Fig 2: Population dynamics of identified morphological states.(A) Population fraction dynamics over time for each morphological cluster with (right) or without (left) AhR inhibitors (top). Population fraction contribution of each cluster at the last time point of the culture (bottom). Comparisons of the population fraction with and without AhR inhibitor were performed using the Chi-Square test for the dependency between the AhRi treatment and a cell’s cluster identity. **: p < 0.001, *: p <0.05 (B) UMAP showing CD34 and CD38 expression levels at different time points, in the presence or absence of AhRi.

Fig 3: Study of the Progenitor Cells within the iPSC-Derived EBs(A) Representative plots of day 9, 11, and 14 EBs of WT and MYB-/- iPSCs, which were enzymatically dissociated, stained for expression of CD34 and CD45, and analyzed by flow cytometry. Hemogenic and nonhemogenic-endothelial cells are CD34+CD45-, HPCs are CD34+CD45+, and differentiated hematopoietic cells are CD34-CD45+.(B) Relative proportion of CD34+CD45+, CD34+CD45-, and CD34-CD45+ populations on day 9, 11, and 14 normalized to the total number of single- and double-positive cells. Mean and SD of four repeats are plotted.(C) Relative proportion of CD34+CD45+ populations on day 9, 11, and 14. Statistical comparisons were done using a paired t test. ns, nonsignificant; *p < 0.05, **p < 0.01.(D) Image of the different colony types. The images on the left show a bright-field image of representative CFU-E, CFU-M, and CFU-GM WT colonies in methylcellulose media at day 14, while the images on the right show the cytospined and eosin and methylene blue-stained cells present within the three different types of colony.(E) Dissociated day 14 EBs were plated into H4434 MethoCult; after 14 days, colonies were scored. The percentage of each type of colony is displayed as mean with SD (WT n = 5, MYB-/- n = 3 for each knockout clone). Presence of erythroid (CFU-E), granulocyte-macrophage (CFU-GM), and macrophage progenitors (CFU-M) can be detected in WT iPSC differentiation, whereas MYB-/- iPSCs display only CFU-M potential. RUNX1-/- and SPI1-/- iPSCs did not generate any hematopoietic colonies. See also Figure S5.

Fig 4: Mechanism of metformin on CML CD34+ cells.A Purified CML CD34+ cells were treated with metformin for 24 and 48 h and subjected to label-free quantitative MS analysis. Heatmap of alternation pathways regulated by metformin was shown. B, C Representative GSEA plots of oxidative pathway and glycolysis post metformin treatment 48 h. D, E OCR and ECAR of CML CD34+ cells in response to metformin. F GO cell compartment (CC) enrichment analysis and volcano plots of proteins regulated by metformin for indicated times (FC > 1.5 and P < 0.05). G, H RT-PCR (G, n = 3) and western blot analysis (H) of ER stress signaling of metformin or vehicle-treated purified CML or normal CD34+ cells. *, **, and *** indicated P value< 0.05, <0.005, and <0.001, respectively. I Purified CML CD34+ cells were treated with imatinib, tunicamycin or tunicamycin plus imatinib for 72 h. Cell apoptosis were measured. Representative flow cytometry plots were shown on the top. n = 4. ** and *** indicated P value <0.005 and <0.001 versus non-treated group, respectively. J GSEA plots of E2F target, G2M checkpoint, MYC target, DNA repair, and combined protein subset named CML stem cell targets are shown.

Fig 5: 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.