Detection of residual pluripotent stem cells in cell therapy products utilizing droplet digital PCR: an international multisite evaluation study.

IF 5.4 2区 医学 Q1 CELL & TISSUE ENGINEERING Stem Cells Translational Medicine Pub Date : 2024-10-10 DOI:10.1093/stcltm/szae058
Satoshi Yasuda, Kiyoko Bando, Marianne P Henry, Silvana Libertini, Takeshi Watanabe, Hiroto Bando, Connie Chen, Koki Fujimori, Kosuke Harada, Takuya Kuroda, Myriam Lemmens, Dragos Marginean, David Moss, Lucilia Pereira Mouriès, Nicole S Nicholas, Matthew J K Smart, Orie Terai, Yoji Sato
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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.

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利用液滴数字 PCR 检测细胞治疗产品中残留的多能干细胞:一项国际多站点评估研究。
由于多能干细胞衍生的肿瘤形成的潜在风险,多能干细胞衍生的细胞治疗产品(CTPs)中残留的未分化多能干细胞(PSCs)是其临床应用的一个主要安全问题。作为健康与环境科学研究所细胞治疗-检测、循环与安全技术委员会(Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee)的一部分,开展了一项国际多学科多点研究,以评估用液滴数字 PCR(ddPCR)方法检测 PSC 衍生 CTP 中残留的未分化 PSC。为了评估 ddPCR 在量化细胞样本中残留 iPSCs 中的应用,在 iPSC 衍生的心肌细胞(CMs)背景中添加了不同数量的诱导多能干细胞(iPSCs),以模拟不同浓度的残留 iPSCs。采用一步反转录 ddPCR(RT-ddPCR)方法测量了几种 iPSC 特异性标记物的 mRNA 水平,并评估了实验室之间和实验室内部的检测性能(精确度、灵敏度和特异性)。RT-ddPCR 检测的可变性最初是通过测量所有参与机构的相同 RNA 样本来评估的。随后,每家机构都独立完成了包括加标步骤在内的整个过程,以确定影响潜在变异性的参数。我们的研究结果表明,针对 ESRG、LINC00678 和 LIN28A 基因的 RT-ddPCR 检测能高度灵敏、稳健地检测 iPSC 衍生 CM 的杂质,而造成实验室间变异的主要原因是 iPSC 加标步骤,而不是 RT-ddPCR。RT-ddPCR测定一般适用于PSC衍生的CTP的肿瘤致性评估,并配有适合每种CTP的适当标记基因。
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来源期刊
Stem Cells Translational Medicine
Stem Cells Translational Medicine CELL & TISSUE ENGINEERING-
CiteScore
12.90
自引率
3.30%
发文量
140
审稿时长
6-12 weeks
期刊介绍: STEM CELLS Translational Medicine is a monthly, peer-reviewed, largely online, open access journal. STEM CELLS Translational Medicine works to advance the utilization of cells for clinical therapy. By bridging stem cell molecular and biological research and helping speed translations of emerging lab discoveries into clinical trials, STEM CELLS Translational Medicine will help move applications of these critical investigations closer to accepted best patient practices and ultimately improve outcomes. The journal encourages original research articles and concise reviews describing laboratory investigations of stem cells, including their characterization and manipulation, and the translation of their clinical aspects of from the bench to patient care. STEM CELLS Translational Medicine covers all aspects of translational cell studies, including bench research, first-in-human case studies, and relevant clinical trials.
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