Integrated Multi-omics Analysis Uncovers Molecular Dysregulation in Glycosylceramide Synthase-Deficient Human Induced Pluripotent Stem Cells
Glycosphingolipids (GSLs) are membrane lipids characterized by highly diverse glycosylation patterns. They are involved in numerous cellular processes and can be linked to diseases. With the aim to study their relevance for early mammalian development, we mutated the UDP-glucose ceramide glucosyltransferase gene (UGCG) encoding the enzyme glucosylceramide synthase (UGCG) in human induced pluripotent stem cells (hiPSCs) by CRISPR/Cas9. The functional impairment of UGCG was confirmed by glycomic profiling. UGCG knock out (KO) hiPSCs displayed normal morphology, growth behaviour, and expression of stem cell markers compared to wildtype (WT) hiPSCs. Furthermore, the KO cells maintained pluripotency, as evidenced by their capacity for in vitro differentiation into all three embryonic germ layers and their ability to form teratomas in vivo. Quantitative proteomic analysis of explanted teratoma derived from WT and UGCG KO hiPSCs revealed differential expression of numerous proteins, with notable enrichment of pathways associated with signal transduction or protein localization and transport. Phosphorylation profiling of key kinases and their targets uncovered reduced activation of STAT family transcription factors in UGCG KO hiPSCs. Complementary transcriptomic profiling combined with Ingenuity Pathway Analysis (IPA) of differentially expressed genes between WT and UGCG KO hiPSCs, as well as their ectodermal derivatives, revealed a predicted positive enrichment of proteins associated with signaling pathways in UGCG KO hiPSCs. Global lipidomic profiling showed a significant increase in sphingomyelin levels in UGCG KO hiPSCs and their ectodermally differentiated derivatives, whereas total ceramide content remained comparable between UGCG KO and WT cells. In summary, despite the absence of overt phenotypic changes, our findings demonstrate that glycosphingolipid deficiency induces multiple molecular perturbations in hiPSCs.