Biotechnology Research Institute for Drug Discovery (BRD), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Molecules. 2017 Jul 10;22(7):1151. doi: 10.3390/molecules22071151.
The use of human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in regenerative medicine is hindered by their tumorigenic potential. Previously, we developed a recombinant lectin-toxin fusion protein of the hPSC-specific lectin rBC2LCN, which has a 23 kDa catalytic domain (domain III) of exotoxin A (rBC2LCN-PE23). This fusion protein could selectively eliminate hPSCs following its addition to the cell culture medium. Here we conjugated rBC2LCN lectin with a 38 kDa domain of exotoxin A containing domains Ib and II in addition to domain III (PE38). The developed rBC2LCN-PE38 fusion protein could eliminate 50% of 201B7 hPSCs at a concentration of 0.003 μg/mL (24 h incubation), representing an approximately 556-fold higher activity than rBC2LCN-PE23. Little or no effect on human fibroblasts, human mesenchymal stem cells, and hiPSC-derived hepatocytes was observed at concentrations lower than 1 μg/mL. Finally, we demonstrate that rBC2LCN-PE38 selectively eliminates hiPSCs from a mixed culture of hiPSCs and hiPSC-derived hepatocytes. Since rBC2LCN-PE38 can be prepared from soluble fractions of culture at a yield of 9 mg/L, rBC2LCN-PE38 represents a practical reagent to remove human pluripotent stem cells residing in cultured cells destined for transplantation.
人多能干细胞（hPSCs）如人胚胎干细胞（hESCs）和人诱导多能干细胞（hiPSCs）在再生医学中的应用受到其致瘤潜力的阻碍。以前，我们开发了一种 hPSC 特异性凝集素 rBC2LCN 的重组凝集素-毒素融合蛋白，它具有 23 kDa 的催化结构域（结构域 III）的外毒素 A（rBC2LCN-PE23）。这种融合蛋白可以在添加到细胞培养基后选择性地消除 hPSCs。在这里，我们将 rBC2LCN 凝集素与外毒素 A 的 38 kDa 结构域连接起来，该结构域除了结构域 III 外还包含结构域 Ib 和 II（PE38）。开发的 rBC2LCN-PE38 融合蛋白在 0.003 μg/mL（24 小时孵育）的浓度下可以消除 50%的 201B7 hPSCs，其活性约比 rBC2LCN-PE23 高 556 倍。在低于 1 μg/mL 的浓度下，对人成纤维细胞、人间充质干细胞和 hiPSC 衍生的肝细胞几乎没有影响或没有影响。最后，我们证明 rBC2LCN-PE38 可以从 hiPSC 和 hiPSC 衍生的肝细胞的混合培养物中选择性地消除 hiPSCs。由于 rBC2LCN-PE38 可以从培养物的可溶性部分以 9 mg/L 的产率制备，因此 rBC2LCN-PE38 代表了一种实用的试剂，可以去除移植前培养细胞中存在的人多能干细胞。
