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. 2018 Feb 5;44(3):362–377.e7. doi: 10.1016/j.devcel.2017.12.005

Figure 6.

Figure 6

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.