Abstract

Lung organoid models derived from mouse and human stem cells have become a powerful tool to study lung development and disease. Especially during the last years, human lung organoid models have become even more relevant for modelling SARS-CoV-2 infection and drug screening. Here, we describe a hiPSC derived lung organoid model and its potential to model viral infection. To generate hiPSC derived lung organoids, the pluripotency of the hiPSC lines was first verified by the expression of Nanog, Oct4, TRA-1-81, TRA-1-60, SSEA-3, SSEA-4 by flow cytometric analysis, Western blot and immunofluorescence. Subsequently, hiPSC were differentiated into CXCR4, CKit, FOXA2 and SOX17-expressing endoderm followed by generation of NKX2.1+ ventral-anterior foregut endoderm. Expression of NKX2.1, SOX9 and SOX2 in the developed foregut spheroids was confirmed by immunofluorescence. After 4-6 weeks, these spheroids formed branched structures containing differentiated cells, such as MUC5A+ goblet cells, SCGB1A1+ club cells and SFTPB+SOX9neg alveolar epithelial type 2 cells (AEC2). To model viral infection, 6 weeks old organoid cultures were infected with wt SARS-CoV2/München-1.2/2020/984. Notably, 72 hours post infection, both viral infection and replication were confirmed by the determination of the virus titer, qPCR and immunofluorescence of SARS-CoV-2 envelope-protein and SARS-CoV-2 nucleo-protein. In summary, we were able to successfully generate and infect hiPSC derived lung organoids, indicating that this system could be used in future functional studies not only to model other viral infections and epithelial repair, but also in the context of lung development and other diseases such as fibrosis and cancer.