PSC 衍生细胞治疗产品 (CTP) 中残留的未分化多能干细胞 (PSC) 是其临床应用的一个主要安全问题，因为存在 PSC 衍生肿瘤形成的潜在风险。作为健康与环境科学研究所细胞治疗追踪、循环的一部分，进行了一项国际多学科多中心研究，以评估液滴数字 PCR (ddPCR) 方法，以检测 PSC 衍生的 CTP 中残留的未分化 PSC

The presence of residual undifferentiated pluripotent stem cells (PSCs) in PSC-derived cell therapy products (CTPs) is a major safety issue for their clinical application, due to the potential risk of PSC-derived tumor formation. An international multidisciplinary multisite study to evaluate a droplet digital PCR (ddPCR) approach to detect residual undifferentiated PSCs in PSC-derived CTPs was conducted as part of the Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee. To evaluate the use of ddPCR in quantifying residual iPSCs in a cell sample, different quantities of induced pluripotent stem cells (iPSCs) were spiked into a background of iPSC-derived cardiomyocytes (CMs) to mimic different concentrations of residual iPSCs. A one step reverse transcription ddPCR (RT-ddPCR) was performed to measure mRNA levels of several iPSC-specific markers and to evaluate the assay performance (precision, sensitivity, and specificity) between and within laboratories. The RT-ddPCR assay variability was initially assessed by measuring the same RNA samples across all participating facilities. Subsequently, each facility independently conducted the entire process, incorporating the spiking step, to discern the parameters influencing potential variability. Our results show that a RT-ddPCR assay targeting ESRG, LINC00678, and LIN28A genes offers a highly sensitive and robust detection of impurities of iPSC-derived CMs and that the main contribution to variability between laboratories is the iPSC-spiking procedure, and not the RT-ddPCR. The RT-ddPCR assay would be generally applicable for tumorigenicity evaluation of PSC-derived CTPs with appropriate marker genes suitable for each CTP.© The Author(s) 2024. Published by Oxford University Press.