Investigation of Epithelial Cell Fate Decisions in the Developing Human Lung Guides the Generation of Human Lung Organoid Model Systems

1 in 10 U.S. infants are born prematurely (www.cdc.gov), before the lungs have fully formed. The primary cause of mortality in these patients is underdeveloped lungs, but even surviving patients have a 40% chance of developing life-long lung problems. Our ability to effectively treat premature babies by speeding up lung development is hindered by the severe lack of knowledge about normal human lung development that is exacerbated by a need for better human cell-based developmental model systems. Mouse studies have shown that the lung develops as a network of epithelial tubes undergo repeated rounds of bifurcation within surrounding mesenchyme. This process, called branching morphogenesis, gives rise to the conducting airways and alveoli, where gas exchange takes place. Bud tip progenitor cells reside at the tips of branching buds during development and give rise to all adult lung epithelial cell types, including conducting airway-specific cells and alveolar cells. Bud tip progenitor cells terminally differentiate into alveolar cells at the very end of development and are not found in adult lungs. However, bud tip progenitors remain immature in premature infants, impeding gas exchange. Despite the importance of bud tip progenitors in premature lungs, it is unknown how this population of specialized progenitors is maintained during development, or what signals prompt these cells to differentiate into specific lung lineages. To address these questions, I first characterized bud tip progenitors in the native developing lung using bulk RNA sequencing and protein staining, and then developed a fetal bud tip progenitor organoid system to show that the growth factors Fibroblast Growth Factor 7 (FGF7), WNT and All-Trans Retinoic Acid (ATRA) are sufficient to maintain the bud tip progenitor cell state in culture. To determine how bud tip progenitors are instructed to give rise to differentiated epithelial cell types, I next employed single-cell RNA sequencing of the lung epithelium to identify signaling pathways active during development. Testing of these pathways in vitro using the bud tip progenitor organoids revealed that transient activation of the SMAD signaling pathway is sufficient to generate a population of cells with transcriptomes and functional properties mirroring adult lung basal stem cells, the resident stem cell of the adult airway. Finally, recapitulation of these developmental events using human pluripotent stem cells (hPSCs) in a dish led to the development of hPSC-derived lung bud tip organoids and efficient derivation of lung basal stem cells from hPSCs, which have many applications for future study of human lung development and disease without the use of donated human tissue.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/153506/1/ajanemil_1.pd