title: Identification of unsafe human induced pluripotent stem cell lines using a robust surrogate assay for pluripotency.
abstract: Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies, and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify "unsafe" hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these "unsafe" hiPSC lines should be interpreted carefully.
authors.affiliation: CSIRO, Materials Science and Engineering, Clayton, Victoria, Australia; Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Polanco
authors.forename: Mirabelle S H
authors.initials: MS
authors.lastname: Ho
authors.forename: Bei
authors.initials: B
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhou
authors.forename: Ernst
authors.initials: E
authors.lastname: Wolvetang
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mason
authors.forename: Christine A
authors.initials: CA
authors.lastname: Wells
authors.forename: Gabriel
authors.initials: G
authors.lastname: Kolle
authors.forename: Sean M
authors.initials: SM
authors.lastname: Grimmond
authors.forename: Ivan
authors.initials: I
authors.lastname: Bertoncello
authors.forename: Carmel
authors.initials: C
authors.lastname: O'Brien
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Laslett
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D013724:Teratoma / Q000473:pathology; D059467:Transcriptome
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C494174:Pou5f1 protein, mouse
keywords: Embryonic stem cells; Fluorescence activated cell sorting; Induced pluripotent stem cells; Pluripotency; Pluripotent stem cells; Reprogramming; Tumor cell purging