Abstract
Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells.
Highlights
Human embryonic stem cells can be differentiated into neural progenitor cells [1] and mesenchymal progenitor cells [2] in vitro. hESC-derived human neural progenitors (hNP) cells and hESCs-derived mesenchymal progenitor (hMP) cells provide an invaluable source of cells to study early human neural and mesodermal development, respectively, as well as cell replacement therapy, drug screening, and toxicological studies
We identify that expression of N-acetyl-D-galactosamine (VVA lectin), Nacetyl-D-glucosamine (DBA lectin) and Fuca1,3-GlcNAc glycans (LTL lectin) is significantly up regulated in hNP cells, compared to hESCs and hMP cells
Human NP cells were derived from WA09 hESCs as described previously [1]. hNP cells can be propagated on MatrigelTM coated 100-mm tissue culture dishes for many passages (Figure 1B) and maintain normal karyotype
Summary
Human embryonic stem cells (hESCs) can be differentiated into neural progenitor (hNP) cells [1] and mesenchymal progenitor (hMP) cells [2] in vitro. hESC-derived hNP cells and hMP cells provide an invaluable source of cells to study early human neural and mesodermal development, respectively, as well as cell replacement therapy, drug screening, and toxicological studies. Human embryonic stem cells (hESCs) can be differentiated into neural progenitor (hNP) cells [1] and mesenchymal progenitor (hMP) cells [2] in vitro. Human NP cells are likely to follow a similar differentiation pathway as in vivo neurogenesis, based on murine studies where early expression of neural markers upon differentiation in vitro can parallel that of mouse neurogenesis [3,4]. Notch has been implicated in directing neural stem cells to a glial cell fate and neural stem cell renewal. While these two findings may seem contradictory, glysosylation of Notch on different serine residues could cause different outcomes [7,8]. The role of the carbohydrate surface is being brought to the forefront
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