N2B27 media formulations influence gastruloid development

Abstract

Gastruloids are 3D aggregates of pluripotent stem cells grown in suspension culture that mimic many aspects of gastrulation and early axial elongation. The N2B27 basal medium in which mouse gastruloids are cultured can either be home-made (HM-N2B27) with materials of known origin, or commercially sourced (NDiff227), where the exact formulation is unknown. In this study, we examined whether these formulations resulted in significant differences in gastruloid development. Our results reveal that while both media enable standard gastruloid elongation, HM-N2B27 gastruloids initiate the elongation process earlier, have a higher number of cells and an increased anterior domain. Despite the maintenance of overall gene expression patterns, RNAseq analysis indicated differences in cell fate specification, with HM-N2B27 gastruloids exhibiting higher expression of spinal cord-related genes, while NDiff227 favours mesodermal differentiation. Furthermore, differential gene enrichment analysis suggests that changes in key signalling pathways underlie the differences between HM-N2B27 and NDiff227 gastruloids. These findings highlight the importance of basal media composition for gastruloid development, underscoring the need for careful media selection during in vitro engineering of stem cell-based embryo models.

D.A.T. was funded in this work by the Biotechnology and Biological Sciences Research Council through a New Investigator Grant (BB/X000907/1) and a Strategic Longer and Larger (sLoLa) Programme Grant (BB/Y00311X/1), and by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC/T002131/1). A.M.A. was funded by an European Research Council Advanced Grant (MiniEmbryoBlueprint 834580) and by the "Maria de Maeztu" Program for Units of Excellence in R&D of the Ministerio de Ciencia, Innovación y Universidades (CEX2018-000792-M). J.D.F. was funded by a "la Caixa" Foundation (100010434) junior leader fellowship project (LCF/BQ/PI23/11970017). A.D. and J.D.F. were funded by European Molecular Biology Organization Postdoctoral Fellowships (ALTF 948-2022 to A.D.; ALTF 605-2022 to J.D.F.). Open Access funding provided by the University of Liverpool. Deposited in PMC for immediate release.