Derivation of a new human embryonic stem cell line, endeavour-1, and its clonal propagation

Here we describe the derivation of a novel human embryonic stem (hES) cell line, Endeavour-1 (E1), its four new clonal lines (E1C1, E1C2, E1C3, E1C4), and their characterization. E1 and its clonal lines are propagated on human fetal fibroblasts (HFFs) derived and grown in a largely serum-free medium. Seven inner cell masses were isolated from 34 donated human embryos (27 survived), and one new hES cell line was obtained. E1 has been in culture for over 1 year and possesses all the typical features of stem cells, i.e., expression of stem cell surface markers (stage-specific embryonic antigens SSEA-3 and SSEA-4, and tumor recognition antigens TRA-1-60 and TRA-1-81), staining for alkaline phosphatase, and the presence of the pluripotent gene marker (nanog). This line shows pluripotency both under in vitro and in vivo conditions. E1 has a normal karyotype (46XX). Using our optimized procedure for cloning, four new clonal lines were derived from E1: E1C1, E1C2, E1C3, and E1C4. These clonal lines show normal characteristics: karyotype of that of the parent line (46XX) except for E1C3, which showed reciprocal translocation involving chromosomes 15 and 17; stem cell surface markers SSEA-4, TRA-1-60, and TRA-1-81; and gene expression for pluripotency (Nanog). All of these clonal lines formed embryoid bodies (EBs) in suspension cultures. After seeding, the EBs differentiated, forming cell lineages derived from all three germ layers as indicated by immunolocalization for the ectodermal marker beta-III tubulin, the mesodermal marker CD34, and the endodermal marker alpha-fetoprotein (AFP). There were subtle differences in the expression of these markers between clones. These clonal lines showed pluripotency in vivo. E1 and its clonal lines can differentiate to definitive endoderm after treatment with activin A, and, as indicated by expression of SOX17, FOXa2, and GATA-4 by RT-PCR, there are some subtle differences between these clonal lines. This may help in selecting clonal lines for specific lineage specification and for developing future cell therapy for various diseases.