAM^low (red) cell fractions and isotype controls (gray). (F) Cloning efficiency of EpCAM^hi CD24^low (blue) and CD24^hi (red) cells with linear regression analysis (r ² = 0.9481, 1/slope = 22.45, n = 7).

Fig. 3.

Lung epithelial cfus are capable of clonal proliferation and self-renewal in vitro. (A) Schematic representation of potential outcomes of mixing experiment. (B) Total colonies that were red, green, or mixed color. (C) Phase contrast image of colonies. (D) Overlay of fluorescence from cocultures using RFP^pos and eGFP^pos EpCAM^hi CD24^low cells with WT EpCAM^neg Sca-1^pos cells. (E) Schematic representation of stromal-free bulk serial passaging. (F) Incidence of cfus (% of cells seeded + SEM, n ≥ 2) of EpCAM^hi CD24^low cells after bulk serial passage in stromal-free cultures with FGF-10 and HGF. (G) Increase in total number of epithelial cfus generated after serial passaging. (H) EpCAM vs. CD104 subsetting of passaged cells (P3). (I) Cloning efficiency of EpCAM^hi CD104^hi (blue) and EpCAM^low CD104^low (red) passaged cells (P3) replated in stromal-free culture with FGF-10 and HGF. (J) Schematic representation of stromal-free clonal serial passaging. (K) Incidence of cfus (mean number of colonies + SEM, n ≥ 3 colonies for each generation) from serial recloning of single colonies.

Fig. 4.

Generation of distinct epithelial colony subtypes. Bright-field images of lobular cystic airway-like colonies (A), dense saccular alveolar-like colonies (B), and colonies with mixed morphologies (C). Fluorescent confocal images of DAPI (blue) (D–F), MUC5AC (green) (G–I), proSP-C (red) (J–L), and overlay of MUC5AC and proSP-C staining of representative colonies (M–O).

Fig. 5.

Multilineage differentiation of mixed lung epithelial cfus. Heat map shows relative real-time RT-PCR gene expression levels in cells harvested from single airway, alveolar, and mixed epithelial colonies. Values represent average ΔCt values of genes relative to 18s RNA control. ND, not detected (Ct <40).

Fig. 6.

Evidence of an epithelial hierarchy. (A) Images of a primary colony that was enzymatically dissociated and the secondary colonies generated after subsequent reseeding of disaggregated cells. (B) Proposed lineage hierarchy of different lung epithelial cfu subsets.