Tissue-like structure formation of mouse embryonic stem cell (mESC) and lineage-specific immunofluorescence in conventional 2D cultures versus 3D bioreactor cultures, compared to mouse embryos. DAPI staining (blue) was used as a nuclear counter stain. Center column: histology of the day E9.5 mouse embryo; primitive gut tube (endoderm/Foxa2), cardiac region (mesoderm/anti-α smooth muscle actin [ASMA]), neural tube (ectoderm/Nestin), and post-mitotic neurons in the neuronal tissue (ectoderm/class III β-Tubulin). Right column: mESC after 10 days in 3D culture are partly differentiated into structures resembling primitive gut (endodermal lineage FoxA2), structures resembling primitive cardium (mesodermal lineage, ASMA), and structures resembling neural tube as well as neuronal-like cells (arrow; ectodermal lineage, Nestin and class III β-Tubulin, respectively), similar in part to that in E9.5 mouse embryo (center column). Left column: differentiated cells in 2D cultures at day 10 are unorganized, and most tissue-like structures found in 3D were absent. Scale bars: 25 μm. Color images available online at www.liebertonline.com/ten.