Simplified in vitro generation of human gastruloids for modelling early development

Abstract The purpose of this study was to optimize the efficiency and cost of human gastruloid formation by testing and adjusting individual parameters using as examples two distinct human pluripotent stem cell lines, both available from the UK Stem Cell Bank. For the first step, commercially sourced culture medium was replaced with a home-made defined recipe, known as ‘N2B27’, into which specific reagents can be titrated. By reducing the concentration of Activin A to 15% of the original protocol, efficient elongation of aggregated embryoid bodies was achieved. Also, titrating initial cell density and delaying the brief culture in GSK3 inhibitor until the onset of cell aggregation in individual wells was advantageous. Efficiency of formation of early gastruloids exhibiting the expected regionalization of the three embryonic germ layers was further enhanced by addition of TGF β -inhibitor. The optimization steps presented here thus provide a simplified, robust and relatively economical protocol for consistent generation of elongated gastruloids from human pluripotent stem cells. This streamlined method improves accessibility and reproducibility, also providing a standardized platform to investigate fundamental principles of early human development. Summary Statement We present an optimized protocol for human gastruloid production that should enhance efficiency, reproducibility and affordability for future in vitro studies into early human post-implantation development.