Generation of alveolar organoids from human induced pluripotent stem cells using a stirred-tank bioreactor.

Introduction: Organoids are increasingly establishing themselves as a valuable, patient- centered alternative to preclinical animal models as they are highly complex models for organ development studies and, in particular, robust disease models for mechanistic investigations and therapy response assessments. Methods: We previously established a very simple and practical protocol for producing induced pluripotent stem cell-derived lung organoids (iPSC-LuOrgs) from pre-differentiated embryoid bodies using a stirred bioreactor system. Here we adapted the protocol to investigate the generation of lung organoids directly from iPSCs in stirred suspension, and performed a comprehensive cellular and molecular characterization, including single-cell RNA sequencing, of the resulting LuOrgs. Results: Direct transfer of iPSCs as a single-cell suspension into the bioreactor and subsequent differentiation led to the formation of lung structures that appeared less differentiated and were predominantly alveolar in nature. Fibroblasts and bronchial epithelial cells were generated only to a limited extent. Discussion: Pre- differentiation of iPSCs, e.g., in embryoid bodies seems to be a necessary prerequisite for the efficient generation of iPSC-LuOrgs, which then exhibit alveolar and bronchial cell types and structures supported by mesenchymal cells.