Enucleation of hESC-derived erythroid cells in vitro. (A) Diameter decreases with time in culture. Data for each day represent diameters of nucleated cells, except “27e” represents diameters of enucleated cells at 27 days. Enucleated cells decrease to less than half the original diameter on day 8. (B) Nuclear-to-cytoplasm ratio decreases with time in culture. Samples significantly different from day 8: *P < .05, **P < .001, #P < .002. (C,E) Erythroid cells derived from human ESCs were cultured in vitro for 4 weeks in Stemline II media with supplements and cocultured with OP9 stromal cells on day 36. On day 42, cells were cytospun and stained with Wright-Giemsa dye (C: original magnification ×200; E: original magnification ×1000). (D,F) Red blood cells from human blood were also cytospun and stained with Wright-Giemsa and compared with hESC-derived erythroid cells (D: original magnification ×200; F: original magnification ×1000). Scale bar represents 10 μm.