Fibroblast-like cells derived from the gonadal ridges and dorsal mesenteries of human embryos as feeder cells for the culture of human embryonic germ cells.

The establishment of optimal hEG culture systems is a major challenge for the field of embryonic germ cell research. It is important to find appropriate feeder cells to support the growth of hEG. The clinical application of human embryonic germ cells cultured on mouse-derived feeder cells is restricted, since human embryonic germ cells cultured on mouse-derived feeder cells are at risk of contamination by heterogeneous proteins or pathogens. In order to avoid this limitation, we have isolated and cultured three human embryonic fibroblast-like cell lines derived from the gonadal ridges and dorsal mesenteries of 5- to 10-week old embryos. These cells expressed basic fibroblast growth factor and leukemia inhibitory factor, both essential for the growth of human embryonic germ cells. We then used the mitomycin-inactivited human embryonic fibroblast-like cells as feeder cells to culture human embryonic germ cells derived from the gonadal ridges and dorsal mesenteries of 5- to 10-week old embryos. Of 21 human primordial germ cell cultures initiated, seven were continuously grown and split for 10 passages with normal and stable human karyotypes. These cells expressed markers characteristic of pluripotent stem cells, including alkaline phosphatase, stage-specific embryonic antigens (SSEA)-1, SSEA-3, SSEA-4, tumor related antigens (TRA)-1-60, TRA-1-81, and the POU transcription factor Octamer-4 (Oct-4). Moreover, the cells possessed the capacity to differentiate into all three primary germ layers (ectoderm, mesoderm, and endoderm). Therefore, we have successfully used the human embryonic fibroblast-like cells derived from gonadal ridges and dorsal mesenteries as feeder cells to culture proliferative, undifferentiated and pluripotent human embryonic germ cells.