title: Temporal analysis of genome alterations induced by single-cell passaging in human embryonic stem cells.
abstract: Simplified culture conditions are essential for large-scale drug screening and medical applications of human pluripotent stem cells (hPSCs). However, hPSCs [ie, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (iPSCs) are prone to genomic instability, a phenomenon that is highly influenced by the culture conditions. Enzymatic dissociation, a cornerstone of large-scale hPSC culture systems, has been reported to be deleterious, but the extent and the timeline of the genomic alterations induced by this passaging technique are still unclear. We prospectively monitored three hESC lines that were initially derived and cultured on human feeders and passaged mechanically before switching to enzymatic single-cell passaging. We show that karyotype abnormalities and copy number variations are not restricted to long-term culture, but can occur very rapidly, within five passages after switching hESCs to enzymatic dissociation. Subchromosomal abnormalities preceded or accompanied karyotype abnormalities and were associated with increased occurrence of DNA double-strand breaks. Our results indicate that enzymatic single-cell passaging can be highly deleterious to the hPSC genome, even when used only for a limited period of time. Moreover, hPSC culture techniques should be reappraised by complementing the routine karyotype analysis with more sensitive techniques, such as microarrays, to detect subchromosomal abnormalities.
authors.affiliation: 1 INSERM , U1040, Montpellier, France .
authors.forename: Qiang
authors.initials: Q
authors.lastname: Bai
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Ramirez
authors.forename: Fabienne
authors.initials: F
authors.lastname: Becker
authors.forename: Véronique
authors.initials: V
authors.lastname: Pantesco
authors.forename: Thierry
authors.initials: T
authors.lastname: Lavabre-Bertrand
authors.forename: Outi
authors.initials: O
authors.lastname: Hovatta
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Lemaître
authors.forename: Franck
authors.initials: F
authors.lastname: Pellestor
authors.forename: John
authors.initials: J
authors.lastname: De Vos
mesh_terms: D059786:Abnormal Karyotype; D002460:Cell Line; D049109:Cell Proliferation; D053903:DNA Breaks, Double-Stranded; D015870:Gene Expression; D015894:Genome, Human; D000066449:Human Embryonic Stem Cells / Q000502:physiology*; D006801:Humans