Maintenance of human ES cells under the completely chemically defined condition. (A) Phase contrast micrographs showing H9 cells cultured with mTeSR1 media for 35 passages on hE-cad-Fc or 11 passages on Matrigel. Scale bars indicate 100 μm. (B) Alkaline phosphatase activity in H9 cells cultured on Matrigel or on hE-cad-Fc under defined conditions (53 passages). (C) H9 cells cultured on hE-cad-Fc-coated surface in mTeSR1 media for 36 passages were identified by immunocytochemistry using an anti-Oct-3/4 antibody. Nuclei were counterstained with DAPI. Scale bar indicates 100 μm. (D) The expression of SSEA-4 was analyzed by FACS after 53 passages on hE-cad-Fc. (E) Immunocytochemical analysis of protein expression in human ES cells cultured in various conditions. The data represent means ± SD of three individual experiments. (F) The expression of genes characteristic of the undifferentiated state was analyzed by RT-PCR, as in Figure 1F. (G) Karyotyping of H9 cells cultured for 30 passages on a hE-cad-Fc-coated surface with mTeSR1 media. (H) Characterization of teratomas from H9 cells cultured on an hE-cad-Fc-coated surface. Hematoxylin and eosin staining of paraffin sections through teratomas identified the differentiation huES cells into various tissues, including immature neuroblastic tissue with neuronal rosettes (a), neuroepithelium with pigment (b), immature sebaceous tissue (c), cartilage (d), columnar epithelium (e), and gut-like epithelial structures (f). Panel g contains neural tissue (1), cartilage (2), bone parenchyma (3), and epithelial tissue (4). Bar indicates 100 μm.
Nagaoka et al. BMC Developmental Biology 2010 10:60 doi:10.1186/1471-213X-10-60