Abstract
Introduction: Alveolar damage is a hallmark of a variety of lung diseases. Human induced pluripotent stem cells (hiPSCs) are increasingly used for generation of alveolar type II epithelial cells (iAEC2) to study alveolar injury and regeneration. The differentiation process is however still inefficient. The aim of the present study was to increase efficiency of iAEC2 generation from hiPSC.
Methods: Using an hiPSC lung progenitor NKX2.1-GFP reporter line, the impact of several compounds, cell seeding densities, and gel matrixes on the differentiation protocol was evaluated using gene expression, immunohistochemistry, flow cytometry and Incucyte® live-cell imaging analyses.
Results: A cell passage ratio of 1:12 in hiPSC-derived definitive endoderm (DE) cells combined with a vitronectin-coated surface, showed superior generation of anterior foregut endoderm (AFE) cells. Combined administration of the BMP inhibitors dorsomorphin and noggin was compared to further improve AFE differentiation. Results demonstrated higher efficiency in generating FOXA2+/ SOX17- AFE cells for dorsomorphin (85.9%) than noggin (47.1%) or when combined (59.5%). Live imaging during hiPSC differentiation revealed that in control cultures, ~7% of ventralized AFE cells expressed NKX2.1-GFP. Future experiments will use live imaging to read-out novel strategies to improve AFE to VAFE differentiation.
Conclusions: We showed that vitronectin-coated surfaces combined with a 1:12 cell passage ratio and dorsomorphin are essential for generation of the AFE lineage. Moreover, we proved the feasibility of real-time tracking of NKX2.1 expression using a reporter cell line combined with live imaging for protocol optimization.
Footnotes
Cite this article as Eur Respir J 2022; 60: Suppl. 66, 2992.
This article was presented at the 2022 ERS International Congress, in session “-”.
This is an ERS International Congress abstract. No full-text version is available. Further material to accompany this abstract may be available at www.ers-education.org (ERS member access only).
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