| Summary: | Background Alveolar organoids may be useful tools in drug discovery for lung diseases, such as chronic obstructive pulmonary disease, and for studying the effects of respiratory viruses, such as severe acute respiratory syndrome coronavirus 2. Induced pluripotent stem cell (iPSC)-derived alveolar organoids offer ethical and cost-effective alternatives to animal testing and primary cell-based methods. In this study, we present generating alveolar organoids from iPSCs and compare the efficiency of generating iPSCs from alveolar type 2 (AT2) and umbilical cord blood (UCB) cells. Methods The protocol started with a two-dimensional culture and transitioned to a three-dimensional culture using Matrigel after the endoderm stage. Organoid cultivation lasted for at least 40 days, and the characteristics of alveolar organoids were assessed using flow cytometry, real-time polymerase chain reaction, and immunostaining. Results iPSCs derived from AT2 cells showed a better ability to generate alveolar organoids than those derived from UCB cells. This difference in the ability of AT2 iPSCs and UCB iPSCs to generate alveolar organoids appeared during the definitive endoderm differentiation stage. AT2 iPSCs showed higher expression of the anterior foregut endoderm marker SOX2 and lung progenitor gene expression markers, such as NKX2.1 and CPM, which are associated with the lung progenitor differentiation stage. Conclusion This protocol successfully generated alveolar organoids from AT2 iPSCs; however, the efficiency of differentiation varied depending on the origin of the iPSCs. This study also found differences in gene expression and developmental potential between iPSCs, which may have contributed to the observed differences in differentiation efficiency.
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