Julie Bekkhus,a,b, Almira Ibragimov,a,b, Sander Johannes Thorbjørnsen Guttorm,c, Igor Meszka,b, Stefan
Krauss,b, and
Steven Ray Wilson,a,b.
a Section of Chemical Life Science, Department of Chemistry, University of Oslo, Norway
b Hybrid Technology Hub, Faculty of Medicine, University of Oslo, Norway
c Metabolomics and Metabolic Molecular Biology, Oslo University Hospital, Norway
Email: juliebek@uio.no
Gastruloids, derived from human Embryonic Stem Cells (hESCs), provide a scalable in vitro model that can be used to study various aspects of early human development. They can also be used to assess the influence of pharmaceuticals and environmental compounds on tissue patterning and germ layer formation. This system overcomes many limitations of animal models while remaining ethically acceptable for developmental studies. Fluoxetine, a widely prescribed selective serotonin reuptake inhibitor (SSRI), is used by approximately 0.76 % of pregnant women1. Although it is not classified as a potential teratogen, the extent to which it modulates early developmental signaling pathways is still not fully understood. In this study, we investigate the effects of fluoxetine on human gastruloid development across a range of concentrations. Gastruloids were exposed to fluoxetine starting one day after aggregation and continuously for the following four days (Figure 1). Daily imaging was performed using an EVOS microscope, and growth rates were quantified with area measurements using machine learning approaches. Quantitative Polymerase Chain Reaction (qPCR) was performed to assess the regulation of genes relevant to early embryonic development. Additionally, immunohistochemistry (IHC) combined with confocal spinning disk microscopy allowed for examination of germ layer organization. Mass Spectrometry (MS) will be used to measure fluoxetine penetration and stability in E6 medium. In collaboration with the Section for Metabolomics and Lipidomics at Oslo University Hospital, single-gastruloid lipidomics are being performed to assess heterogeneity within and between conditions.ospital to evaluate H Our results demonstrate a dose-dependent effects of fluoxetine on gastruloid growth, with 20 uM fluoxetine being lethal. At sublethal concentrations, qPCR revealed downregulation of mesodermal markers and upregulation of neural lineage markers, suggesting a shift from mesodermal to neuroectodermal fate. This shift may reflect modulation of Wnt signaling by fluoxetine, which is consistent with prior reports of Wnt pathway sensitivity to SSRIs at moderate levels2. In conclusion, our findings indicate that fluoxetine can alter early tissue patterning in human gastruloids in a dose-dependent manner, highlighting the model’s sensitivity to pharmacological perturbations of development.
Figure 1: Pretreated human Embryonic Stem Cells (hESCs) from 2D cultures were aggregated to form gastruloids. From 1 day post-aggregation (dpa), gastruloids were exposed to fluoxetine or maintained in control E6 medium, with daily media changes until 5 dpa. Illustration adapted from van den Brink et al.(3)
References
Molenaar, Nina M., et al. J. Affect. Disord, 2020, https://doi.org/10.1016/j.jad.2019.12.014
Warkus, Erica LL, and Yusuke Marikawa. ToxSci, 2018, https://doi.org/10.1093/toxsci/kfy143
van den Brink, Susanne C., and Alexander van Oudenaarden. Trends Cell Biol., 2021, https://doi.org/10.1016/j.tcb.2021.06.007