Abstract
Lung epithelial progenitors differentiate into alveolar type 1 (AT1) and type 2 (AT2) cells. These cells form the air-blood interface and secrete surfactant, respectively, and are essential for lung maturation and function. Current protocols to derive and culture alveolar cells do not faithfully recapitulate the architecture of the distal lung, which influences cell fate patterns in vivo. Here, we report serum-free conditions that allow for growth and differentiation of mouse distal lung epithelial progenitors. We find that Collagen I promotes the differentiation of flattened, polarized AT1 cells. Using these organoids, we performed a chemical screen to investigate WNT signaling in epithelial differentiation. We identify an association between Casein Kinase activity and maintenance of an AT2 expression signature; Casein Kinase inhibition leads to an increase in AT1/progenitor cell ratio. These organoids provide a simplified model of alveolar differentiation and constitute a scalable screening platform to identify and analyze cell differentiation mechanisms.
Highlights
Lung alveolar progenitor cells differentiate at saccular stages (E17 in mouse, 26 weeks in human) into flattened alveolar type 1 (AT1) cells, constituting the surface for gas exchange, and secretory type 2 (AT2) cells, which prevent alveolar collapse by secreting pulmonary surfactant (Morrisey and Hogan, 2010; Chao et al, 2015)
Differentiation of progenitors into AT1 and AT2 cells is regulated by multiple signals including WNT (Wingless and Int-1 ) and FGF (Fibroblast Growth Factor) from the mesenchyme (Volckaert and De Langhe, 2015; Li et al, 2018), mechanical forces (Li et al, 2018), epigenetic modifications (Wang et al, 2016), and the extracellular matrix (ECM) (Kim et al, 2018)
Mouse fetal alveolar epithelial progenitors are maintained in 3D serumfree cultures
Summary
Lung alveolar progenitor cells differentiate at saccular stages (E17 in mouse, 26 weeks in human) into flattened alveolar type 1 (AT1) cells, constituting the surface for gas exchange, and secretory type 2 (AT2) cells, which prevent alveolar collapse by secreting pulmonary surfactant (Morrisey and Hogan, 2010; Chao et al, 2015). Despite the central role of AT1 cells in lung development (Zepp et al, 2021), AT1 cell isolation and generation in vitro have been longstanding obstacles, and the efficiency of AT1 cell generation in vitro, or the properties of the cells obtained in these cultures, have not been analyzed in detail, limiting the relevance of these models for cell differentiation studies These shortcomings highlight the need for an alveolar organoid model with morphological and cellular features as observed in vivo, which would accelerate the identification of differentiation-p romoting factors. To help address these questions, we developed a serum-free, rapid, and scalable distal lung progenitor organotypic culture system that recapitulates both AT1 cell differentiation and the endogenous tissue architecture of the alveolizing lung, which will facilitate mechanistic investigations of alveolar development
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