Introduction and objectives Regeneration of healthy lung tissue in patients with end-stage respiratory disease (ESRD) would cure disease, rather than treating symptoms. For this a detailed understanding of lung development is needed and the mouse has been used extensively as an in vivo genetically-modifiable model. Differentiation and validation of human induced pluripotent stem cells (hiPSCs) is entirely based on mouse literature. The most important epithelial stem cell population in developing lungs is found in distal branching tips, and these Sox9+ lung epithelial stem cells (LESCs) generate all epithelial lineages. Our objective was to develop a self-renewing, genetically-modifiable epithelial in vitro culture system from human embryonic LESCs and differentiate them into alveolar and bronchiolar cells.
Methods Human embryonic LESCs were characterised using genome-wide transcriptional analysis (RNAseq) and immunohistochemistry (5–20 post-conceptional weeks). LESCs were microdissected and self-renewing expansion in 3D organoid culture was established empirically. Using RNAseq and immunohistochemistry, we assessed the similarities between cultured and fresh LESCs. Genetic stability was evaluated by karyotyping. Organoids were differentiated in vitro, or in vivo using xenotransplantation into bleomycin-injured adult mouse lungs or kidney capsule. Gene editing was done using CRISPR-Cas9 to delete SOX9.
Results RNAseq of LESCs identified broad-scale transcriptional differences between mouse and human embryonic lung stem cells. Human LESCs were successfully expanded for over 10 months as karyotypically-stable 3D organoids using a combination of 7 signalling molecules. The LESC stem cell markers, transcriptome and organoid morphology were maintained throughout the culture period. Bronchiolar and alveolar differentiation was achieved in vitro and in vivo. Moreover, xenotransplantation of organoids into bleomycin-injured adult mouse lungs was extremely efficient. Knocking out SOX9 led to a loss-of-self-renewing phenotype.
Conclusions Our novel genetically-modifiable human embryonic lung culture system enables for the first time the in vitro study of human lung development and disease modelling. We anticipate that this work will transform lung regenerative medicine by guiding the development of improved protocols for hiPSC differentiation and manipulation of adult stem cells in vivo, with benefits for patients with ESRD. Additionally, our protocol for xenotransplantation following lung injury provides the first method for efficient future cell therapy.