title: Ablation of undifferentiated human embryonic stem cells: exploiting innate immunity against the Gal alpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) epitope.
abstract: Although undifferentiated human embryonic stem cells (hESCs) are tumorigenic, this capacity is lost after differentiation, and hESCs are being widely investigated for applications in regenerative medicine. To engineer protection against the unintentional transplantation of undifferentiated cells, we generated hESCs carrying a construct in which the alpha1,3-galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitope. The low-variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti-Neu5Gc, were not observed.
authors.affiliation: Department of Gene Function and Development, Roslin Institute, Roslin, Midlothian, United Kingdom.
authors.forename: Zoe
authors.initials: Z
authors.lastname: Hewitt
authors.forename: Helen
authors.initials: H
authors.lastname: Priddle
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Thomson
authors.forename: Davina
authors.initials: D
authors.lastname: Wojtacha
authors.forename: Jim
authors.initials: J
authors.lastname: McWhir
mesh_terms: D000017:ABO Blood-Group System; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D003001:Cloning, Molecular; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000276:immunology*; D005700:Galactosyltransferases / Q000235:genetics*; D006801:Humans; D007113:Immunity, Innate; D007621:Karyotyping; D051379:Mice; D016513:Mice, SCID; D009844:Oligosaccharides / Q000276:immunology*; D016366:Open Reading Frames; D012016:Reference Values; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D000017:ABO Blood-Group System; D017931:DNA Primers; D009844:Oligosaccharides; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase