Induction of Primordial Germ Cell Like Cell from Mouse Epiblast Stem Cells in Three-Dimensional Culture

Generation of primordial germ cell-like cells (PGCLCs) from pluripotent stem cells in vitro serve as key intermediates in the modeling of germline development. While previous studies have predominantly focused on the induction of PGCLCs from epiblast-like cells (EpiLCs), recent studies suggest that BMP4 signaling can also drive PGCLC specification from epiblast stem cells (EpiSCs). However, the efficiency of PGCLC induction from EpiSCs remains suboptimal and underexplored. We hypothesized that the dimensional structure of the culture environment significantly influences the differentiation efficiency. To evaluate PGCLC induction efficiency, we used Blimp1-mVenus×Stella-ECFP (BVSC) transgenic reporter embryonic stem cells. FACS analysis revealed that the proportion of Blimp1-mVenus⁺/Stella-ECFP⁺ double-positive PGCLCs was significantly higher in the 3D aggregate culture compared to the 2D monolayer system. Our findings demonstrate that a 3D culture environment enhances the efficiency of PGCLC induction from mouse EpiSCs compared to a 2D monolayer system. These results highlight the importance of culture dimensionality in optimizing germ cell differentiation protocols and provide a useful framework for further studies on germline development.