RPS19^+/p.Q126X iPSCs exhibit panhematopoietic defects with the Ery lineage most strongly affected. (A) Relative ratio of hematopoietic cells released by WT and RPS19^+/p.Q126X EBs at day 14 of differentiation. Cell numbers are normalized to those produced by the WT1 clone, which was analyzed consistently in all experiments. Cultures containing roughly equal numbers of EBs were analyzed. WT and RPS19^+/p.Q126X EBs were of similar size. **P = .002; n = 5 separate experiments. (B) Quantification of hematopoietic lineages in the experiment from panel A. MK (CD41⁺/CD235⁻), Ery (CD41⁺/CD235⁺), My (CD41⁻/CD235⁻, CD45⁺) cells were identified by flow cytometry in WT and RPS19^+/p.Q126X EB cultures at day 14 of differentiation. **P < .01; n = 5 separate experiments. (C) Relative frequency (%) of each lineage present in day 14 EB cultures represented in experiments from panels A and B. The proportion of Ery cells was decreased in cultures derived from RPS19^+/p.Q126X iPSCs (RPS19^+/p.Q126X vs WT1: 2% vs 19%, n = 5, P < .01). MPP refers to the CD41⁺/CD235⁺ primitive multipotential progenitor population that arises transiently in day 7 to 8 EB cultures and disappears gradually as the cells differentiate into mature lineages.^25,27  (D) Representative flow cytometry analysis showing reduced proportion of Ery cells (CD235⁺/CD41⁻) produced by RPS19^+/p.Q126X iPSCs compared with WT1 control in day 14 EB cultures. (E) Hematopoietic progenitor assay. CD43⁺ progenitors released from day 8 EBs were seeded in triplicate into methylcellulose containing EPO, IL-3, SCF, and GM-CSF. CFU-GM and Ery colonies were enumerated 7 to 9 days after plating. The Ery progenitors were reduced in the RPS19^+/p.Q126X samples compared with WT1 control (*P < .05). The bars in the graphs (A-B) represent mean values. For iPSC lines examined in 3 or more independent experiments, the error bars show SD. For lines examined twice, individual data points from each experiment are shown as open squares. MK, megakaryocytic; My, myeloid.