Abstract
We developed two human-induced pluripotent stem cell (hiPSC)/human embryonic stem cell (hESC)-specific glycan-recognizing mouse antibodies, R-10G and R-17F, using the Tic (JCRB1331) hiPSC line as an antigen. R-10G recognizes a low-sulfate keratan sulfate, and R-17F recognizes lacto-N-fucopentaose-1. To evaluate the general characteristics of stem cell glycans, we investigated the hiPSC line 201B7 (HPS0063), a prototype iPSC line. Using an R-10G affinity column, an R-10G-binding protein was isolated from 201B7 cells. The protein yielded a single but very broad band from 480 to 1236 kDa by blue native gel electrophoresis. After trypsin digestion, the protein was identified as podocalyxin by liquid chromatography/mass spectrometry. According to Western blotting, the protein reacted with R-10G and R-17F. The R-10G-positive band was resistant to digestion with glycan-degrading enzymes, including peptide N-glycanase, but the intensity of the band was decreased significantly by digestion with keratanase, keratanase II, and endo-β-galactosidase, suggesting the R-10G epitope to be a keratan sulfate. These results suggest that keratan sulfate-type epitopes are shared by hiPSCs. However, the keratan sulfate from 201B7 cells contained a polylactosamine disaccharide unit (Galβ1-4GlcNAc) at a significant frequency, whereas that from Tic cells consisted mostly of keratan sulfate disaccharide units (Galβ1-4GlcNAc(6S)). In addition, the abundance of the R-10G epitope was significantly lower in 201B7 cells than in Tic cells.
Highlights
In the last decade, many interesting findings have been reported in the field of stem cells
We examined the glyco-epitope profiles of the crude extracts of 201B7 cells, as described previously in the antibodies raised recognized both hiPSCs (Tic) cells, which were used as antigens to raise anti-iPSCs in mice [6,8]
The predominant portion of these glyco-epitopes on podocalyxins isolated from Tic cells and 201B7 cells appear to be expressed on O-linked glycans and/or glycosaminoglycans and not on Nlinked glycans, 5 potential N-glycosylation sites are present in the mature protein
Summary
Many interesting findings have been reported in the field of stem cells. Human-induced pluripotent stem cells (hiPSCs) are already used as tools for drug development and disease modeling, and their most important potential application is the generation of cells and tissues that can be used for regenerative medicine [1]. Stem cell research is obviously one of the most fascinating and exciting areas of contemporary biology, including glycobiology [2]. Significant alterations in the cellular glycoform may occur during differentiation and glycans could serve as specific stem cell markers and cellular differentiation biomarkers. At the time when hiPSCs were first generated from differentiated cells, SSEA4 [3,4] and TRA1-60 [5] were widely used as pluripotent cell markers
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