Lectin binding studies of human embryonic stem cell and human embryonic stem cell derived neural progenitor cell surfaces
Venable, Alison Melina
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Human embryonic stem cells (hESCs) present a novel way to study developmental processes in vitro. Also, specific differentiation into cell types such as neurons is important due to the potential for therapeutic repair. Yet, fundamental studies have often been precluded in stem cell research perhaps due to the promotion of the cell’s potential in ameliorating a number of diseases. The purpose of this research is to examine the glycan expression of hESCs and hESC derived neural progenitors to foster understanding of glycoconjugates that may aid or denote pluripotency and neural differentiation specifically. The cell surface was screened because eukaryotic cells are endowed with a diverse array of glycoconjugates that promotes cell to cell communication. These specific oligosaccharides on the cell surfaces can also act as tags to delineate discriminating cell phenotypes. The goal was to examine the cell surfaces for glycan expression and to possibly determine new biomarkers for pluripotency, and neural differentiation using a panel of biotinylated lectins combined with flow cytometry analysis and immunocytochemistry. In the first study, SSEA-4, a pluripotency marker for hESCs was chosen to sort hESCs and then these were tested using the lectins. The glycan signature of hESCs shows that there are a number of carbohydrates and carbohydrate linkages present on the surface including alpha-linked mannose (ConA), galactosyl(beta 1-3)N-acetylgalactosamine (PNA), terminal galactose and N-acetylgalactosamine (RCA), GalNAc beta 4-Gal (WFA), N-acetylglucosamine (TL) and sialic acid alpha (2,6) GalNAc (SNA). There was no expression of alpha-linked N-acetylgalactosamine (DBA) and fucose alpha-linked to mannose (LTL). In the second study, nestin was chosen to denote multipotent hNPs, and using the same methodology, there was a dramatic increase of VVA, MAA, and PHA-L binding on hNP surfaces suggesting the presence of N-acetyl D-galactosamine, alpha 2,3-linked Neu5Ac, and complex N-type glycans containing beta(1,6) linked branches respectively. These lectins may be indicative of differentiation events but more specifically, of neurogenesis and may indicate a neural stem cell phenoype.