SOX9 inactivation affects the proliferation and differentiation of human lung organoids

The regulation of the transcription factor sex-determining region Y-box transcription factor 9 (SOX9) in lung development has been described in mouse, but the same principles apply to human lung development is unknown due to a lack of appropriate experimental approaches and models.

Here, we used gene editing technology to inactivate SOX9 in human embryonic stem cells that were then induced to differentiate into lung organoids to investigate the role of SOX9 in human lung epithelium development.

Complete knockout of the transactivation domain of SOX9 by gene editing resulted in indels in both alleles of SOX9. SOX9^-/- hESCs could be induced to differentiate into lung progenitor organoids. In vitro long-term expansion showed that SOX9 inactivation did not affect the differentiation of pulmonary epithelial cells, but promoted apoptosis and reduced proliferative capacity in the organoids. When lung progenitor organoids were transplanted under the kidney capsule of immunodeficient mice, expression of the club cell marker secretoglobin family 1A member 1 (SCGB1A1) was detected in SOX9^-/- transplants but was absent in wild-type (WT) transplants. The maturation of goblet cells was also affected by SOX9 inactivation, as evidenced by the presence of mucin 5 AC (MUC5AC) in the cytoplasm of SOX9^-/- grafts as compared to WT grafts in which most MUC5AC was secreted into the lumen. In vivo lung orthotopic transplantations showed that SOX9 inactivation had a limited effect on the differentiation of alveolar cells and lung regeneration in injured mice.

SOX9 modulates the proliferative capacity of lung epithelium but is not an indispensable transcription factor in the regulation of human lung epithelium development.