Optimizing the in vitro colony-forming assay for more efficient delineation of the interaction between lung epithelial stem cells and their niche

The use of in vitro 3D organoid/colony forming assay (CFA); which mimics the in vivo environment have provided insight into the mechanisms by which lung stem cells maintain and repair the lung. In recent years, the use of CFA has markedly expanded. However, variations among laboratories in lung cell isolation methods, media used, type, origin, and processing methods of mesenchymal cells used as feeders for the epithelial colonies, and terms utilized to describe and quantify the growing colonies, have caused difficulty in reproducing results among different labs. In this study, we compared several previously described methods for lung cell isolation and culture media, to identify their influence on retrieved cells and growing colonies. We also characterized the effect of freeze/thaw, and propagation of fibroblasts on their ability to support epithelial colonies. Importantly, we suggested markers to identify fibroblast subtypes that offer the best support to alveolar stem cell proliferation. Then, we used our optimized assay to confirm the in vitro identity of recently described epithelial progenitors. We also tested the effect of hyperoxia on lung stem cells, and examined the expression of the receptors for the SARS-COV-2 virus entry into epithelial cells, on our organoids. In summary, our findings facilitate CFA standardization, help understand how niche cell variations influence growing colonies, and confirm some of the recently described lung stem cells. and the effect of their modulation on lung stem cell activation, proliferation, and differentiation In this follow up study, we first examined several aspects of the stem and niche cell collection and culture methods, aiming to increase the efficiency and physiological aspects of the assay. We then used the optimized assay to characterize various lung fibroblast subtypes with regards to their in vitro ability to support alveolar stem cell proliferation and differentiation in light of the recent in vivo characterizations Furthermore, we utilized the assay to identify aspects of the hyperoxia effect on lung stem cells. Then, we examined the similarity between our in vitro-grown colonies, and the previously described lineage-negative epithelial progenitors (LNEP), and alveolar epithelial progenitors (AEP) Finally, in light of the current COVID-19 pandemic, and the urgent need to conduct more basic research to understand various aspects of the function of the causative virus; COV-SARS-2, and the lung epithelial cells response to it, we examined the expression of the virus's entry and priming receptors, Ace2 and Tmprss2 respectively, on our organoids.