Basic FGF Stimulated the Growth of Premigratory and Postm igratory PGCs
To more thoroughly define the functions of bFGF in the in vitro PGC culture system, we plated 11.5 dpc PGCs in the presence of bFGF under a variety of conditions. As previously shown, bFGF stimulated the accumulation of PGCs plated on mitotically inactivated STO fibroblast feeder layers (Fig. 1A). We have previously shown that the optimal stimulatory effect of bFGF in this system occurs at a bFGF concentration of 1 ng/ml. Given the important role that LIF plays in PGC culture, and as bFGF can rapidly increase the accumulation of LIF RNA by STO feeder layers (unpublished results), we questioned whether the effect of bFGF on PGCs was indirect, acting primarily through the feeder layer. buy ventolin inhalers
To determine the necessity of the feeder layer to mediate the bFGF effect, PGCs obtained from 11.5 dpc embryos were cultured on gelatinized tissue culture plastic in the absence of a STO feeder layer. While PGC survival under these conditions is poor, bFGF still exhibited a strong positive effect toward PGCs (Fig. 1B), indicating that the bFGF effect is independent of the STO feeder layer. In these crude cell suspensions, PGCs account for less than 2% of all cells (unpublished results). Under these conditions, the somatic cells that accompany the PGCs form a monolayer during 24-48 h of culture. As these somatic cells could also indirectly mediate the effects of bFGF, we plated immunomagnetically purified PGCs (more than 80% free of somatic cells) on STO feeder cells. Again, bFGF stimulated PGC growth under these conditions (Fig. 1C). Together, these results demonstrate that bFGF stimulates PGC growth either in the absence of the fibroblast feeder layer, or stimulates highly purified PGCs in the presence of a feeder layer.
FIG. 1. Analysis of the effect of bFGF on PGC growth in culture. PGCs obtained from 11.5 dpc embryos were cultured in 96-well microtiter plates in the absence (open bars) or presence (closed bars) of 1 ng/ml recombinant human bFGF, and the number of PGCs was determined by alkaline phosphatase staining. A) A single-cell suspension of urogenital ridges was plated on STO feeder layers. B) PGCs were cultured in gelatinized wells in the absence of feeder cells. C) PGCs at 11.5 dpc were immunomagnetically purified and cultured on STO feeder cells.