Fig. 4: Embryonic CD24⁺ PreProBs emerging in response to KMT2A::AFF1 display self-renewal capacity.

A Schematic illustration of the replating assay in semi-solid media. B Semi-solid in vitro cultures of CD24⁺ PreProBs control and KMT2A::AFF1^Vav-Cre+ fetal livers (FLs) from E18.5. Scoring and replating was done weekly. Total number of colonies/1.000 cells for individual embryos are shown. Means ± SD (4 experiments). C Frequencies of B (CD19⁺B220⁺) (blue) and myeloid (CD11B⁺GR1⁺) (red) cells detected by flow cytometry at different time points in the replating culture of KMT2A::AFF1^Vav-Cre+ CD24⁺ PreProBs. Mean percentages of total CD45⁺ cells (left) and representative cytospin (right) of cells at 2^nd (top) and 3^rd (bottom) replating. D Schematic illustration of in vivo assay. E, F LSK and CD24⁺ PreProBs from control and KMT2A::AFF1^Vav-Cre+ were transplanted into sub-lethally irradiated NSG mice and donor cells assessed in peripheral blood at the indicated time-points. E Mean frequencies of CD45.2 donor cells displayed as percentages of total CD45⁺ cells. F Lineage distribution (B/ Myeloid/ T) of reconstituted KMT2A::AFF1⁺ PreProBs and LSK mice shown as mean percentages of total CD45.2⁺ cells (n_LSK=  1–3; n_PreProB = 1–9). G Photos (left) and violin plot of white blood cell (WBC) counts (right) of recipient´s spleens at final readout, 2-8 months after transplantation. Immunophenotype (CD24⁺ PreProBs or LSK) and genotype of donor cells are indicated. H Immunophenotype of recipient´s bone marrow (BM) transplanted with CD24⁺ PreProBs from KMT2A::AFF1^Vav-Cre+ embryos. Cells were gated for donor CD45.2⁺ cells and negative for lineage markers. CD19 and IL7R are displayed for individual mice. Numbers show percentage of total CD45.2⁺ cells. I UMAP visualizations of flow cytometry data from transplanted KMT2A::AFF1^Vav-Cre+ embryos, displaying different B progenitors. Combined UMAP of CD45.2⁺7-AAD^-Lin^- cells (4 PreProBs, 2 LSKs) (top, left), one KMT2A::AFF1⁺ LSK (top, right) and three different KMT2A::AFF1⁺ CD24⁺ PreProB recipients (bottom). Down sampled to 30.000 cells/mouse. **p ≤ 0.01; ****p ≤ 0.0001.