Increased myelopoiesis from PTPN11 p.E76K heterozygous iPSCs. (A) EBs generated from WT or PTPN11 p.E76K (SHP-2) heterozygous iPSCs were cultured in a multilineage cytokine cocktail to support myeloerythroid maturation (see supplemental Methods). The iPSC lines are numbered according to the individual donor followed by the clone from that individual. The relative proportion of myeloid (My; CD45⁺CD18⁺), erythroid (Ery; CD235⁺), and megakaryocytic (Meg; CD41⁺CD42⁺) cells produced by each clone in three independent experiments is shown. The PTPN11-mutant clones produced increased proportions of myeloid cells (P = .008) and reduced proportions of erythroid cells (P = .001). (B) Hematopoietic progenitors released from day 8 EBs were enumerated in methylcellulose colony assays. Each bar represents the results of 3 separate experiments, each with triplicate colony assays. Myeloid colonies produced by PTPN11 p.E76K iPSCs were increased (P = .004), and erythroid colonies were decreased (P = .001). (C) Myeloid colonies from methylcellulose assays. Scale bar represents 200 µm. (D) Hematopoietic progenitors from day 8 monolayer differentiation cultures of PTPN11 p.E76K or WT iPSCs were suspended at 25 000 cells/mL in medium containing stem cell factor, GM-CSF, and IL-3 and were quantified at the indicated time points. By day 18, all cells exhibited myeloid morphology, similar to those represented in panel (E). (E) May-Grünwald-Giemsa-stained cells from WT and PTPN11 p.E76K myeloid colonies represented in panels (B) and (C). Scale bars represent 20 µm.