title: Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.
abstract: Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×10⁴ RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.
authors.affiliation: Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Kuroda
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yasuda
authors.forename: Shinji
authors.initials: S
authors.lastname: Kusakawa
authors.forename: Naoya
authors.initials: N
authors.lastname: Hirata
authors.forename: Yasunari
authors.initials: Y
authors.lastname: Kanda
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Suzuki
authors.forename: Masayo
authors.initials: M
authors.lastname: Takahashi
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Nishikawa
authors.forename: Shin
authors.initials: S
authors.lastname: Kawamata
authors.forename: Yoji
authors.initials: Y
authors.lastname: Sato
mesh_terms: D015197:Carcinogenicity Tests / Q000379:methods*; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D012333:RNA, Messenger / Q000032:analysis; D016601:RNA-Binding Proteins / Q000032:analysis / Q000096:biosynthesis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D012680:Sensitivity and Specificity
chemical_list: C000715187:Lin28A protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins