P9 - Expanding the glycoanalytical toolbox for understanding the role of glycans in pluripotent stem cell biology

Profiling of the glycome is crucial to tackle glycan complexity and diversity, and to uncover the role of individual glycan structures in health and disease. A highly precise and scalable technology for glycan profiling is multiplexed capillary gel electrophoresis coupled to laser induced fluorescencedetection (xCGE-LIF). Recently, we successfully translated existing xCGE-LIF based N-glycan profiling strategies to the analysis of sialylated and neutral glycosphingolipids (GSLs). In the first funding period, we developed a software package for automated data analysis, and together with P7, we expanded our GSL-glycan database by ganglioside-derived glycans with and without 9-Oacetylated sialic acids. The achieved methodological advances enabled us (i) to identify the GSL glycan Lc4 as novel pluripotency marker for human pluripotent stem cells (hPSCs), (ii) to uncover changes in the GSL pattern of murine splenic T cells and dendritic cells under inflammatory conditions, and (iii) to significantly progress work in projects P1, P4, P6 and P7 of this research unit by providing glycan profiling and glycoproteomics. We will continuously expand our glycoanalytical toolbox, e.g. towards N-glycans decorated with modified sialic acids and to further provide our technologies to the consortium. To enable studies on the biological function of GSLs during early human developmental processes, we targeted the UDP-glucose ceramide glucosyltransferase (UGCG) gene in human induced pluripotent stem cells (hiPSCs) by CRISPR-Cas9. UGCG catalyses the de novo synthesis of glucosylceramide, the precursor of all globo-, lacto- and ganglio-series GSLs. The obtained UGCG-/- hiPSCs were completely devoid of glucosylceramide-based GSLs, but remained characteristics of pluripotency. However, distinct phenotypes became apparent upon differentiation and we will apply the UGCG-deficient hiPSC model in the following to study the biological meaning of GSLs, and in particular of gangliosides, during hPSC differentiation. A specific focus will be laid on understanding how GSLs contribute to cellular signalling processes of hPSC. We expect a deeper understanding on the role of GSLs for hPSC differentiation, which is urgently required to improve in vitro differentiation approaches of hPSCs for future medical applications.