Establishment of embryonic gem cells and their differentiation into germ cells.

点击翻译按钮获取中文摘要

In this study, we found that 30-33 dpc embryo would be suitable for primordial germ cell collection in cynomolgus monkey. We were able to culture alkaline phosphatase positive cells isolated from genital-ridges for certain period. But EG (embryonic germ) cell line was difficult to establish. Therefore we modified cell isolation procedure, culture condition and origin of feeder cells to establish EG cell line.First, we tried mechanical dissection instead of trypsin digestion when we isolate PGC from genital-ridges and mesenteries. Cells recovered by mechanical dissection seems to proliferate more than that prepared by trypsin digestion. In addition, we used monkey embryonic fibloblast as feeder cells. Monkey embryonic fibloblast seems to provide better condition than mouse embryonic fibloblast or STO cell line for the maintenance of PGC. Addition of hLIF (human leukemia inhibitory factor), bFGF (basic fibroblast growth factor), forskolin, and SCF (stem cell factor) improved PGC maintenance and alkaline phosphatase positive cells were cultured for several months. These cells were also positive for PAS staining, which is characteristic for migrating PGC. Unfortunately, these cells did not form colonies, which is characteristic to EG cell line. We also tried to establish GS (germline stem) cell, which is committed to the germ cell linage. Some cells were maintained for certain period but colony formation was not observed. Our result strongly suggested that PGC can be cultivated and maintained for several months, however, different condition from mouse and human would be necessary to establish EG and GS cell line in cynomolgus monkey.