Undifferentiated hESCs lack hematopoietic markers. Undifferentiated hESCs were analyzed for their morphological, histochemical, and cell surface phenotypes. (A) Undifferentiated H1 cells, maintained in feeder-free culture with MEF-CM. Scale bar, 500 μm. (B) Histochemical detection of alkaline phosphatase activity and (C) immunodetection of SSEA-4 in undifferentiated H1 cells. Scale bars, 50 μm. (D-F) Flow cytometric analysis of cell surface marker expression of SSEA-4, CD45, and CD34 on undifferentiated hESCs. Positively stained cells, shown as shaded histograms, were determined based on isotype controls, shown as open histograms. Horizontal bars indicate mean percentages of positive cells ± SEMs from 9-13 independent experiments using both H1 and H9 cell lines. (G) Schematic of the hematopoietic differentiation protocol. hES indicates human embryonic stem cell; MEF-CM, mouse embryonic fibroblast conditioned medium; BMP-4, bone morphogenetic protein 4; and CFU, colony-forming unit.