SSEA-1-positive fibronectin is secreted by cells deviated from the undifferentiated state of human induced pluripotent stem cells
SSEA-1-positive fibronectin is secreted by cells deviated from the undifferentiated state of human induced pluripotent stem cells
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SSEA-1阳性纤连蛋白是由偏离人类诱导多能干细胞未分化状态的细胞分泌的
DOI：
10.1016/j.bbrc.2020.06.074
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发表时间：
2020
影响因子：
3.1
通讯作者：
Tateno Hiroaki
中科院分区：
文献类型：
--
作者：
Watanabe Tomoko;Saito Sayoko;Hiemori Keiko;Kiyoi Kayo;Mawaribuchi Shuuji;Haramoto Yoshikazu;Tateno Hiroaki
人诱导多能干细胞（hiPSC）的质量控制对于有效和稳定地生产用于移植的hiPSC衍生的细胞治疗产品是重要的。在细胞培养过程中，hiPSC自发地经历形态变化并失去多能特性。这种细胞被称为偏离细胞，其从hiPSC的未分化状态改变，并表达早期分化标志物阶段特异性胚胎抗原1（SSEA-1）。在这项研究中，我们寻找可溶性SSEA-1+糖蛋白分泌的偏离细胞产生的hiPSC培养在细胞培养基中含有热灭活的补充剂。从SSEA-1+偏离细胞的细胞培养上清液获得的糖蛋白通过O-聚糖结合凝集素富集，并用抗SSEA-1抗体印迹。通过LC-MS/MS分析和免疫沉淀结合蛋白质印迹鉴定由抗SSEA-1抗体特异性检测的大于250 kDa的单一蛋白条带为纤连蛋白（FN），表明FN是SSEA-1的载体蛋白。然后，我们构建了一个夹心酶联免疫吸附试验，以检测SSEA-1 + FN分泌的偏离细胞。该FN-SSEA-1测试被证明是灵敏和特异的，允许非破坏性检测混合细胞群体中的SSEA-1+偏离细胞，检测下限为100个细胞/mL。开发的测定法可以为用于再生医学的hiPSC的质量控制提供标准技术。
Quality control for human induced pluripotent stem cells (hiPSCs) is important for efficient and stable production of hiPSC-derived cell therapy products to be used for transplantation. During cell culture, hiPSCs spontaneously undergo morphological changes and lose pluripotent properties. Such cells are termed deviated cells, which are altered from the undifferentiated state of hiPSCs, and express the early differentiation marker stage-specific embryonic antigen 1 (SSEA-1). In this study, we searched for soluble SSEA-1+glycoproteins secreted from deviated cells generated by culturing hiPSCs in cell culture medium containing heat-inactivated supplements. Glycoproteins obtained from cell culture supernatants of SSEA-1+deviated cells were enriched by an O-glycan binding lectin and blotted with anti-SSEA-1 antibody. A single protein band at >250 kDa specifically detected by anti-SSEA-1 antibody was identified as fibronectin (FN) by LC-MS/MS analysis and immunoprecipitation combined with western blotting, indicating that FN is a carrier protein of SSEA-1. We then constructed a sandwich enzyme-linked immunosorbent assay to detect SSEA-1+FN secreted from deviated cells. This FN-SSEA-1 test proved to be both sensitive and specific, allowing for non-destructive detection of SSEA-1+deviated cells within mixed cell population, with a lower limit of detection of 100 cells/mL. The developed assay may provide a standard technology for quality control of hiPSCs used for regenerative medicine.