Generation of Primordial Germ Cell-like Cells from hESCs Using BMP4 and hAFSC-Conditioned Medium

TL;DR

This paper describes a new method to generate primordial germ cell-like cells from human embryonic stem cells using BMP4 and a special conditioned medium.

Contribution

A novel, reproducible protocol for generating PGC-LCs from hESCs without embryoid bodies.

Findings

The method uses BMP4 and hAFSC-4 conditioned medium to produce DDX4/DAZL-positive PGC-LCs.

Adherent culture avoids embryoid body formation, improving reproducibility.

The protocol offers a physiologically relevant model for human germ cell development.

Abstract

The differentiation of human embryonic stem cells (hESCs) into primordial germ cell-like cells (PGC-LCs) provides a robust in vitro model to study human germline specification. Here, we present a simple, reproducible, and cost-effective protocol for generating DEAD-box helicase 4 (DDX4)/VASA and Deleted in Azoospermia-Like (DAZL)-positive PGC-LCs from hESCs using a combination of bone morphogenetic protein 4 (BMP4) and conditioned medium (CM) derived from Stage-Specific Embryonic Antigen-4 (SSEA4)-positive human amniotic fluid stem cells (hAFSC-4). Importantly, unlike conventional protocols that rely on embryoid body formation, our method employs adherent cultures for germ cell differentiation. This approach enhances reproducibility by avoiding the spontaneous and stochastic variability inherent to embryoid body formation. This protocol provides a reproducible and physiologically relevant platform for studying human germ cell development in vitro.

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