Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZC Leiden, the Netherlands.
Sequencing Analysis Support Core, Leiden University Medical Center, 2333 ZC Leiden, the Netherlands.
Cell Rep Methods. 2023 May 23;3(6):100488. doi: 10.1016/j.crmeth.2023.100488. eCollection 2023 Jun 26.
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 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 gametogenesis.
目前从人类多能干细胞（hPSC）生成人类原始生殖细胞样细胞（hPGCLC）的方法效率可能较低，并且难以生成足够的 hPGCLC 来优化配子发生。我们提出了一种分化方法，该方法使用稀释的基底膜提取物（BMEx）和低浓度的 BMP4 在可扩展的 2D 细胞培养中有效诱导 hPGCLC 分化。我们表明，BMEx 覆盖物增强了 BMP/SMAD 信号传导，诱导了管腔形成，并增加了关键的 hPGCLC-祖细胞标记物的表达，如 TFAP2A 和 EOMES。在使用 BMEx 覆盖物方法生成的 hPGCLCs 中，能够在人胎儿卵巢重建培养中上调更多成熟的生殖细胞标记物，如 DAZL 和 DDX4。这些发现强调了 BMEx 在 hPGCLC 分化过程中的重要性，并证明了 BMEx 覆盖物方法在研究人类 PGC 和羊膜形成以及探索实现配子发生的下一步骤方面具有潜力。
