Figure 5.

PGCs in Defined Medium Can Be Cultured Clonally in Activin, but Not BMP4

(A) Ovotransferrin (OT) replaces chicken serum in culture medium. PGCs (1,000) were cultured in FA medium with the addition of 0.2% fetal bovine serum (FBS), 0.2% chicken serum (CS), and 10 μg/ml human transferrin (HuT) or 10 μg/ml OT for 10 days. Error bars, SEM; ^∗p < 0.05 and ^∗∗∗p < 0.001 with respect to FA samples. Each cell treatment was assayed in three independent experiments on a male and a female PGC line.

(B) PGCs can be propagated in Activin or BMP4 in defined medium conditions. PGC number over 14 days in culture (500 were plated on day 1) is shown. Each cell treatment was assayed on six different PGC lines (four male and two female) in three independent experiments. Error bars, SEM; ^∗∗p < 0.01 and ^∗∗∗p < 0.001 with respect to FABot sample.

(C) PGCs express pluripotency and germ cell markers in Activin- or BMP4-defined culture medium. RT-PCR analysis of cDNA prepared from a PGC line cultured in FABot, FAot, or FBot is shown. FAcs and HiS are shown as positive controls. Image represents one of two independent experiments using two male PGC lines. CEF, chick embryonic fibrobast; cES, chick embryonic stem cell.

(D) Activin is sufficient for derivation and clonal growth of PGCs. Blood was isolated from single embryos (stage 16 HH) and cultured for 3 weeks. PGCs were counted and cultures containing >50,000 cells were scored as positive. Cells from a male or a female PGC line derived in FABot were diluted to one cell/2 μl in Fot, and single cells were plated and cultured for 3 weeks in FABot, FAot, or FBot. PGCs were counted and cultures containing >50,000 cells were scored as positive. ^∗∗∗p < 0.001 with respect to FBot samples using two-tailed Fisher’s exact test.

(E–G) FGF2, insulin, and Activin can be replaced by the corresponding downstream effectors. PGCs (500), cultured in FABot, were seeded in a well and the indicated growth factors were removed. Doxycycline (dox) was added and cells were propagated for 10 days (A and B) or 12 days (C) and then counted.

(E) PGCs containing a tet-inducible constitutively active MEK1 construct proliferate in the absence of FGF2.

(F) PGCs containing a tet-inducible constitutively active AKT construct proliferate in the absence of insulin.

(G) PGCs containing constitutively active SMAD3 and tet-inducible constitutively active SMAD5 constructs proliferate in the absence of Activin and BMP4.

Each cell treatment was assayed using two different PGC lines (one male and two female) in three independent experiments. ^∗∗∗p < 0.001 and ^∗∗p < 0.01 with respect to the indicated samples.