Characterization and functional analyses of iPSC-derived megakaryocyte-lineage cells. (A) Wright-Giemsa stain of megakaryocytes produced via AhR antagonism. (B) Ploidy analysis of iPSC-derived megakaryocytes demonstrating endoreplication. (C) Quantitative PCR analysis comparing undifferentiated iPSCs vs iPSC-derived megakaryocytes for a spectrum of megakaryocyte-specific markers. Expression is normalized to β-actin levels. Data are the average of 3 independent experiments ± SD. (D) FACS analysis comparing platelets in whole blood vs iPSC megakaryocyte–derived platelets. Mature megakaryocytes were grown on an OP9 stromal layer for 3 to 5 days to allow for terminal megakaryocyte differentiation and platelet production. Cultures were initially gated using GPIIb (CD41a) expression and demonstrated that iPSC megakaryocyte–derived platelets were similar to platelet populations from whole blood with respect to size (FSC), granularity (SSC), as well as the expression of GPIX and GPIb, 2 subunits of the GPIb/V/IX complex, that are characteristic of platelets.