Extended Data Figure 2: Rationale for selecting candidate transcription factors for library screening.

a, Heatmap of the expression profile of HSC-specific transcription factors (TFs) in haemogenic endothelium (CD34⁺FLK1⁺CD43⁻CD235A⁻) versus fetal-liver HSCs (CD34⁺CD38⁻CD90⁺CD45⁺). Twelve HSC-specific transcription factors enriched in fetal-liver HSCs relative to haemogenic endothelium (blue box) were cloned individually into a Dox-inducible lentiviral vector. b, The expression level of SOX17, a marker of haemogenic endothelium, was 2.4-fold higher in haemogenic endothelium (N = 7) than fetal-liver HSCs (N = 10). *P < 0.001. c, The library was supplemented with genes identified in previous screens. Candidates in red were drawn from a previous screen by the Rafii group³; blue from the Rossi group²; green from the Daley group⁷. The final library of 26 candidates is shown. d, Diagram of the Dox-ON pInducer-21 lentiviral vector used in this study (top). rtTA3 and eGFP are driven by EF1α-promoter; infection efficiency is indicated by the GFP signal. e, GFP analysis by FACS and fluorescence 3 days after infection of haemogenic endothelium cells. Routinely, over 50% transduction efficiency was achieved. For transplantation, haemogenic endothelium cells infected at day 3 EHT were incubated for 24 h and injected into mice. Dox was provided for 2 weeks in vivo after transplantation into sub-lethally irradiated immune-deficient NSG mice. f, Scheme for screening the 26 transcription factors library, and resulting haematopoietic chimaerism. hPSC-derived haemogenic endothelium was cultured for an additional 3 days in EHT medium, then infected with the library of 26 transcription factors. Infected cells (100,000) were injected intrafemorally into sub-lethally irradiated (250 rad) NSG mice, which were treated with doxycycline for 2 weeks to induce transgene expression in vivo. g, FACS analysis of bone marrow and thymus of an engrafted recipient is shown. Human CD45⁺ cells from bone marrow were analysed for CD33⁺ myeloid cells, CD19⁺ B cells, and CD3⁺ T cells as indicated. Thymic cells were analysed for human CD4 and CD8, with percentages of single and double-positive cells indicated. A photograph of an engrafted thymus is shown (bottom right). Data shown as mean ± s.d.