In vitro differentiation of ES cells. The phenotype of mutant ES cells was tested by 2-step in vitro differentiation. (A) Pictures of representative embryoid bodies at day 8 of differentiation. DKO ES cells form EBs, but lack macroscopic hemoglobinization as can be seen in wild-type and SKO EBs. (B) EBs were disrupted and replated to enumerate erythroid potential. Graphs show number of primitive (left) and definitive (right) erythroid colonies per 100 000 replated EB cells. Red bar indicates the average of colony numbers. (C) Pictures of representative primitive and definitive red blood cell (rbc) colonies of wild-type and mutant ES cells. ΔmHS−3.5ΔmHS−25/6 ES cells did not produce colonies. Below are pictures of cells from the erythroid colonies after cytospin and May-Grünwald-Giemsa/benzidine staining. Primitive erythroid cells of wild-type and SKO cells have a normal morphology. Cytospins from wild-type and SKO definitive erythroid colonies show cells at various stages of differentiation. Nonhemoglobinized progenitors are indicated with black arrows, and hemoglobinized (brown) erythroid cells are indicated by red arrows.