In vitro studies on PGC or PGC-like cells in cultured yolk sac cells and embryonic stem cells of the mouse

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 laye r under the conditions mentioned above-upon the embryonic stem (ES) cells ( nl) 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 i n a modified Dulbecco's modified Eagle's medium (DMEM-plus/20: the postfix represents the concentration of FBS added in percentage), many cells exhibi ted strong immunoreactivities to the monoclonal antibodies 4C9 and 2C9 whic h are known to react with PGC specifically. Both the 4C9- and the 2C9-posit ive cells were sensitive to the treatment with busulfan added in vitro, sup porting 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 SCP, LIF, bFGF and TNF-a lpha (DMEM-NT/20). When the R1 cells were cultured in the yolk sac-conditio ned DMEM/NT-20 medium on the laminin substratum, the entire colonies were f aintly stained with 4C9 but not with 2C9, At times solitary ES cells migrat ed out from the colonies, and reacted strongly with 4C9. When yolk sec cell s and R1 cells were cultured on the two sides of a collagen-coated membrane , the yolk sec cells being feeder cells, some R1 cell colonies were intense ly 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 c ells which either had been destined to become the germ line cells or had al ready acquired cellular characteristics close to PGCs. It was tentatively c oncluded that the R1 cell population contained, as judged from the immunore activities, germ-cell-like cells, and that the yolk sac cells and/or their secretory products might facilitate the proliferation of, or the conversion of nl cells to, the germ-cell-like cells.