Description
Glycosphingolipid (GSL) patterns change during the differentiation of mesenchymal stem cells (MSCs). Research by Hohenwallner et al. shows that standard 2D cultures of single-donor human adipose-derived MSCs reveal diverse ganglioside species linked to specifi c cellular states and lineage commitments (1). Additionally, ganglioside-rich lipid rafts are crucial for multilineage differentiation and signal transduction(2). However, GSL profi les from traditional 2D cultures may not capture the biological variability or three-dimensional microenvironments, especially for more primitive umbilical-cord-derived MSCs (UC-MSCs). This study aims to fi ll that gap by profi ling the glycosphingolipidome of UC-MSCs from multiple donors in 3D-spheroid culture. UC-MSCs (N=3 donors) were differentiated into adipogenic, chondrogenic, and osteogenic lineages using a 3D spheroid culture system. To facilitate lipid extraction from dense spheroids, we employed a mechanical dissociation method with a Beatbox homogenizer. Primary profi ling utilized the Orbitrap IQ-X in positive and negative modes. We also established an online 2D-LC-HRMS (HILICxRP) platform linked to EAD-ZenoTOF for high-resolution analysis of isomers and double-bond characterization. Multivariate analysis, revealed high inter-donor consistency with distinct GSL fi ngerprints for each cell lineage across all biological replicates. Preliminary data show clear lineage clustering, suggesting biologically signifi cant glycolipidomic differences. These results indicate that the 3D spheroid model can yield lineage-specifi c glycolipidomic profi le across diverse human donors. This studydemonstrates the effi cacy of cold-mechanical homogenization for the dissociation of 3D spheroids and validates lineage markers across three donors, establishing a foundation for quality control in stem cell therapies. Moreover, the developed 2D-LC-EAD workfl ow serves as a platform for addressing isomeric ambiguities.
| Period | 25 Feb 2026 |
|---|---|
| Event title | Joint DoSChem Retreat – 2026 |
| Event type | Seminar/Workshop |
| Location | Rust, AustriaShow on map |
| Degree of Recognition | National |