Fig 2: ETV6-RUNX1 “proB” Cells Exhibit Multilineage Priming and Potential(A) RNA-seq data from ETV6-RUNX1-expressing iPSCs overlaid (haloed) on the existing PCA map calculated from primary samples (Figure 4D). Control hPSC-derived data (haloed) are shown for comparison (left). PCA also including reverted ETV6-RUNX1 IPS (pink) (right). PC1 and PC2 are shown; each dot represents one sample. The ellipses show the 90% density function for that cell type. Two different ETV6-RUNX1 cell lines (no. 2.1 and no. 2.8) used in thre experiments. Reverted clone, n = 2, in one experiment.(B) Comparison of gene expression in MIFF3/reverted ETV6-RUNX1 (RC) (green) and ETV6-RUNX1 hIPSC (purple) proB cells. Each dot represents one sample. Bars show mean fragments per kilobase of transcript per million fragments mapped (FPKM) value ± SD, n = 4–5.(C) Gene set enrichment analysis (GSEA) of STAT5A target gene expression comparing ETV6-RUNX1 hIPSC (red) with control proB cells at D31 of differentiation. NES, normalized enrichment score. FDR, false discovery rate.(D) GSEA as in (C). Lineage affiliations of gene sets used are indicated above (Laurenti et al., 2013).(E) Single-cell qPCR data of control MIFF3 and ETV6-RUNX1+ proB cells from D31. Each column represents a single cell. Colored by CT value. Genes labeled in red are myeloid, blue are lymphoid, and green are B cell genes. Only cells expressing GADPH and ETV6-RUNX1 are shown. A total of 37–56 cells investigated per population, n = 2–3.(F) Survival of MIFF3/reverted ETV6-RUNX1 (RC) (green) and ETV6-RUNX1 Venus (V)+ hIPSC (purple)-derived single proB cells (CD34+CD19+) grown in liquid culture supplemented with myeloid cytokines (percentage of wells with ≥3 cells at 14 days). Mean ± SD, n = ETV6-RUNX1 = 4; MIFF3/reverted = 6, in two experiments.(G) Myeloid differentiation potential MIFF3/reverted ETV6-RUNX1 (RC) (green) and ETV6-RUNX1 Venus (V)+ (purple)-derived single proB cells (CD34+CD19+) grown in liquid culture supplemented with myeloid cytokines (percentage of wells with ≥20 cells at 14 days). Mean ± SD, n = ETV6-RUNX1 = 4; MIFF3/reverted = 6, in two experiments.(H) Cytospin of macrophages generated from ETV6-RUNX1+ proB cells.(I) Agarose gel of DH7JH rearrangements in myeloid cells derived from liquid culture of ETV6-RUNX1 proB cells (clones no. 2.1 and no. 2.8) and primitive ETV6-RUNX1+ CD34+ cells. Marker lanes (M) 1 kb+ (Invitrogen). DH7JH recombination predicted band 100–130 bp (van Dongen et al., 2003). See also Figure S5.

Fig 3: Identification of a CD19–IL-7R+ Progenitor(A) Fluorescence-activated cell sorting (FACS) analysis of CS17 FL showing CD19−IL-7R+ progenitor co-expressing CD34 and CD45RA. Adult BM (right) is shown for comparison. Cells were gated CD45+LIN−CD19–. Further gating as indicated. Mean percentage of total CD45+CD34+ cells.(B) Kinetics of IL-7R progenitor emergence during development as percentage of total CD45+CD34+ cells. Each dot represents one biological sample. Mean with range. Statistical comparison versus CB control.(C) B cell potential of IL-7R+KIT+ FL progenitors was tested by co-culture on MS-5 for 2 weeks. Percentage of CD19+ B cells of total CD45+ cells is shown (left) with representative FACS analysis (right). CD34+ cells were used as control. Mean ± SD, n = 2–3.(D) Agarose gel of IGH DJ rearrangements in IL-7R+ FL progenitor. Top: DH7JH recombination assay. Bottom: DH1-6JH recombination assay. Label for lanes 1–6 is shown to the right. Marker lanes (M). Predicted DH7JH recombination between 100 and 130 bp. Predicted DH1-6JH rearrangements between 110 and 290 bp, n = 1 (van Dongen et al., 2003).(E) GM and erythroid potential of IL-7R+KIT+ FL progenitor in semi-solid media. Data show distribution of colony types as percentage of plated cells versus CD34+ cells as controls. Mean ± SD, n = 2 per developmental stage.(F) Myeloid potential of IL-7R+KIT+ FL progenitor in liquid culture (20–40 cells/well). Graph shows percentage of wells expanded to >50 cells (left). Cytospin of macrophages generated from FL IL-7R+KIT+ progenitor (right). Mean ± SD, n = 3.See also Figure S2.

Fig 4: HCL cells are sensitive to venetoclax-induced cell death in vitro. (A) PBMCs were obtained by Ficoll-Paque centrifugation from peripheral blood of eight HCL patients, incubated with venetoclax for 24 h, and cell death was analyzed by flow cytometry. Leukemic cells or T + NK cells were discriminated according to CD19 expression and forward side scatter, and next gated to determine binding of Annexin V. Results from a representative sample (HCL#8) are shown. (B) HCL cell death induced by venetoclax for the whole cohort evaluated. (C) T + NK cell death induced by venetoclax for the whole cohort evaluated. (D) Comparison of cell death induced in HCL cells and T + NK cells. For all the group graphics, each symbol represents a single patient (n = 8, **p < 0.01, ***p < 0.001, ****p < 0.0001, paired one-way ANOVA using correction for (B) and Friedman test followed by Dunn’s multiple comparisons test for (C, D), ns, not significant).

Fig 5: Flow cytometry analysis from approximately day 31 of co-cultureCells were harvested after 3 weeks of MS5 co-culture and stained for surface markers.(A) Cells were gated for singlets and scatter and then for viability (7AAD) and CD45 expression as indicated in the figure (top panel). Expression of CD19 and CD34 is shown within the 7AAD-CD45+ fraction (second panel, left). The CD19 positive fraction is further gated for expression of CD10 and CD20 (right). Counting beads were used in this experiment and can be seen in the scatter plot in the top middle panel.(B) Cells were gated for singlets and scatter (as in Figure 8A, plots not shown). Contour plot of viability (7AAD) and CD45 expression as shown in the figure (left). Further gating on 7AAD-CD45+ cells for B lymphoid (CD19) and, within the CD19- gate, myeloid (CD11b and CD14) markers are shown to the right.