KIT D816V ESCs recapitulate the KIT^highand erythroid phenotype of patient KIT D816V iPSCs. (A) Flow cytometry analysis of KIT D816V and control ESC-derived hematopoietic cells 11 days after CD34⁺ MACS selection (upper panels). Prominent CD235α⁺ and CD45⁺/KIT^high populations are observed in KIT D816V ESC–derived cells. Cytospin preparations of the same samples show nucleated erythrocytes (red arrowheads) (lower panels). Scale bars, 50 μm. (B) Quantification of hematopoietic cell populations (n = 4) derived from KIT D816V clone 1 and D816V clone 2 ESCs and control ESCs. KIT D816V ESCs show prominent CD45⁺/KIT⁺ and CD235⁺ populations. The CD45⁺/KIT^high MC populations were not statistically significant different for KIT D816V and control. (C) Quantitative RT-PCR data for KIT D816V and control ESCs and corresponding HPCs 11 days after CD34⁺ enrichment by MACS. Gene expression values were subjected to bidirectional clustering and are shown in heat map format (red and blue, high and low gene expression, respectively). (D) CFU assay for hematopoietic cells obtained from KIT D816V and control ESCs. Bars show mean ± standard deviation of 3 independent experiments. *P < .05, Welch’s t test. G, granulocyte; GM, granulocyte, macrophage; M, macrophage.