Selective Removal of Undifferentiated Human Embryonic Stem Cells Using Magnetic Activated Cell Sorting Followed by a Cytotoxic Antibody
Selective Removal of Undifferentiated Human Embryonic Stem Cells Using Magnetic Activated Cell Sorting Followed by a Cytotoxic Antibody
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DOI：
10.1089/ten.tea.2011.0311
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发表时间：
2012-05-01
影响因子：
4.1
通讯作者：
Choo, Andre
中科院分区：
文献类型：
--
作者：
Schriebl, Kornelia;Satianegara, Gernalia;Choo, Andre
使用来自人胚胎干细胞（hESC）的分化细胞最相关的问题之一是存在残留的未分化hESC，因为它们具有形成畸胎瘤的风险。开发了一种新的细胞-细胞分离方法，该方法消除了形成畸胎瘤的hESC，以确保更安全的细胞治疗。通过将抗体（IgM或IgG）与独特的细胞毒性抗体mAb 84组合，使用磁激活细胞分选（MACS）选择性去除未分化的hESC，然后选择性杀死残留的hESC，可以实现分化的hESC的所需纯度。在使用不同比例（5%-50% hESC）的未分化hESC和人成纤维细胞池的病例研究中显示了这种分离策略的适用性和稳健性，以反映分化细胞群中污染hESC的不同情况。值得注意的是，在MACS步骤后去除了97.2%-99.7%的hESC，在mAb 84处理步骤后去除了99.1%-100%的hESC，这通过双染色流式细胞术和RT-qPCR分析证实。这些体外研究结果在体内严重联合免疫缺陷（SCID）小鼠模型中得到进一步验证。重要的是，我们观察到在MACS步骤后注射细胞28周后，9只SCID小鼠中有8只没有畸胎瘤形成，而在所有对照组中均观察到畸胎瘤。因此，MACS与独特的细胞毒性抗体mAb 84的组合构成了成功和安全的细胞治疗不可或缺的工具。
One of the most pertinent concerns of using differentiated cells derived from human embryonic stem cells (hESC) is the presence of residual undifferentiated hESC, because they carry a risk of teratoma formation. A new cell-cell separation approach that eliminates teratoma-forming hESC in order to ensure safer cell therapy was developed. By combining antibodies (IgMs or IgGs) for the selective removal of undifferentiated hESC using magnetic activated cell sorting (MACS) followed by selective killing of residual hESC with the unique cytotoxic antibody mAb 84, the required purity of differentiated hESC can be achieved. The applicability and robustness of this separation strategy is shown here in a case study using pools of undifferentiated hESC and human fibroblast cells at different ratios (5%-50% hESC) to reflect the different scenario of contaminating hESC in a differentiated cell population. Notably, 97.2%-99.7% of the hESC were removed after the MACS step and 99.1%-100%, after the mAb 84 treatment step, which was confirmed by double-staining flow cytometry and RT-qPCR analysis. These in vitro findings were further validated in an in vivo severe combined immunodeficiency (SCID) mouse model. Importantly, we observed the absence of teratoma formation in eight out of nine SCID mice 28 weeks postinjection of cells after the MACS step, whereas teratomas were observed in all of the controls. Thus, the combination of MACS with the unique cytotoxic antibody mAb 84 constitutes an indispensible tool for successful and safe cell therapy.