Nakagawa S, Saburi S, Yamanouchi K, Tojo H, Tachi C
Department of Animal Resource Sciences, School of Agriculture and Life Sciences, University of Tokyo, Japan.
Arch Histol Cytol. 2000 Jul;63(3):229-41. doi: 10.1679/aohc.63.229.
The present study aims: 1) to determine those conditions which promote the proliferation of primordial germ cells (PGCs) of the migratory phase in the yolk sac; and 2) to examine the effects of yolk sac cells as a feeder layer under the conditions mentioned above upon the embryonic stem (ES) cells (R1) with high potential for entering the germ line in vivo in chimeras. In murine yolk sac cells obtained on Day 10.5-11.5 of pregnancy and cultured in a modified Dulbecco's modified Eagle's medium (DMEM-plus/20: the postfix represents the concentration of FBS added in percentage), many cells exhibited strong immunoreactivities to the monoclonal antibodies 4C9 and 2C9 which are known to react with PGC specifically. Both the 4C9- and the 2C9-positive cells were sensitive to the treatment with busulfan added in vitro, supporting the supposition that they were PGCs. The respective numbers of the 4C9- and the 2C9-positive cells increased approximately 4 and 12 times when they were cultured in DMEM-plus/20 fortified with SCF, LIF, bFGF and TNF-alpha (DMEM-NT/20). When the R1 cells were cultured in the yolk sac-conditioned DMEM-NT/20 medium on the laminin substratum, the entire colonies were faintly stained with 4C9 but not with 2C9. At times solitary ES cells migrated out from the colonies, and reacted strongly with 4C9. When yolk sac cells and R1 cells were cultured on the two sides of a collagen-coated membrane, the yolk sac cells being feeder cells, some R1 cell colonies were intensely stained as a whole with either the 4C9 or the 2C9 antibody, suggesting that these colonies might be composed of cells clonally derived from stem cells which either had been destined to become the germ line cells or had already acquired cellular characteristics close to PGCs. It was tentatively concluded that the R1 cell population contained, as judged from the immunoreactivities, germ-cell-like cells, and that the yolk sac cells and/or their secretory products might facilitate the proliferation of, or the conversion of R1 cells to, the germ-cell-like cells.
1）确定促进卵黄囊中迁移期原始生殖细胞（PGC）增殖的条件；2）研究在上述条件下，作为饲养层的卵黄囊细胞对在嵌合体中具有高体内生殖系进入潜能的胚胎干细胞（ES细胞，R1）的影响。在妊娠第10.5 - 11.5天获得的小鼠卵黄囊细胞，并在改良的杜尔贝科改良伊格尔培养基（DMEM-plus/20：后缀表示添加的胎牛血清浓度，以百分比计）中培养，许多细胞对已知与PGC特异性反应的单克隆抗体4C9和2C9表现出强免疫反应性。4C9阳性细胞和2C9阳性细胞对体外添加白消安的处理均敏感，支持它们是PGC的推测。当4C9阳性细胞和2C9阳性细胞在添加了干细胞因子（SCF）、白血病抑制因子（LIF）、碱性成纤维细胞生长因子（bFGF）和肿瘤坏死因子-α（TNF-α）的DMEM-plus/20（DMEM-NT/20）中培养时，它们各自的数量分别增加了约4倍和12倍。当R1细胞在层粘连蛋白基质上的卵黄囊条件培养基DMEM-NT/20中培养时，整个集落用4C9染色较淡，但用2C9未染色。有时单个ES细胞从集落中迁移出来，并与4C9发生强烈反应。当卵黄囊细胞和R1细胞在胶原包被膜的两侧培养，卵黄囊细胞作为饲养细胞时，一些R1细胞集落整体被4C9或2C9抗体强烈染色，这表明这些集落可能由克隆来源于干细胞的细胞组成，这些干细胞要么已注定成为生殖系细胞，要么已获得接近PGC的细胞特征。初步得出结论，从免疫反应性判断，R1细胞群体中含有类生殖细胞，并且卵黄囊细胞和/或其分泌产物可能促进类生殖细胞的增殖，或使R1细胞转化为类生殖细胞。
