Alveolar epithelial-like cell differentiation in a dynamic bioreactor: a promising 3D-approach for the high-throughput generation of lung cell types from human induced pluripotent stem cells

Purpose: Human induced pluripotent stem cell (hiPSC)-derived lung cell types such as alveolar epithelial cells are promising for toxicological and pharmaceutical in vitro screenings. Reproducible differentiation processes are highly demanded, but protocols which are suitable for the high-throughput generation of lung cell types from hiPSCs are lacking. Methods: In this study, a new approach for the hiPSC-differentiation in alveolar epithelial-like cells type 2 under dynamic 3D-conditions in a suspension bioreactor is presented. Gene and protein expression analyses of key markers during the embryonal lung development have been performed in comparison to cells differentiated under static 2D-conditions to evaluate the differentiation efficacy of the new bioreactor-based approach. Finally, the resulting cells were infected by SARS-CoV-2 pseudotypes to demonstrate their functionality and suitability for e.g. COVID-19 drug development. Results: The dynamic bioreactor is suitable to differentiate hiPSCs in spheroids, which express relevant lung markers in each developmental stage on gene and protein level. The 3D method is able to significantly increase the expression of some markers in comparison to conventional 2D differentiation. 3D-differentiated alveolar epithelial-like cells express functional SARS-CoV-2 receptors and can display the viral infection. Conclusion: The presented dynamic 3D-differentiation is a promising, new approach to generate alveolar epithelial-like cells from hiPSCs as cell source for in vitro lung models.