title: Excess of O-linked N-acetylglucosamine modifies human pluripotent stem cell differentiation.
abstract: O-linked-N-acetylglucosamine (O-GlcNAc), a post translational modification, has emerged as an important cue in controlling key cell mechanisms. Here, we investigate O-GlcNAc's role in the maintenance and differentiation of human pluripotent stem cells (hPSC). We reveal that protein expression of O-GlcNAc transferase and hydrolase both decreases during hPSC differentiation. Upregulating O-GlcNAc with O-GlcNAc hydrolase inhibitors has no significant effect on either the maintenance of pluripotency in hPSC culture, or the loss of pluripotency in differentiating hPSC. However, in spontaneously differentiating hPSC, excess O-GlcNAc alters the expression of specific lineage markers: decrease of ectoderm markers (PAX6 by 53-88%, MSX1 by 26-49%) and increase of adipose-related mesoderm markers (PPARγ by 28-100%, C/EBPα by 46-135%). All other lineage markers tested (cardiac, visceral-endoderm, trophectoderm) remain minimally affected by upregulated O-GlcNAc. Interestingly, we also show that excess O-GlcNAc triggers a feedback mechanism that increases O-GlcNAc hydrolase expression by 29-91%. To the best of our knowledge, this is the first report demonstrating that excess O-GlcNAc does not affect hPSC pluripotency in undifferentiated maintenance cultures; instead, it restricts the hPSC differentiation towards specific cell lineages. These data will be useful for developing targeted differentiation protocols and aid in understanding the effects of O-GlcNAc on hPSC differentiation.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
authors.forename: Julien Jean Pierre
authors.initials: JJ
authors.lastname: Maury
authors.forename: Ken Kwok-Keung
authors.initials: KK
authors.lastname: Chan
authors.forename: Lu
authors.initials: L
authors.lastname: Zheng
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Andre Boon-Hwa
authors.initials: AB
authors.lastname: Choo
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
chemical_list: D017351:N-Acetylglucosaminyltransferases; D006867:Hydrolases; D000117:Acetylglucosamine