title: A novel in vitro method for detecting undifferentiated human pluripotent stem cells as impurities in cell therapy products using a highly efficient culture system.
abstract: Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%-0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process.
authors.affiliation: Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo, Japan; Division of Cellular & Gene Therapy Products, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Tano
authors.affiliation: Division of Cellular & Gene Therapy Products, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yasuda
authors.affiliation: Division of Cellular & Gene Therapy Products, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Kuroda
authors.affiliation: National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: Hirohisa
authors.initials: H
authors.lastname: Saito
authors.affiliation: Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Umezawa
authors.affiliation: Division of Cellular & Gene Therapy Products, National Institute of Health Sciences, Tokyo, Japan; Department of Quality Assurance Science for Pharmaceuticals, Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan; Department of Cellular & Gene Therapy Products, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; Department of Translational Pharmaceutical Sciences, Graduated School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yoji
authors.initials: Y
authors.lastname: Sato
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D064987:Cell- and Tissue-Based Therapy; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D007797:Laminin; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology*
chemical_list: D003470:Culture Media; D007797:Laminin