N2B27 media formulations influence gastruloid development

Balayo, T.; Lunn, S.; Pascual-Mas, P.; Fiuza, U.-M.; Vasudevan, A.; Frenster, J. D.; Galloon, H. Y.; Peirats, R. F.; Martinez Arias, A.; Dias, A.; Turner, D. A.

doi:10.1101/2025.03.15.643474

bioRxiv · 10.1101/2025.03.15.643474

N2B27 media formulations influence gastruloid development

Balayo, T.·Lunn, S.·Pascual-Mas, P.·Fiuza, U.-M.·Vasudevan, A.·Frenster, J. D.·Galloon, H. Y.·Peirats, R. F.·Martinez Arias, A.·Dias, A.·Turner, D. A.

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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 the standard gastruloid elongation, HM-N2B27 results in gastruloids that start the elongation process earlier, have higher number of cells and an increased anterior domain. RNAseq analysis showed significant 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 underline 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. Summary statementIn this work we explored the cellular and molecular sensitiveness of the mouse gastruloid model system to variations in N2B27 media formulations.

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Balayo, T., Lunn, S., Pascual-Mas, P., Fiuza, U.-M., Vasudevan, A., Frenster, J. D., Galloon, H. Y., Peirats, R. F., Martinez Arias, A., Dias, A., Turner, D. A.. 2025-03-16. N2B27 media formulations influence gastruloid development. https://doi.org/10.1101/2025.03.15.643474

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