Identification of Unsafe Human Induced Pluripotent Stem Cell Lines Using a Robust Surrogate Assay for Pluripotency


  • Author contributions: J.C.P.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; M.S.H.H.: conception and design and collection and/or assembly of data; B.W., Q.Z., and G.K.: collection and/or assembly of data; E.W.: provision of study material; E.M.: data analysis and interpretation; C.A.W.: data analysis and interpretation and manuscript writing; S.M.G.: collection and/or assembly of data and data analysis and interpretation; I.B.: conception and design; C.O.B.: conception and design, manuscript writing, and data analysis and interpretation; A.L.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, provision of study material, manuscript writing, financial support, and final approval of manuscript.


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. STEM Cells 2013;31:1498–1510