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

在本研究中，我们发现30-33 dpc的胚胎适合用于食蟹猴的原始生殖细胞收集。从生殖器脊分离的碱性磷酸酶阳性细胞能够培养一定时间。但胚胎生殖细胞系的建立较为困难。为此，我们改进了细胞分离方法、培养条件和饲养细胞来源，建立了EG细胞系。首先，当我们从生殖器脊和肠系膜分离PGC时，我们尝试机械解剖而不是胰蛋白酶消化。通过机械解剖恢复的细胞似乎比通过胰蛋白酶消化制备的细胞增殖得更多。此外，我们使用猴胚胎成纤维细胞作为饲养细胞。猴子胚胎成纤维细胞似乎比小鼠胚胎成纤维细胞或STO细胞系提供了更好的维持PGC的条件。添加hLIF（人白血病抑制因子）、bFGF（碱性成纤维细胞生长因子）、forskolin和SCF（干细胞因子）可改善PGC维持，碱性磷酸酶阳性细胞培养数月。这些细胞PAS染色也呈阳性，这是PGC迁移的特征。不幸的是，这些细胞没有形成集落，这是EG细胞系的特征。我们还尝试建立GS（种系干细胞），这是致力于生殖细胞谱系。部分细胞维持了一定时间，但未见集落形成。本研究结果表明，PGC可以培养并维持数月，但要在食蟹猴体内建立EG和GS细胞系，需要小鼠和人不同的条件。

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.