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.
Highlights
Cell therapy products (CTPs) are expected to offer promising treatments for serious and life-threating diseases for which no adequate therapy is currently available
We focused on laminin-521 as a cell culture matrix, which permits survival of dissociated human pluripotent stem cells (hPSCs) without a Rho-associated protein kinase (ROCK) inhibitor [7]. mTeSR1 medium is conventionally used to culture dissociated human induced pluripotent stem cells (hiPSCs) on dishes coated with laminin-521 [7], and other hPSC media besides mTeSR1 have not been fully characterized with laminin-521
A method to detect residual undifferentiated hPSCs contained in CTPs is required to evaluate product quality during manufacturing processes
Summary
Cell therapy products (CTPs) are expected to offer promising treatments for serious and life-threating diseases for which no adequate therapy is currently available. An increasing number of CTPs derived from human pluripotent stem cells (hPSCs), i.e. induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs), are being developed for regenerative medicine/cell therapy because of their infinite self-renewal capacity and their ability to differentiate into various types of cells. Quality assessment of CTPs is critical to ensure their safety and efficacy for clinical application [1]. The presence of residual undifferentiated cells in CTPs derived from hPSCs is one of the most serious concerns for tumorigenicity because the undifferentiated hPSCs have a capacity to form teratoma in animals [1,2,3,4]. Establishment of a detection method for residual undifferentiated cells is necessary for the safety and quality assessment of CTPs derived from hPSCs
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