Figure 4. Differentiation of CD47-null EB-like clusters.

(A) EB-like clusters were cultured in the presence of serum for 6 days to induce substrate adhesion and then transferred in to the indicated lineage specific media for 36 h and stained with smooth muscle actin antibody (red) to detect mesodermal cells. (B) Differentiated EB-like clusters were stained with the ectoderm neural markers glial fibrillary acidic protein (GFAP, green), neuron-specific beta III tubulin (TUJI, red). (C) Differentiated EB-like clusters were stained with anti-a-fetoprotein (red) to detect ectodermal cells. In all panels DAPI was used to visualize nuclei (blue). (D–F) A single clone isolated from a CD47-null EB-like cluster in serum-free medium was expanded and then differentiated in the indicated lineage-specific medium for 7 days and stained for the indicated markers. Blue = DAPI, green in (F) = actin.