Division of Advanced Technology and Development, BML, Inc., Saitama, Japan.
Department of Laboratory Medicine, International Medical Center, Saitama Medical University, Saitama, Japan.
Allergol Int. 2018 Apr;67(2):234-242. doi: 10.1016/j.alit.2017.08.008. Epub 2017 Sep 14.
Several methods have been developed to detect allergen-specific IgE in sera. The passive IgE sensitization assay using human IgE receptor-expressing rat cell line RBL-2H3 is a powerful tool to detect biologically active allergen-specific IgE in serum samples. However, one disadvantage is that RBL-2H3 cells are vulnerable to high concentrations of human sera. Only a few human cultured cell lines are easily applicable to the passive IgE sensitization assay. However, the use of human induced pluripotent stem cells (iPSCs) to generate human mast cells (MCs) has not yet been reported.
The nuclear factor-kappa B (NF-κB)-responsive luciferase reporter gene was stably introduced into a human iPSC line 201B7, and the transfectants were induced to differentiate into MCs (iPSC-MCs). The iPSC-MCs were sensitized overnight with sera from subjects who were allergic to cedar pollen, ragweed pollen, mites, or house dust, and then stimulated with an extract of corresponding allergens. Activation of iPSC-MCs was evaluated by β-hexosaminidase release, histamine release, or luciferase intensity.
iPSCs-MCs stably expressed high-affinity IgE receptor and functionally responded to various allergens when sensitized with human sera from relevant allergic subjects. This passive IgE sensitization system, which we termed the induced mast cell activation test (iMAT), worked well even with undiluted human sera.
iMAT may serve as a novel determining system for IgE/allergens in the clinical and research settings.
已经开发出几种方法来检测血清中的过敏原特异性 IgE。使用表达人 IgE 受体的大鼠细胞系 RBL-2H3 的被动 IgE 致敏测定法是一种强大的工具，可用于检测血清样本中具有生物活性的过敏原特异性 IgE。然而，一个缺点是 RBL-2H3 细胞易受高浓度人血清的影响。只有少数人培养细胞系易于应用于被动 IgE 致敏测定法。然而，尚未报道使用人诱导多能干细胞 (iPSC) 来生成人肥大细胞 (MCs)。
核因子-kappa B (NF-κB)-响应荧光素酶报告基因被稳定引入人 iPSC 系 201B7 中，并将转染细胞诱导分化为 MCs(iPSC-MCs)。iPSC-MCs 用对雪松花粉、豚草花粉、螨虫或室内灰尘过敏的受试者的血清进行过夜致敏，然后用相应过敏原的提取物进行刺激。通过β-己糖胺酶释放、组胺释放或荧光素酶强度来评估 iPSC-MCs 的激活。
iPSC-MCs 稳定表达高亲和力 IgE 受体，并在与相关过敏受试者的人血清致敏时对各种过敏原产生功能性反应。即使使用未稀释的人血清，这种我们称之为诱导肥大细胞激活测试 (iMAT) 的被动 IgE 致敏系统也能很好地工作。
iMAT 可作为 IgE/过敏原在临床和研究环境中的新型测定系统。
