Hematopoietic differentiation of patient-specific KIT D816V iPSCs. (A) KIT D816V mutation in iPSCs by allele-specific PCR (upper panel). KIT D816V mutation in iPSCs by Sanger sequencing (lower panels). Control 1-3, iPSCs without mutation; D816V 1-3, iPSCs with mutation; M, molecular weight marker; W, water control; -, HMC-1.1 cell line; +, HMC-1.2 cell line. (B) Hematopoietic differentiation protocol. EBs were formed from iPSCs (Ba-b) and differentiated toward the hematopoietic lineage (Bc-d). Scale bars, 500 μm. (C) Representative flow cytometry analysis of KIT D816V and control iPSC-derived hematopoietic cells from patients 1 through 3. The plots represent synchronized hematopoietic-differentiation experiments and show patient-specific phenotypes. Gates 1, 2, and 3 are for CD45⁺/KIT^high, CD45⁺/KIT⁺, and CD235α⁺ cells, respectively. (D) Quantification of CD45⁺/KIT^high populations for KIT D816V and control iPSC-derived cells from all 3 patients (P1, P2, and P3). Flow cytometry was performed between days 15 and 31 of hematopoietic differentiation (n = 4-21). *P < .0001. (E) Quantification of CD45⁺/KIT⁺ populations by flow cytometry, as in (D), between days 15 and 49 of differentiation (n = 4-30). *P ≤ .0003. (F) Quantification of CD235α⁺ population by flow cytometry, as in (D), between days 15 and 49 of differentiation (n = 4-30). *P < .0001. (G) CFU assay for KIT D816V and control iPSC-derived hematopoietic cells. Colony numbers and phenotype were evaluated 14 days after seeding. Bars indicate average colony numbers ± standard deviation of ≥3 independent experiments, with the exception of patient 2 control iPSCs (n = 2). *P = .02. (H) Quantitative RT-PCR data for patient 1–derived KIT D816V and control iPSCs and corresponding HPCs. Results are from 2 and 4 independent hematopoietic-differentiation experiments for control and KIT D816V HPCs, respectively. Gene expression values were subjected to bidirectional hierarchical clustering and are shown in heat map format (red and blue represent high and low gene expression, respectively). Statistical analysis was performed with Welch’s t test.