SOX9 inactivation affects the proliferation and differentiation of human lung organoids

Abstract

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

SOX9 maintains human foetal lung tip progenitor state by enhancing WNT and RTK signalling

“…Strong depletion of gRNAs targeting MYBL2 and CTNNB1 (Fig 1E) was consistent with MYBL2 being a central factor regulating cell proliferation and the importance of WNT signalling for tip progenitor cell growth (Musa et al , 2017; Hein et al , 2022). gRNAs targeting SOX9 were moderately depleted suggesting a role in tip progenitor self‐renewal, consistent with previous studies (Chang et al , 2013; Rockich et al , 2013; Li et al , 2021). By contrast, depletion of SOX2 did not markedly influence progenitor self‐renewal, consistent with a recent knockout study (Sun et al , 2021).…”

An organoid CRISPRi screen revealed that SOX9 primes human fetal lung tip progenitors to receive WNT and RTK signals

“…1E) was consistent with MYBL2 being a central factor regulating cell proliferation and the importance of WNT signalling for tip progenitor cell growth (Hein et al, 2021;Musa et al, 2017). gRNAs targeting SOX9 were moderately depleted suggesting a role in tip progenitor self-renewal, consistent with previous studies (Chang et al, 2013;Li et al, 2021;Rockich et al, 2013). By contrast, depletion of SOX2 did not markedly influence progenitor self-renewal, consistent with a recent knock-out study (Sun et al, 2021).…”

Neonatal lung-derived SSEA-1+ cells exhibited distinct stem/progenitor characteristics and organoid developmental potential

“…Further, Sox9 expression has been observed in the club-like stem/progenitor cells that the cell population was activated and differentiated into alveolar lineages in response to injury ( Kathiriya et al., 2020 ). And Sox9 inactivation affected the proliferative capacity of human lung progenitor cells ( Li et al., 2021 ). Thus, decreased Sox9 expression in adult pulmonary SSEA-1 + cells might result in decreased developmental activity or be maintained in a quiescent state.…”