Figure 2.

Induction of PGCs by BMP4/BMP8B-expressing COS cells. (A) Diagram showing coculture of epiblast masses with COS cells. Mouse embryos (F₁ hybrid of 129 SvEv × C57BL/6) at E6.0–6.25 were isolated, in which the extraembryonic ectoderm (red; xe) and epiblast (light blue; ep) are enclosed by endoderm (dark blue; xn for extraembryonic endoderm and en for embryonic endoderm). The extraembryonic ectoderm and proximal epiblast cells were removed with a sharp tungsten needle (red dashed line). The epiblast masses with visceral endoderm were then transferred onto pIRES or pBmp4/Bmp8b COS cells. (B) Percentage of embryos containing PGCs. About 12% of the control epiblast masses (3/25) formed PGCs after coculture with pIRES COS cells, whereas 13 of 20 epiblast masses (65%) contained PGCs after coculture with pBmp4/Bmp8b COS cells. (C) Average number of PGCs per PGC-containing embryo. The average number of PGCs was 3.3 ± 2.3 (mean ± SEM) for epiblast masses cocultured with pIRES COS cells, whereas 34.2 ± 7.2 PGCs were detected in epiblast masses cocultured with pBmp4/Bmp8b COS cells. (D) An example of an epiblast mass cocultured with pIRES COS cells after staining for ALP. No obvious PGC-like cells were observed. (E) An example of an epiblast mass cocultured with pBmp4/Bmp8b COS cells. More than 30 PGCs (red arrows) were found in a cluster and its vicinity. These cells have typical PGC characteristics (in terms of size and shape, having strong ALP staining in the cell membrane, and with a darkly stained spot in the cytoplasm). χ² was used for statistical analysis. (Bar = 40 μm in D and E.)