title: Human embryonic stem cells passaged using enzymatic methods retain a normal karyotype and express CD30.
abstract: Human embryonic stem cells (hESCs) are thought to be susceptible to chromosomal rearrangements as a consequence of single cell dissociation. Compared in this study are two methods of dissociation that do not generate single cell suspensions (collagenase and EDTA) with an enzymatic procedure using trypsin combined with the calcium-specific chelator EGTA (TEG), that does generate a single cell suspension, over 10 passages. Cells passaged by single cell dissociation using TEG retained a normal karyotype. However, cells passaged using EDTA, without trypsin, acquired an isochromosome p7 in three replicates of one experiment. In all of the TEG, collagenase and EDTA-treated cultures, cells retained consistent telomere length and potentiality, demonstrating that single cell dissociation can be used to maintain karyotypically and phenotypically normal hESCs. However, competitive genomic hybridization revealed that subkaryotypic deletions and amplifications could accumulate over time, reinforcing that present culture regimes remain suboptimal. In all cultures the cell surface marker CD30, reportedly expressed on embryonal carcinoma but not karyoptically normal ESCs, was expressed on hESCs with both normal and abnormal karyotype, but was upregulated on the latter.
authors.affiliation: Division of Gene Function and Development, Roslin Institute, Roslin, Midlothian, Scotland. Alison.Thomson@bbsrc.ac.uk
authors.forename: Alison
authors.initials: A
authors.lastname: Thomson
authors.forename: Davina
authors.initials: D
authors.lastname: Wojtacha
authors.forename: Zoë
authors.initials: Z
authors.lastname: Hewitt
authors.forename: Helen
authors.initials: H
authors.lastname: Priddle
authors.forename: Virginie
authors.initials: V
authors.lastname: Sottile
authors.forename: Alex
authors.initials: A
authors.lastname: Di Domenico
authors.forename: Judy
authors.initials: J
authors.lastname: Fletcher
authors.forename: Martin
authors.initials: M
authors.lastname: Waterfall
authors.forename: Néstor López
authors.initials: NL
authors.lastname: Corrales
authors.forename: Ray
authors.initials: R
authors.lastname: Ansell
authors.forename: Jim
authors.initials: J
authors.lastname: McWhir
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D002871:Chromosome Banding; D017364:Collagenases / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D015894:Genome, Human / Q000187:drug effects*; D006801:Humans; D020014:K562 Cells; D007621:Karyotyping; D017730:Ki-1 Antigen / Q000378:metabolism*; D008954:Models, Biological; D014357:Trypsin / Q000494:pharmacology
chemical_list: D017730:Ki-1 Antigen; D004533:Egtazic Acid; D014357:Trypsin; D017364:Collagenases