Outline of successive culture steps for erythroid differentiation by hES cells. (A) A flow chart of sample collection and analyses performed. (B) Clumps of undifferentiated ES cells were cultured in EB medium for 14 days in suspension, which gave rise to cystic embryoid bodies (EBs). EBs then were plated on matrigel-coated plates in the GEM medium for up to 21 days, during which time nonadherent cells were generated. Nonadherent cells then were further expanded as described in “Materials and methods.” Images were obtained using a Leica DMIL inverted microscope (Leica, Heidelberg, Germany) with a Leica C PLAN 4×/0.10 numeric aperture (NA) (top 2 panels) or 10×/0.22 NA (bottom 2 panels) objective and a PixeLINK megapixel FireWire camera (model PL-A662; PixeLINK, Ottawa, ON, Canada) with PixeLINK Capture software version 1.0.