Figure 3.

RUNX1 R174Q mutation but not RUNX1 deletion affects the output of granulomonocytes. AII_1, AII_2 are iPSC clones of 2 patients from pedigree A; D(a) and D(b) are 2 clones of 1 patient from pedigree D; and C1, C2 and C3 are control iPS cell lines. Cell numbers were normalized in all experiments to those produced by the C2 clone. (A-D) CD34⁺CD43⁺ cells were sorted at day 14 and tested for their colony-forming potential in a methylcellulose assay. The histograms show (A) 1 representative experiment of 3 for pedigree A and (B) 1 representative experiment of 7 for pedigree D, each in triplicate. Error bars represent ± SD of triplicate. (C) Photos of granulomonocytic colonies (CFU-GM). Scale bar = 100 μm. (D) Area of more than 30 CFU-GM colonies obtained from each iPSC line was analyzed by AxioVisio 4.6 software. (E-H) Analysis of hematopoietic populations generated at day 21 from progenitors sorted at day 14. (E) May-Grünwald-Giemsa staining of monocytes and/or macrophages (CD14^highCD15^low) and granulocytes (CD15^high) derived from iPSCs. Scale bar = 10 μm. (F) FACS analysis of the GM cells in 1 representative experiment. (G-H) Absolute numbers of generated monocytes and/or macrophages (CD14^highCD15^low) and granulocytes (CD15^high) normalized to 1 × 10² plated progenitors. (G) The histograms show average of the cell number obtained in 5 independent experiments for monocytes and/or macrophages and in 3 for granulocytes for pedigree A. (H) The histograms show average of the cell number obtained in 4 independent experiments for pedigree D. Error bars represent ± SD of the average. (I) qRT-PCR analysis of NR4A3 expression level in CD34⁺CD43⁺ cells in both A (left; n = 4) and D (right; n = 4) pedigrees. Data are normalized to PPIA transcript, and expression is compared with control C3. Error bars represent ± SD of triplicate. *P < .05; **P < .01; ***P < .001; Student t test. ns, nonsignificant.