Malgorzata Zawadzkaa,b, Kristina Sæterdal Kømurcua,b, Igor Meszkab, Helena Hruškováa, Stefan Kraussb,
Steven Ray Wilson,a,b, and Hanne Røberg-Larsena,b
a Department of Chemistry, University of Oslo, Norway
b Hybrid Technology Hub-Centre of Excellence, Institute of Basic Medical Sciences, Faculty of Medicine,
University of Oslo, Norway
Email: m.e.zawadzka@kjemi.uio.no
Human gastruloids are three-dimensional, stem cell-derived models that replicate aspects of the gastrulation process, offering a valuable platform for studying early human embryonic development and related disease mechanisms¹. This study focuses on detecting and quantifying oxysterols involved in key developmental signaling pathways², such as the Hedgehog and Wnt pathways.
Due to the low abundance and structural complexity of oxysterols, and the limited material from individual gastruloids, we optimized a Girard T-charge-tagging method suitable for limited sample oxysterol analysis. This approach was combined with on-line solid-phase extraction and liquid chromatography–mass spectrometry (LC–MS)3. Using this adapted workflow, we successfully identified and quantified 24S-hydroxycholesterol and 26-hydroxycholesterol, and detected a yet-to-be identified dihydroxycholesterol.
For quantification, oxysterol levels were normalized against total protein content. Individual gastruloids were lysed in isopropanol, and the resulting liquid fraction was used for oxysterol analysis, while the precipitated protein pellets were solubilized in aqueous buffer for quantification with BCA assay. Also, proteomics profiling revealed the presence of proteins associated with oxysterol metabolism and developmental signaling, such as Smoothened and GLI⁴.
Overall, our findings demonstrate that oxysterol profiling at the single-gastruloid level is feasible using a modified LC–MS workflow. This establishes a foundation for integrated multi-omics approaches to investigate developmental signaling pathway activity in gastruloid models. Ongoing work aims to extend this analysis to individual human gastruloids. Future research will focus on examining individual human gastruloids at various developmental stages.
Figure. Graphical abstract of the workflow for oxysterol analysis in single gastruloids.
References
1. Amel, A., Rossouw, S. & Goolam, M. Gastruloids: A Novel System for Disease Modelling and Drug Testing. Stem Cell Rev.
Rep. 19, 104–113 (2023).
2. Griffiths, W. J. & Wang, Y. Sterols, Oxysterols, and Accessible Cholesterol: Signalling for Homeostasis, in Immunity and During Development. Front. Physiol. 12, 723224 (2021).
3. Kømurcu, K. S. et al. Mass spectrometry reveals that oxysterols are secreted from non-alcoholic fatty liver disease induced organoids. J. Steroid Biochem. Mol. Biol. 232, 106355 (2023).
4. Li B. Communication codes in developmental signaling pathways. Development 146, dev170977 (2019).