Fig. 1.

In vitro induction of MLL-AF4 in iPSC-derived hematopoietic cells. (A) The scheme of in vitro differentiation of iPSCs into hematopoietic cells. (B) Morphological change of iPSCs during the in vitro differentiation. (Scale bar, 1 μm.) (C) Colony-forming assay of iPSC-derived hematopoietic cells harvested at day 12 of in vitro differentiation. BFU-E, burst-forming unit-erythroid; GEMM, colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte; GM, colony-forming unit-granulocyte, macrophage; G, colony-forming unit-granulocyte. All are shown at 40× magnification. (D) Phenotypic analysis of iPSC-derived hematopoietic cells harvested at day 12 of in vitro differentiation. (E) The serial replating potential of iPSC-derived hematopoietic cells with or without (w/o) Dox induction. FACS-sorted GFP⁺ and GFP⁻ cells that were immediately seeded on methocult were labeled as “w/o DOX” group. Data are shown as mean ± SD of three independent experiments. (F) Flow cytometry analysis of MLL-AF4–induced iPSC-derived hematopoietic cell harvested at day 12 of in vitro differentiation. MLL-AF4 and rtTA plasmids were transfected followed by 72-h induction of MLL-AF4 with the addition of 2 μg⋅ml⁻¹ doxycycline. (G) Principle component analysis of RNA-Seq data from in vitro-derived HSPCs from iPSC (iPSC-HSPCs, CD34 iPSC, or MN iPSC derived) without MLL-AF4 transfection (CD34_w/o MA4; MN_w/o MA4) or with MLL-AF4 transfection (CD34 + MA4; MN + MA4), compared with the peripheral blood mobilized CD34⁺ HSC (mobHSC), and the publicly available dataset for common myeloid progenitor (CMP) and lymphoid-primed multipotent progenitor (LMPP). (H) GSEA signatures of in vitro-derived iPSC-HSPCs without MLL-AF4 transfection (w/o MA4) or with MLL-AF4 transfection (+MA4). P < 0.05 and false discovery rate (FDR) <0.25 were considered significant conditions. (I) Heat map showing relative gene expression of regulatory genes identified as HSC specific in the indicated cell types.