Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay

Current methods to generate human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) can be inefficient, and it is challenging to generate sufficient hPGCLCs to optimize in vitro game-togenesis. We present a differentiation method that uses diluted basement membrane extract (BMEx) and low BMP4 concentration to efficiently induce hPGCLC differentiation in scalable 2D cell culture. We show that BMEx overlay potentiated BMP/SMAD signaling, induced lumenogenesis, and increased expression of key hPGCLC-progenitor markers such as TFAP2A and EOMES. hPGCLCs that were generated using the BMEx overlay method were able to upregulate more mature germ cell markers, such as DAZL and DDX4, in human fetal ovary reconstitution culture. These findings highlight the importance of BMEx during hPGCLC differen-tiation and demonstrate the potential of the BMEx overlay method to interrogate the formation of PGCs and amnion in humans, as well as to investigate the next steps to achieve in vitro gametogenesis.

Automated summary

Current methods for generating human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) are inefficient, making it challenging to produce enough hPGCLCs for in vitro gametogenesis optimization. In this study, a differentiation method using diluted basement membrane extract (BMEx) and low BMP4 concentration was developed, which efficiently induced hPGCLC differentiation in scalable 2D cell culture. The overlay of BMEx enhanced BMP/SMAD signaling, induced lumenogenesis, and increased the expression of key hPGCLC-progenitor markers. These findings highlight the significance of BMEx in hPGCLC differentiation and its potential for studying PGC and amnion formation, as well as investigating further steps towards achieving in vitro gametogenesis.