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.

Keywords: CRISPR/Cas9; Differentiation; Lung organoids; Proliferation; SOX9.

Fig. 1

Generation of SOX9^−/− hESCs and differentiation of human lung organoids. A Schematic structures of the SOX9 gene and protein. gRNA sites (gRNA1 and gRNA2) are indicated. DIM, dimerization domain; HMG, high-mobility group domain; K2/PQA/TA, transactivation domains. B Sequenced genotypes of SOX9 wild-type (WT) and knockout (SOX9^−/−) clones. The targeted mutation in each allele (Al-1 and Al-2) is indicated. The protospacer adjacent motif (PAM) is shown in red and gRNA sequences are shown in blue. Deletions in SOX9 are indicated on the right side. C Morphology of SOX9 wild-type (WT) and knockout (SOX9^−/−) hESCs. Scale bars, 500 μm. D mRNA expression levels of pluripotency-related genes. E Schematic of the directed differentiation protocol for generating alveolar organoids from hESCs. F Representative bright-field images of the differentiation time course of WT and SOX9^−/− cell lines. Scale bars, 500 μm. G mRNA expression levels of differentiation markers (n = 3, representative of 3 separate experiments). *p < 0.05, **p < 0.01 (unpaired 2-tailed Student’s t test). POU5F1, embryonic stem cell marker; FOXA2 and SOX17, definitive endoderm markers; NKX2.1, lung progenitor marker; SOX2, embryonic stem cell and proximal airway progenitor marker; SOX9, distal progenitor marker. H Immunofluorescence labeling of NKX2.1, SOX2, SOX9, and Ac-TUB in day 21 WT and SOX9^−/− organoids. Scale bars, 50 μm. I Quantification of NKX2.1, SOX2 and SOX9 positive cells in H. NKX2.1: n = 12 (WT), n = 10 (SOX9^-/-); SOX2, SOX9: n = 16 (WT), n = 14 (SOX9-/-). *p < 0.05, ****p < 0.0001 (unpaired 2-tailed Student’s t test).

Fig. 2

SOX9^−/− does not affect the maturation of AT1 and AT2 cells. A Representative bright-field images of SOX9 wild-type (WT) and knockout (SOX9^−/−) organoids on day 31 or 65. Scale bars, 500 μm. B mRNA expression levels of AT2 markers (SP-B, SP-C, and LAMP3) and AT1 marker (AGER, AQP5, PDPN) in organoids on days 31 and 65 relative to H9 hESCs. Data represent mean ± SEM (n = 3 independent experiments). C, D Immunofluorescence labeling of AT2 markers (SP-C) and AT1 markers (PDPN and AQP5) in organoids on day 31 (C) and day 67 (D). Scale bars, 50 μm

Fig. 3

SOX9^−/− does not affect the maturation of airway organoids. A Schematic of the protocol for differentiation of organoids from day 21 NKX2.1⁺ lung progenitors. B Bright-field images of SOX9 wild-type (WT) and knockout (SOX9^−/−) airway organoids on days 35 and 48. Scale bars, 500 μm. C mRNA expression levels of differentiation markers (n = 3, representative of 3 separate experiments). NKX2.1, LP marker; P63, basal cell marker; FOXJ1, ciliated cell marker; SCGB1A1, club cell marker; MUC5AC, goblet cell marker. D Immunofluorescence labeling of airway markers in organoids on day 35. Scale bars, 500 μm (left); 500 μm (right, WT); 100 μm (right, SOX9^−/−)

Fig. 4

SOX9^−/− reduces proliferation and promotes apoptosis in lung organoids. A Bright-field images of day 98 SOX9 wild-type (WT) and knockout (SOX9^−/−) alveolar organoids and day 123 WT alveolar organoids. Scale bars, 500 μm. B mRNA expression levels of markers in day 98 WT and SOX9^−/− alveolar organoids (n = 3, representative of 3 separate experiments). C Immunofluorescence labeling of cell proliferation marker Ki67 (green) in day 98 WT and SOX9^−/− alveolar organoids. Scale bars, 100 μm (left); 50 μm (right). D Number of Ki67-positive cells in day 98 WT (n = 6) and SOX9^−/− (n = 5) alveolar organoids. ***p < 0.001 (unpaired 2-tailed Student’s t test). E Immunofluorescence labeling of apoptosis marker cleaved caspase-3 (green) and epithelial marker E-cadherin in day 98 WT and SOX9^−/− alveolar organoids. Scale bars, 100 μm (left); 50 μm (right). F Number of cleaved caspase-3–positive cells in day 98 WT (n = 9) and SOX9^−/− (n = 5) alveolar organoids. *p < 0.05 (unpaired 2-tailed Student’s t test). G Schematic of the protocol for single-cell passaging of day 21 NKX2.1⁺ lung progenitors. (H) Bright-field images of day 41 (single cell passaged once [P1]) and day 59 (single cell passage twice [P2]) airway organoids. Scale bars, 500 μm. I, J mRNA expression levels of TSP1 during alveolar organoid (I) or airway organoid (J) differentiation (n = 3)

Fig. 5

SOX9^−/− promotes the emergence of SCGB1A1⁺ club cells and inhibits the maturation of MUC5AC⁺ goblet cells after long-term engraftment under kidney capsule. A Schematic of the experimental design. B Images of organoids 5 months after transplantation. C–H Immunofluorescence labeling of vascular marker CD31, epithelial marker E-cad and lung lineage markers in SOX9 wild-type (WT) and knockout (SOX9^−/−) organoids transplanted under kidney capsule. Scale bars, 50 μm (C, D, and F, right panels of each sample; E, right panel of WT); 100 μm (C, D, and F, left panels of each sample; E, SOX9^−/− and left panel of WT; G; H).

Fig. 6

SOX9^−/− does not affect the differentiation of alveolar cells or their role in promoting the functional recovery of injured mice. A Schematic of the experimental design. B–D Immunofluorescence labeling of human mitochondria marker MAB1273, AT2 marker pro–SP-C, AT1 marker human PDPN (HU-PDPN), and distal progenitor marker SOX9 in transplanted SOX9 wild-type (WT) and knockout (SOX9^−/−) lung progenitor organoids. White arrowhead in B indicates cells coexpressing MAB1273 and pro–SP-C; white arrowhead in C indicates cells coexpressing HU-PDPN and pro–SP-C. n = 4 (WT), n = 5 (SOX9^-/-). Scale bars, 100 μm (B, left panels of each sample); 50 μm (B, right panels of each sample; C; D). E Hematoxylin and eosin (H&E) and Masson’s trichrome staining of transplanted WT and SOX9^−/− lung progenitor organoids. Scale bars, 500 μm (top); 100 μm (bottom). F Arterial blood gas analysis. Partial oxygen pressure (pO₂), partial carbon dioxide pressure (pCO₂), and oxygen saturation (sO₂) were measured in WT and SOX9^−/− organoid-transplanted mice. Data represent mean ± SEM (n = 3)