The discovery of the Cystic fibrosis (CF) gene in 1989 has paved the way for incredible progress in treating the disease such that the mean survival age of individuals living with CF is now ~58 years in Canada. Recent developments in gene targeting tools and new cell and animal models have re-ignited the search for a permanent genetic cure for all CF. In this review, we highlight some of the more recent gene therapy approaches as well as new models that will provide insight into personalized therapies for CF.
Human PSC (hPSC) differentiations can capture developmental phenotypes and processes and are useful for studying fundamental biological mechanisms driving tissue morphogenesis and cell lineage development. Here, we show for the first time the temporal development of lung cell lineages using hPSC that model developmental milestones observed in primary tissue, the generation of renewable fetal lung epithelial organoids, and the functional utility of the lung models at different differentiation stages for Cystic fibrosis disease modeling. We first show the presence of hPSC-derived lung progenitor cells reminiscent of early trimester lung development and can capture a population enriched with basal stem cells that generates renewable airway organoids. Maturation and polarization in air liquid interface (ALI) generates additional epithelial cell lineages found in adult lung tissues including pulmonary neuroendocrine, brush, mature basal, ciliated and secretory cell types. Finally, pseudotime analysis of the integrated datasets from the fetal and ALI stages reveal the developmental trajectories of the cells as they emerge during differentiation. Overall, hPSC differentiation can capture aspects of human lung development and potentially provide important insight into congenital causes of diseases.
Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air‐liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic‐activated cell sorting system, and the generation of adult‐like airway epithelia through air‐liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air‐liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture
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