Mimicking the niche of lung epithelial stem cells and characterization of several effectors of their in vitro behavior

Abstract

The niche surrounding stem cells regulate their fate during homeostasis and after injury or infection. The 3D organoid assay has been widely used to study stem cells behavior based on its capacity to evaluate self-renewal, differentiation and the effect of various medium supplements, drugs and co-culture with supportive cells. We established an assay to study both lung and trachea stem cells in vitro. We characterized their proliferation and differentiation spectrum at baseline then evaluated the effect of co-culturing with fibroblasts and endothelial cells and/or treating with several biologically relevant substances as possible contributors to their niche. We found that lung epithelial (but not tracheal basal) stem cells require co-culture with stromal cells to undergo clonal proliferation and differentiation. Fibroblasts were more efficient than endothelial cells in offering this support and the pattern of support varied based on the tissue origin of the stromal cells. Treating distal lung epithelial or basal stem cells with FGF2, FGF9, FGF10, LIF as well as ALK5 and ROCK inhibitors increased their colony formation efficiency and resulted in variable effects on colonies number, size and differentiation spectrum. This model and findings pave the way for better understanding of lung stem cell niche components and factors that can manipulate lung stem cell behavior.

Effects of the common polymorphism in the human aldehyde dehydrogenase 2 (ALDH2) gene on the lung

“…We therefore isolated MTECs from all groups and compared them using the 3D stem cell colony forming assay [29]. Parallel culture wells were treated with vehicle, H 2 O 2 (as a source of ROS and oxidative stress), and/or the Aldh2 agonist, Alda-1.…”

“…6g). Because fibroblasts from individuals with the ALDH2*2 polymorphism are known to be more vulnerable to injury than WT fibroblasts [39] and because fibroblasts are an essential component of the lung stem cell niche [29], we also investigated whether fibroblasts isolated from the lungs of WT, Aldh2*2 Tg, and Aldh2 −/− mice have a different influence when co-cultured with WT tracheal basal stem cells in the colony forming assay. Co-culture with fibroblasts increased the CFE of WT tracheal basal stem cells, but no significant difference was observed among the three fibroblasts groups (Fig.…”

“…Tissue sections (6-μm thickness) were prepared and immunostained as described previously [29]. The primary antibodies used included rabbit K5 (Covance, Princeton, NJ, USA), goat CC10 (SCGB1A1) and Pro-surfactant protein C (SPC) (Santa Cruz Biotechnology), TTF1 (Abcam), mouse MUC5AC (Thermo Scientific), and mouse acetylated β -tubulin (Sigma) for the identification of basal, club (Clara), alveolar type II, goblet, and ciliated cells.…”

“…Collection of MTEC and whole lung epithelium, isolation of lung fibroblasts and their co-culture in Matrigel were essentially performed as described previously [29]. The medium used, treatment with H 2 O 2 and/or Alda-1, quantification and processing of colonies at the end point were performed in a manner similar to the human organoid culture described above.…”

Organoids as a model system for studying human lung development and disease

“…For example, basal cells are present throughout the human airways but are confined to the trachea in mice [27,28], and mucin-secreting goblet cells are common in human airways but rare in mice [70]. To this end, human lung epithelial cell lines that capture some phenotypic traits [72]. A number of newly identified factors that positively regulate human basal cell marker expression in bronchospheres have recently been described, suggesting that additional pathways may also control basal cell expansion in humans [49].…”