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

SUMMARY Current human primordial germ cell like cells (hPGCLCs) differentiation methods from human pluripotent stem cells (hPSCs) are inefficient, and it is challenging to generate sufficient hPGCLCs to optimize in vitro gametogenesis. We present a new differentiation method that uses diluted basement membrane extract (BME) and low BMP4 concentration to efficiently induce hPGCLC differentiation in scalable 2D cell culture. We show that BME overlay potentiated BMP/SMAD signaling, induced lumenogenesis and increased expression of key hPGCLC-progenitor markers such as TFAP2A and EOMES. These findings highlight the importance of (factors in) BME during hPGCLC differentiation, and demonstrate the potential of the BME-overlay method to interrogate the formation of PGCs and amnion in humans as well to investigate the next steps to achieve in vitro gametogenesis. HIGHLIGHTS hPGCLCs can be generated efficiently in 2D from hPSCs with treatment of BMP4 and BME overlay BME overlay method is highly scalable, cost-effective and simple to perform hPGCLCs differentiate together with amniotic ectoderm- and mesoderm-like cells from a TFAP2A+/CDX2+/EOMES+/GATA3+ common progenitor population BME overlay enables robust hPGCLC formation by potentiating BMP/SMAD signaling in the common progenitor population