Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki Ward, Kawasaki City, Kanagawa, 210-9501, Japan.
Department of Quality Assurance Science for Pharmaceuticals, Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan.
Sci Rep. 2021 Jun 1;11(1):11407. doi: 10.1038/s41598-021-90928-7.
Highly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.
高度敏感地检测残留未分化多能干细胞对于源自人诱导多能干细胞（hiPSC）的细胞处理治疗产品的质量和安全性至关重要。我们之前报道了一种腺病毒（Ad）载体和腺相关病毒载体的产生，它们具有自杀基因，可诱导 Caspase 9（iCasp9），这使得在 hiPSC 衍生的心肌细胞培养物中灵敏地检测未分化的 hiPSC 成为可能。在这项研究中，我们研究了这些载体是否也允许在 hiPSC 衍生的神经祖细胞（hiPSC-NPC）制剂中检测未分化的 hiPSC，这些细胞有望治疗神经退行性疾病。为了检测未分化的 hiPSC，通过免疫染色和流式细胞术确定多能干细胞标志物的表达。使用永生化 NPC 作为模型，发现与测试的载体相比，Ad 载体在检测未分化的 hiPSC 方面最为有效。此外，我们发现 Ad 载体以 iCasp9 依赖的方式杀死大多数 hiPSC-NPC，从而使流式细胞术能够以比以前报道的（0.1%）更低的浓度（0.002%）检测到混合的未分化 hiPSC。这些数据表明，Ad 载体选择性地消除 hiPSC-NPC，从而能够灵敏地检测 hiPSC。这种细胞毒性病毒载体有助于确保用于治疗的 hiPSC-NPC 的质量和安全性。
