Fig 2: Direct targets of SOX17 in hPGCLCs and DE.a, Cumulative distribution function plot showing the functional prediction of SOX17 and PRDM1. The ChIP peaks of SOX17/PRDM1 in hPGCLCs were assigned to genes with TSS within 100 kb of the peak summits. A regulatory potential score was calculated for each gene based on the distance between the peak summit and the TSS96. The genes were then divided into upregulated, downregulated and unchanged according to their expression patterns upon SOX17 or PRDM1 overexpression. Cumulative distribution function plot was generated for each group with genes ranked by decreasing regulatory potential. A one-tailed Kolmogorov-Smirnov test was used to determine the statistical significance between the differentially expressed groups and the unchanged group. Note that upregulated genes (but not downregulated genes) upon SOX17 induction have a significantly higher tendency to be bound by SOX17. In contrast, genes downregulated upon PRDM1 overexpression tend to be bound by PRDM1.b, GO biological process terms that were enriched in SOX17 direct up targets (red dots in Fig. 3d)c, Expression heatmap of SOX17 direct up target genes. Shown are the genes which were (1) upregulated both by SOX17 alone (log2(fold change) >1 and adjusted p-value <0.05 between Dex-treated and non-treated 12h PreME aggregates); (2) upregulated by cytokines (log2(fold change) >2 and adjusted p-value <0.05 between day 2 hPGCLCs and PreME); and (3) downregulated in SOX17 KO (log2(fold change) >1 and adjusted p-value <0.05 between SOX17 KO day 2 aggregate and WT day 2 aggregate)20.d, Top eight TFs with binding sites (ReMap2020) enriched in hPGCLC-specific and DE-specific SOX17 peaks.e, Genome browser snapshots showing the epigenetic landscape of DE-specific (CER1 and LEFTY2) and hPGCLC-specific (NANOS3 and PDPN) SOX17-bound gene targets.f, Heatmap showing expression of genes associated with the top enriched motifs (Fig. 3h) and the top enriched TF binding sites (in d) in hPGCLC-specific and DE-specific SOX17 peaks.g, UniProtKB Keywords that were enriched in PRDM1 direct down targets (blue dots in Fig. 3i).

Fig 3: RNA sequencing of tracheobronchial basal cells from children and adults(A) Cluster diagram showing the expression of 32 differentially expressed genes with a false discovery rate (FDR) < 0.01 and a log2 fold change > 1.2 in three pediatric (months of age/sex; 2.5M, 11M, and 14M) and three adult (years of age/sex; 58F, 61F, and 62F) freshly fluorescence-activated cell-sorted EpCAM+/PDPN+ basal cell samples. Gene order is based on hierarchical clustering based on the similarity in overall expression patterns. Red represents relative expression higher than the median expression and blue represents lower expression.(B) Pathway analysis was performed on the three pediatric and three adult freshly fluorescence-activated cell-sorted EpCAM+/PDPN+ basal cell samples using gene set enrichment analysis (GSEA) to interrogate Hallmark pathways from MSigDB. For pathways with FDR < 0.05, normalized enrichment scores are shown. A negative score (blue) represents upregulation of the pathway in the pediatric samples; a positive score (red) represents upregulation in the adult samples.(C) Cluster diagram showing the expression of 987 differentially expressed genes with a false discovery rate (FDR) < 0.01 and a log2 fold change > 1.2 in three pediatric (months of age/sex; 3M, 30M, and 83M) and three adult (years of age/sex; 58F, 60F, and 69M) cultured basal cell samples. Cells were cultured on 3T3-J2 mouse embryonic fibroblast feeder layers for two or three passages in epithelial cell culture medium containing Y-27632. Gene order is based on hierarchical clustering based on the similarity in overall expression patterns. Red represents relative expression higher than the median expression and blue represents lower expression.(D) Pathway analysis was performed on the three pediatric and three adult cultured basal cell samples using gene set enrichment analysis (GSEA) to interrogate Hallmark pathways from MSigDB. For pathways with FDR < 0.05, normalized enrichment scores are shown. A negative score (blue) represents upregulation of the pathway in the pediatric samples; a positive score (red) represents upregulation in the adult samples.

Fig 4: Altered proliferation and differentiation potential in pediatric airway basal cells compared to adult basal cells(A) Immunofluorescence images showing tracheosphere cultures derived from two pediatric and two adult basal cell cultures. Sections were stained with primary antibodies against keratin 5 (KRT5; purple), MUC5AC (yellow), and a nuclear counterstain (blue). Scale bar = 50 µm.(B) Proportion of EpCAM+/PDPN+ basal cells that formed colonies within 7 days after sorting single cells into 96-well plates in epithelial cell culture medium containing Y-27632 (mean ± SD; n = 3 donors/age group; n = 167–192 cells per donor; p = 0.048, two-tailed unpaired t-test). Brightfield microscopy images show a confluent pediatric well and an adult colony after 7 days of culture. Scale bar = 50 µm.(C) Population doubling analysis of the growth of pediatric and adult basal cell cultures in epithelial growth medium without Y-27632. Pediatric samples outperformed adults at passage 5 (p = 9.8 × 10-5, two-tailed unpaired t-test).(D) Ki67 immunofluorescence staining of passage one pediatric and adult basal cells cultured on feeder layers in epithelial cell culture medium without Y-27632 for 3 days before fixation. Ki67 positivity was quantified as a proportion of positive nuclei/total DAPI stained nuclei (mean ± SD; n = 3 donors/age group; mean of 1402 cells counted per donor, range 810 – 1600; p = 0.045, two-tailed unpaired t-test). The upper panel is an adult culture and the lower panel a pediatric culture.

Fig 5: Induction of hPGCLCs by CRISPR activation of key cis-regulatory elements.a, Generation of day 4 embryoid bodies from hESC lines harbouring the Dox-inducible CRISPRa transgene with the indicated sgRNA combinations. Note that co-activation of (1) SOX17 and PRDM1; or (2) TFAP2C, SOX17 and PRDM1, cis-regulatory elements led to the formation of NANOS3-tdTomato-positive hPGCLCs in the absence of BMP4. Experiment was repeated independently for 3 times with similar results.b, Validation of CRISPRa-induced hPGCLCs by RT-qPCR of key hPGC genes. Average of technical replicates, with individual replicates shown as data points and number of replicates indicated in the figure. PCR was replicated 3 times with similar results.c, Induction of hPGCLCs from hESCs, PreME and ME with or without activation of SOX17 enhancers. FACS analysis of day 4 EBs shows that the activation of SOX17 enhancers and the addition of BMP4 synergistically increased the efficiency of hPGCLC induction from hESCs and PreME, but not from ME. Orange boxes gate PDPN- and NANOS3-positive hPGCLCs.d, A model elucidating the key role of SOX17 enhancers in human germline competence.