Fig. 3.

iHSPCs but not primary HSPCs undergo the leukemic transformation during the long-term engraftment period. (A) Chimerism of human cells in the bone marrow of recipient mice over 16 wk posttransplant. (B) Phenotypic analysis of engrafted human cells in the bone marrow (BM) of recipient mice with long-term engraftment. Gated human CD45⁺ cells were analyzed for myeloid (CD33) and lymphoid (CD10) (Mye/Lym) lineage distribution, percentage of progenitor (CD34⁺CD38⁻, CD7⁺CD45RA⁺), and B cell (CD19) and T cell (CD3) compartments (B/T). (C) Engraftment of primary HSPCs with (+MA4) or without (w/o MA4) transfection of MLL-AF4 in the peripheral blood (PB), spleen, and BM of recipient mice after 12–16 wk of transplantation. (D) Phenotypic comparison of engrafted human CD45⁺ cells in the BM of MLL-AF4 transfected transplants or nontransfected transplants. (E) Profiling of B cell leukemia-associated mutations in the in vitro-derived iPSC-HSPCs without MLL-AF4 transfection (CD34 vitro_w/o MA4; MN vitro_w/o MA4) or with MLL-AF4 transfection (CD34 vitro_MA4; MN vitro_MA4), and in vivo-derived human CD45⁺ cells engrafted in the BM of primary recipients over 9–16 wk posttransplant with MLL-AF4 transfection. CD34_wk9, CD34-iPSC derived HSPCs transfected with MLL-AF4 were transplanted and harvested for human CD45⁺ cells at 9 wk posttransplant; the same meaning for MN-iPSC group. HSPC + MA4_wk16, BM cells harvested from engrafted recipients transplanted with primary HSPCs with MLL-AF4 induction at 16 wk posttransplant; MNCs, peripheral blood-derived mononuclear cells; mobHSC, mobilized HSCs. (F) PCA analysis of RNA-Seq data from in vivo-derived human CD45⁺CD34⁺ cells in the bone marrow of primary recipient mice transplanted with MLL-AF4 transfected primary HSPCs at 16 wk posttransplant (MA4 treated HSPC_vivo), compared with the MLL-AF4 transfected iPSC-HSPCs (CD34-iPSC derived) before transplantation (MA4-treated iPSC-HSPC_vitro) or 16 wk posttransplant (MA4-treated iPSC-HSPC_vivo), and mobilized HSC (mobHSC). (G) GO analysis of in vivo-derived human CD45⁺ cells from engrafted iHSPCs (16 wk posttransplant) compared with the in vitro-derived iPSC-HSPCs. Significantly deregulated genes enriched in hematopoietic cell lineage signature were indicated. (H) GO analysis of enriched features of MLL-AF4 transfected primary HSPCs compared with MLL-AF4 transfected iPSC-HSPCs. In vivo-derived cells of both groups were harvested at 16 wk posttransplant. Gray bars indicate the enriched features containing genes that are mostly down-regulated, and the opposite for the black bars. All RNA-Seq data were generated from three independent biological replicates and analyzed by the average FPKM (fragments per kilobase of transcript per million mapped reads) value.