Protocol For Differentiating Pluripotent Stem Cells Into Primordial Germ Cell-like Cells.

Abstract

Using human pluripotent stem cell differentiation in vitro has significantly broadened the understanding of lineage specification events that occur during early development in vivo, a period that is extremely challenging to investigate. One of the first embryonic lineages to be specified in the human embryo is the germ cell lineage, marked by the induction of the primordial germ cells (PGCs). The PGCs are the sole founder population of the gametes later in life, and their specification is a critical first step towards the organism's fertility. Here, a robust protocol for generating PGC-like cells (PGCLCs) from human induced pluripotent stem cells (hiPSCs) is described, starting with the initial evaluation of the pluripotent state and cellular characteristics of the hiPSCs to ensure effective PGCLC differentiation. For this, the effects of different cell densities, plate coatings, and hiPSCs used for PGCLC differentiation are discussed. Finally, a workflow for characterization and quantification of hPGCLCs, using immunofluorescence and flow cytometry, is provided. The differentiation protocol focuses on conditions that are easy to establish and test in most laboratories and can be adjusted as necessary. Moreover, this protocol is fast, allowing determination of whether hiPSCs are suitable for PGCLC differentiation. The protocol aims to promote consistency and compatibility in hPGCLC outcomes across different hiPSC lines and culture platforms, ensuring robust yields that support reliable characterization and further optimization of downstream differentiation steps toward successful in vitro gametogenesis in humans.