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

在本研究中，我们发现30-33DPC胚胎适合于食蟹猴原始生殖细胞的采集。我们从生殖器脊分离出碱性磷酸酶阳性细胞，培养了一段时间。但EG(胚胎生殖细胞)细胞系的建立比较困难。为此，我们改进了细胞分离程序、培养条件和饲养层细胞的来源，建立了EG细胞系。首先，我们尝试用机械分离代替胰酶消化从生殖器脊和肠系膜分离PGC。机械解剖回收的细胞似乎比胰酶消化法制备的细胞增殖更多。此外，我们还使用了猴胚胎成纤维细胞作为饲养细胞。猴胚胎成纤维细胞似乎比小鼠胚胎成纤维细胞或STO细胞系更有利于PGC的维持。加入人白血病抑制因子(HLIF)、碱性成纤维细胞生长因子(BFGF)、Forsklin和干细胞因子(SCF)可改善PGC的维持，碱性磷酸酶阳性细胞培养数月。这些细胞的PAS染色也呈阳性，这是移行PGC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。我们还尝试建立了致力于生殖细胞系的GS(生殖系干细胞)细胞。部分细胞维持一定时间，但未观察到集落形成。我们的结果有力地表明，食蟹猴PGC可以培养和维持几个月，但建立食蟹猴EG和GS细胞系需要不同的人和鼠的条件。

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.