(F1091) DIFFERENTIATION OF LUNG EXPANDABLE EPITHELIAL PROGENITORS IN AIR-LIQUID INTERFACE OR IN 3D CULTURE FOR FUNCTIONAL MODELING OF ALVEOLI STRUCTURE FORMATION

Abstract: The regeneration of human respiratory epithelium is a continuous and complex process. Cultivating cells from the distal regions of the lungs that form pulmonary alveoli has been particularly challenging due to their intricate, highly organized structure and the rapid onset of differentiation or replicative senescence in vitro. Recent advancements have enabled the differentiation of lung progenitors into a mosaic of surfactant-producing alveolar type 2 (AT2) cells and gas-exchanging alveolar type 1 (AT1) cells. This process relies on the activation of Wnt/Yap signaling and the inhibition of TGFβ pathways to achieve a more mature phenotype.
However, the molecular and cellular mechanisms underlying alveolar development remain poorly understood, and reproducible in vitro models that accurately mimic the biology and pathology of the respiratory system are still lacking.
Our lab established a protocol for generating human embryonic stem cell (hESC)-derived expandable lung epithelial progenitors (ELEPs) that express NK2 Homeobox 1 (NKX2.1) and prosurfactant proteins B and C, forming airway and alveolar structures in 3D cultures or in vivo. ELEPs can also be derived from human-induced pluripotent stem cells (iPSCs) from fibroblasts. We aimed to differentiate ELEPs into mature alveolar-type cells under air-liquid interface (ALI) or 3D culture conditions using various factors and inhibitors. Differentiation was evident morphologically and at the protein level, including SPC processing, caveolin-1 induction, and nascent extracellular matrix protein production (e.g., collagen I) in ALI cultures.
Prolonged differentiation under these conditions activated ER stress pathways, leading to senescence and transitional AT2-AT1 phenotypes, which are typical in lung pathologies. In contrast, 3D cultures prolonged cell viability and supported protein production as well as the formation of structures resembling the alveolar cell network. These findings suggest that our progenitor cells can serve as a valuable in vitro model of human respiratory epithelium for studying development, disease mechanisms, and potential applications in preclinical drug screening.

Funding Source: This work was supported by the Czech Science Foundation (grant no. GA23-06675S), MZČR-RVO (FNBr, 65269705) and by Masaryk University (grant no. MUNI/A/1598/2023).

Clinical Trial ID number: