SR-bFGF maintains hESCs in an undifferentiated state. Human ESCs (H9 cell line) were cultured over 14 passages in either MEF-CM (left column) or in SR-bFGF (right column). (A-B) Double alkaline phosphatase (AP) and Oct4 immunostaining. In the undifferentiated area of the colonies, nuclei were costained for Oct4 (green) and DAPI (blue), resulting in a merged color (aqua). The AP (red) was detected in the cytoplasm regions. Bar = 200 μm in row 1 and 20 μm in row 2. (C) Expression of intracellular Oct4 and cell surface markers SSEA-4, Tra-1-60, and Tra-1-81 was analyzed by flow cytometry. Dotted lines indicate isotype controls. (D) Proliferation capacity. (E) Karyotype (21 passage in SR-bFGF culture).