Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
Biochem Biophys Res Commun. 2021 May 21;554:13-18. doi: 10.1016/j.bbrc.2021.03.078. Epub 2021 Mar 25.
Human induced pluripotent stem cells (hiPSCs) are important starting materials for cell therapy products (CTPs) used for transplantation. During cell culture, hiPSCs often spontaneously undergo morphological changes and lose pluripotency. Such cells are called 'deviated cells', which are deviated from the undifferentiated state of hiPSCs, lack the expression of hiPSC markers and become positive for the early differentiation marker SSEA1 (stage-specific embryonic antigen 1, Lewis X glycan). Previously, we identified fibronectin (FN) as a predominant carrier protein of SSEA1 secreted from deviated cells, but not hiPSCs. A sandwich assay using antibodies (Abs) against FN and SSEA1 was developed for non-destructive quantitative evaluation of deviated cells present in hiPSC cultures. In this study, a novel technology was developed to specifically eliminate deviated cells using an anti-FN Ab along with a near-infrared (NIR) photoabsorber, IRDye700DX N-hydroxysuccinimide ester (IR700), which has been used for cancer photoimmunotherapy. The anti-FN Ab conjugated with the IR700 dye (IR700-αFN) bound to and induced the death of deviated cells upon NIR irradiation. In contrast, IR700-αFN failed to stain the hiPSCs, and IR700-αFN/NIR had little or no effect on survival. Finally, IR700-αFN/NIR irradiation induced selective removal of deviated cells from a mixed culture with hiPSCs, demonstrating that the proposed method is suitable for the removal of unwanted deviated cells present in hiPSC culture for the production of CTPs.
人诱导多能干细胞（hiPSCs）是用于移植的细胞治疗产品（CTPs）的重要起始材料。在细胞培养过程中，hiPSCs 经常自发地发生形态变化并失去多能性。这些细胞称为“偏离细胞”，它们偏离了 hiPSCs 的未分化状态，缺乏 hiPSC 标志物的表达，并对早期分化标志物 SSEA1（阶段特异性胚胎抗原 1，Lewis X 聚糖）呈阳性。以前，我们发现纤连蛋白（FN）是偏离细胞分泌的 SSEA1 的主要载体蛋白，但 hiPSCs 则没有。我们开发了一种使用针对 FN 和 SSEA1 的抗体（Abs）的夹心测定法，用于对 hiPSC 培养物中存在的偏离细胞进行非破坏性定量评估。在这项研究中，开发了一种新的技术，使用针对 FN 的 Ab 与近红外（NIR）光吸收剂 IRDye700DX N-羟基琥珀酰亚胺酯（IR700）一起特异性消除偏离细胞，IR700 已用于癌症光免疫治疗。与 IR700 染料缀合的抗 FN Ab（IR700-αFN）在 NIR 照射下与偏离细胞结合并诱导其死亡。相比之下，IR700-αFN 不能染色 hiPSCs，并且 IR700-αFN/NIR 对生存几乎没有影响或没有影响。最后，IR700-αFN/NIR 照射诱导从与 hiPSCs 的混合培养物中选择性去除偏离细胞，表明所提出的方法适合于从 hiPSC 培养物中去除 CTPs 生产中不需要的偏离细胞。
