Defining the Window of Germline Genesis In Vitro from Murine Embryonic Stem Cells1

Bone morphogenetic protein (BMP) signaling is critical for germline establishment during mouse embryogenesis. To exploit its importance for induction of germline precursors in vitro, mouse embryonic stem cells (mESCs) were cultured as embryoid body (EB) aggregates with combinations of BMP2, BMP4, and BMP8B for 3–10 days. At Day 10 of culture, well-delineated clusters of POU5F1-positive (POU5F1+) cells were visible in BMP4-treated and BMP2-treated EBs; these were rarely detected in untreated and BMP8B-treated cultures. Quantitative mRNA analysis revealed that a significant elevation of markers associated with primordial germ cell development had occurred in the presence of BMP4 by Day 10, including late germline markers such as Ddx4 (Mvh). Reasoning that germline specifi-cation was established by Day 10, we surveyed earlier time points for altered levels of germline marker mRNAs. A peak of early markers, Prdm1 (Blimp1), Ifitm3 (Fragilis), and Dppa3 (Stella), was measured in Day 3 to Day 4 EBs grown in BMP4, followed by a decrease at Day 5. In contrast, other markers, Pou5f1, Nanog, Dazl, and Ddx4, progressively increased from Day 3 to Day 5. Transforming growth factor beta superfamily signaling components Acvr1 (ALK2), Smad1, and Smad5 remained relatively constant. Isolated POU5F1+ cells fro