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Fig. 1 | Stem Cell Research & Therapy

Fig. 1

From: SOX9 inactivation affects the proliferation and differentiation of human lung organoids

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).

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