Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay
Summary
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 gametogenesis. 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 differentiation 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. © 2023 The Authors.
| Authors | Overeem AW, Chang YW, Moustakas I, Roelse CM, Hillenius S, Helm TV, Schrier VFV, Gonçalves MAFV, Mei H, Freund C, Chuva de Sousa Lopes SM |
|---|---|
| Journal | Cell reports methods |
| Publication Date | 2023 Jun 26;3(6):100488 |
| PubMed | 37426764 |
| PubMed Central | PMC10326346 |
| DOI | 10.1016/j.crmeth.2023.100488 |