Leyla Gasimli1, Anne Marie Hickey1, Bo Yang2, Guoyun Li2, Mitche dela Rosa3, Alison V Nairn3, Michael J Kulik4, Jonathan S Dordick5, Kelley W Moremen3, Stephen Dalton4, Robert J Linhardt6. 1. Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. 2. Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. 3. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA. 4. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA. 5. Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Materials Science and Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. 6. Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: linhar@rpi.edu.
Abstract
BACKGROUND: Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. METHODS: Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis. RESULTS: Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. CONCLUSIONS: Differentiation of embryonic stem cells markedly changes the proteoglycanome. GENERAL SIGNIFICANCE: The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation.
BACKGROUND: Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. METHODS:Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis. RESULTS: Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. CONCLUSIONS: Differentiation of embryonic stem cells markedly changes the proteoglycanome. GENERAL SIGNIFICANCE: The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation.
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