We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-β-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.
Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galβ1 - 3GlcNAc (type 1) or Galβ1 - 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galβ1 - 4GlcNAc(6S)β1 - 3Galβ1 - 4GlcNAc(6S)β1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galβ1 - 3GlcNAcβ1 - 3Galβ1 - 4GlcNAcβ1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125-1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galβ1 - 3GlcNAc(6S)β1 - 3Galβ1 - 4GlcNAc(6S)β1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galβ1 - 3GlcNAcβ1 - 3Galβ1 - 4GlcNAcβ1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only "type 2-type 2 keratan sulfate" but also "type 1-type 2 keratan sulfate", significantly.
Abstract. To test the possibility that micromolar formaldehyde, a metabolite of methanol derived from aspartame, exerts cytotoxicity, its effect on rat thymocytes was examined under the in vitro condition using a flow cytometer. Incubation of thymocytes with formaldehyde at 100 mM or more for 24 h significantly increased the populations of shrunken cells and cells with hypodiploid DNA. The peak blood concentration of methanol in human subjects administered abuse doses of aspartame has been reported to exceed 2 mg / dL (625 m M). It would increase the population of thymocytes undergoing apoptosis if formaldehyde at 100 m M or more appears in the blood after administration of aspartame.
Background: Carbohydrate epitopes are often used as markers for characterization of hiPS cells. Results: A mouse IgG1 antibody (R-17F) was raised using hiPS cells as an antigen. Conclusion: R-17F recognizes lacto-N-fucopentaose I on glycolipid and exhibits a cytotoxic effect on hiPS/ES cells. Significance: R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS/ES cells, which are a risk factor for carcinogenesis.
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