Extended Data Figure 4: Identification of transcription factors that confer multi-lineage haematopoiesis in vivo.

a, SNP analysis of engrafted blood cells compared with hiPSC and cord blood MNCs. SNP array genotyping was conducted to confirm the origin of engrafted human cells from hiPSCs in representative mice. SNP genotypes for human CD45⁺ cells taken from bone marrow of engrafted mice, original hPSCs and reference cord blood MNCs were clustered, as shown, showing concordance of original hiPSCs and human cells recovered from bone marrow of engrafted mice. b, Tabular presentation of SNP data: concordance >99% indicates identity between cell types. Green highlight shows that original hPSC line (34hiPSC or H9 hESC) corresponds with human CD45⁺ cells in engrafted mice. Comparison with different hPSC lines or cord blood MNCs did not achieve 99% concordance, validating the SNP array as a means of defining origin of cells. c, Transgene detection in engrafted cells of primary recipients. CD33⁺ myeloid cells, CD19⁺ B cells, and CD3⁺ T cells were isolated from the human CD45⁺ population of bone marrow at 10 weeks from five independent mice. Genomic DNA of each cell type was analysed by PCR to detect integrated lentivirus. Identification number of recipients is shown (numbers 1, 5, and 6 were engrafted with hiPSC-derived haemogenic endothelium; numbers 2 and 3 were engrafted with hESC-derived haemogenic endothelium). L, left femur (injected side); R, right femur; +, positive control (lentiviral vector with each transcription factor). −, negative control (lentiviral vector without transcription factor). d, Overlap between transcription factors that conferred in vivo engraftment from hiPSC- and hESC-derived cells injected into mice and in vitro multi-lineage CFU potential. Transcription factors detected from genomic DNA PCR from in vitro colony screening and in vivo engraftment screening are shown. For in vivo screening, multiple cell lines (iPSC and ESC) were used. Factors detected by in vivo screening were overlapped with those detected by in vitro screening (RUNX1 and LCOR). Consistently, MYB and RORA were detected by in vitro screening as previously reported⁷. Overall, RUNX1, LCOR, SPI1, ERG, HOXA5, HOXA9, and HOXA10 (defined seven transcription factors) were identified in individual experiments with different PSC lines in vivo. e, Factor-minus-one approach to define essential transcription factors for engraftment. Haemogenic endothelium was infected with combination of seven transcription factors minus one each, as indicated, then transplanted into NSG mice. At the 8 week time point, engraftment of human CD45⁺ cells in bone marrow was determined by FACS. Each panel indicates a representative result of GFP vector: all seven transcription factors, and seven minus RUNX1, ERG, SPI1, LCOR, HOXA5, HOXA9, or HOXA10 as indicated in the panel. Reduction of chimaerism was seen when RUNX1, ERG, LCOR, HOXA5, or HOXA9 were removed. In contrast, omitting SPI1 or HOXA10 had a negligible effect on engraftment.