title: A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.
abstract: 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.
authors.affiliation: Research Center for Glycobiotechnology, Ritsumeikan University, Noji-Higashi 1-1-1, Kusatsu, Shiga 525-8577, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Kawabe
authors.forename: Daiki
authors.initials: D
authors.lastname: Tateyama
authors.forename: Hidenao
authors.initials: H
authors.lastname: Toyoda
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hashii
authors.forename: Hiromi
authors.initials: H
authors.lastname: Nakao
authors.forename: Shogo
authors.initials: S
authors.lastname: Matsumoto
authors.forename: Motohiro
authors.initials: M
authors.lastname: Nonaka
authors.forename: Hiroko
authors.initials: H
authors.lastname: Matsumura
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Hirose
authors.forename: Ayaha
authors.initials: A
authors.lastname: Morita
authors.forename: Madoka
authors.initials: M
authors.lastname: Katayama
authors.forename: Makoto
authors.initials: M
authors.lastname: Sakuma
authors.forename: Nobuko
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Miho Kusuda
authors.initials: MK
authors.lastname: Furue
authors.forename: Toshisuke
authors.initials: T
authors.lastname: Kawasaki
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D045744:Cell Line, Tumor; D053595:Embryonic Stem Cells / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000276:immunology*; D007632:Keratan Sulfate / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007632:Keratan Sulfate