Abstract：
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.
摘要：
PSC衍生的细胞治疗产品（CTP）中残留的未分化多能干细胞（PSC）的存在是其临床应用的主要安全性问题。由于PSC衍生的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-TRAcking的一部分，进行了一项国际多学科多点研究，以评估液滴数字PCR（ddPCR）方法来检测PSC衍生的CTP中残留的未分化PSC。流通与安全技术委员会。为了评估ddPCR在定量细胞样品中的残留iPSC中的用途，将不同数量的诱导多能干细胞(iPSC)掺入iPSC衍生的心肌细胞(CM)的背景中以模拟不同浓度的残留iPSC.进行一步逆转录ddPCR（RT-ddPCR）以测量几种iPSC特异性标志物的mRNA水平并评估测定性能（精度，灵敏度,和特异性)在实验室之间和内部。最初通过在所有参与设施中测量相同的RNA样品来评估RT-ddPCR测定变异性。随后，每个设施独立进行整个过程，结合加标步骤，辨别影响潜在变异性的参数。我们的结果表明，靶向ESRG的RT-ddPCR，LINC00678和LIN28A基因对iPSC衍生的CM的杂质提供了高度灵敏和强大的检测，并且对实验室之间的变异性的主要贡献是iPSC加标程序，而不是RT-ddPCR。RT-ddPCR测定通常适用于PSC衍生的CTP的致瘤性评估,具有适合于每个CTP的适当标记基因。
