From Stem Cells to Post-gastrulation Amnioids (PGAs): A Protocol to Model Human Extra-Embryonic Development

Successful embryonic development is a collaborative process: the embryo relies on the support of extra-embryonic and maternal tissues. However, understanding early human development is challenging due to the ethical and technical limitations. The generation of stem cell-based models that mimics embryonic and/or extra-embryonic tissues provides in vitro platforms to study human embryogenesis. We recently developed a 3D embryonic stem cell-derived model that mimics the post-gastrulation amnion and extra-embryonic tissues. These structures, which we call post-gastrulation amnioids (PGAs), capture early human amnion development up to the formation of a mature fluid-filled amniotic sac-like membrane composed of two cell layers of inner amniotic ectoderm and an outer extra-embryonic mesoderm. PGAs gradually expand in culture and exhibit functional traits of human amnion. PGAs are highly reproducible and can be scaled for high throughput studies. Here, we describe a step-by-step protocol for PGA generation and their morphological characterization by immunofluorescence staining as well as a co-culture assay to begin to understand interactions between extra-embryonic and embryonic cells.