Fig 2: SOX17 knockout impairs arterial specification and definitive hematopoiesis(A) Schematic diagram of hematopoietic development and in defined conditions. D, day of differentiation.(B) HB-CFC potential of wild-type or SOX17-/- H9 cells (means ± SDs, for 2 independent experiments performed in duplicate). **p < 0.01, t test.(C) Flow cytometric analysis of day 4 HE.(D and E) Flow cytometric analysis of day 5 HE. Graphs show the percentages and total number of cells generated from 104 hESCs (means ± SDs, n = 3 experiments). **p < 0.01 and ***p < 0.001, t test.(F) Schematic diagram of experiments. Wild and SOX17-/- cells were purified using CD31 magnetic-activated cell sorting (MACS) on day 4 and plated on OP9 or OP9-DLL4 for 5 days.(G) Hematopoietic colony-forming potential of day 4 HE after 5 days of culture on OP9 or OP9-DLL4 (means ± SDs, n = 2 experiments). ****p < 0.0001, 2-way ANOVA, Tukey’s multiple comparisons test.(H) Flow cytometric analysis of T cell differentiation.(I) Graphs show the total number of T cells generated from 104 CD43+ cells (means ± SDs, n = 3 experiments). ****p < 0.0001, 2-way ANOVA, Tukey’s multiple comparisons test.See also Figure S1.

Fig 3: SOX17 induction promotes specification of DLL4+CXCR4+ AHE with superior lympho-myeloid potential on day 5 of differentiation(A) Schematic diagram of experiments.(B) Flow cytometric analysis of HE on day 5 of differentiation in DOX+ and DOX- conditions.(C and D) Graphs show the percentages and total number of cells generated from 104 hESCs (means ± SDs and triplicated independent experiments). *p < 0.05, **p < 0.01, and ***p < 0.001, 2-way ANOVA, Sidak’s multiple comparisons test.(C) DOX effect on HE formation on day 5 of differentiation (day 5 HE).(D) DOX treatment enhances specification of DLL4+CXCR4+ arterial type HE.(E) Limiting dilution assay to determine the frequency of hemogenic progenitors in DLL4+CXCR4- and DLL4+CXCR4+ HE cultures with or without DOX.(F) CFC potential of HPs collected after 5 days of culture of indicated day 5 HE subset (means ± SDs, n = 2 experiments performed in duplicate). **p < 0.01 and ****p < 0.0001, 2-way ANOVA, Tukey’s multiple comparisons test.(G) Graphs show the total number of T cells produced from 104 HPs collected after 5 days of culture of indicated day 5 HE subset (means ± SDs, n = 2–3 experiments). *p < 0.05 and **p < 0.01, 1-way ANOVA, Tukey’s multiple comparisons test.See also Figure S3.

Fig 4: SOX17 enhances arterial specification and definitive lympho-myeloid potential of HE at day 4 of differentiation(A) Schematic diagram of experiments.(B) Effect of SOX17 overexpression during days 2–3 on HB-CFCs (means ± SDs, n = 2 experiments). Graph shows HB-CFCs per 104 cells collected on day 3 of differentiation.(C) Expression of arterial markers and Venus reporter in iSOX17 cells on day 4 of differentiation with or without DOX. Scale bars, 200 µm.(D and E) SOX17 overexpression increases the percentages and total numbers of VEC+ cells and AHE on day 4 of differentiation from 104 hESCs. Results are means ± SDs, n = 3 experiments; **p < 0.01 and ****p < 0.0001, 2-way ANOVA, Sidak’s multiple comparisons test.(F) CFC potential of HP collected following culture of day 4 HE on OP9 or OP9-DLL4 for 5 days (means ± SDs, n = 2 experiments performed in duplicate). *p < 0.05, **p < 0.01, and ****p < 0.0001, 2-way ANOVA, Tukey’s multiple comparisons test.(G) Graphs show the total number of T cells produced from 104 HPs collected following culture of day 4 HE on OP9 or OP9-DLL4 for 5 days (means ± SDs, n = 3 experiments). *p < 0.05, ***p < 0.001, and ****p < 0.0001, 2-way ANOVA, Sidak’s multiple comparisons test.See also Figure S2.

Fig 5: Model of SOX17 action in HESOX17 upregulates expression of genes associated with NOTCH signaling and binds directly to the CDX2 promoter, upregulating CDX2 expression. These molecular events lead to the upregulation of HOXA gene expression and establishment of AHE with robust lympho-myeloid potential and DLL4+CXCR4+ phenotype resembling AHE at the AGM region.