Co-colonization and Co-culture of Lung Alveoli Epithelial Stem Cells and Their Endothelial Niche Cells.

The maintenance of lung function relies crucially on the homeostatic replacement and post-injury regeneration of the lung alveolar epithelium (Hogan et al., Cell Stem Cell 15(2):123-138, 2014). Dysfunctions in these processes contribute to the pathogenesis of numerous lung diseases (Hogan et al., Cell Stem Cell 15(2):123-138, 2014; Matthay et al., J Clin Invest 122(8):2731-2740, 2012). While identifying stem and progenitor cells in the lung epithelium has significantly enriched our understanding of endogenous replacement and regenerative mechanisms (Hogan et al., Cell Stem Cell 15(2):123-138, 2014), it is clear that epithelial cells interact closely with mesenchymal components which create a micro-environmental niche that is vital for regulating both homeostatic replacement and post-injury regeneration of epithelial cells. Specific subsets of alveolar type II cells (AT2) behave as epithelial stem cells of the distal lung. We have identified a CD44^high subpopulation of AT2 cells that are preferentially located near macro-blood vessels and manifest stem cell characteristics (Chen et al., Stem Cell Rep 19(6):890-905, 2024; Am J Physiol Lung Cell Mol Physiol 313(1):L41-l51, 2017). In addition, the macro-blood vessels endothelial cells (ECs) function as niche components to support the CD44^high AT2s. Here, we describe the method to identify the CD44^high AT2 cells by immuno-fluorescence and co-culture of CD44^high AT2 cells with lung endothelial cells-their potential niche component-in 3D organoid culture.