Fig. 1.
Induction of two distinct Flk1pos hematopoietic precursor populations from mouse ESCs using activin A, BMP4 and VEGF. (A) To the left are shown T-EGFP and Flk1 expression in day (d) 3.25 embryoid bodies (EBs) cultured in the absence (no induction) or presence (d3.25 presort) of inducers. The d3.25 population was segregated into d3.25 T+ F+ and d3.25 T+ F– fractions by cell sorting (T, brachyury; F, Flk1). The d3.25 T+ F– cells were cultured as aggregates for 48 hours and the d5.25 F+ and F– fractions isolated (shown to the right). (B) Hematopoietic progenitor potential of the d3.25 T+ F+ and d5.25 F+ populations aggregated for 24 hours and cultured in methylcellulose cultures. Primitive erythroid colonies (EryP) were scored at day 5 of culture, whereas the macrophage (Mac), erythroid-macrophage (eMac), granulocyte-macrophage (GM) and mixed (Mix) colonies were counted at days 7-8. Data are expressed as the mean of six independent experiments; error bars indicate ±s.d. ***, P<0.0002. (C) qRT-PCR analysis of T expression in all populations shown in A. d3.25 T+ F+_RE indicates d3.25 T+ F+ cells reaggregated for 24 hours; d5.25 T+ F+_RE indicates d5.25 F+ cells reaggregated for 24 hours. Data are presented as expression relative to that of the housekeeping gene Actb and are the mean of three independent experiments; error bars indicate ±s.d.