Simplified in vitro generation of human gastruloids for modelling early development

The purpose of this study was to optimize the efficiency, cost and reproducibility of human gastruloid formation by testing and adjusting individual parameters using four human pluripotent stem cell lines available from the UK Stem Cell Bank. First, 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 brief exposure to 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 TGFβ-inhibitor. The optimization steps presented 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 an adaptable platform to investigate fundamental principles of early human development.