Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany.
PLoS One. 2013 Jul 30;8(7):e70543. doi: 10.1371/journal.pone.0070543. Print 2013.
Embryonic stem cells (ES) and induced pluripotent stem (iPS) cells represent promising tools for cell-based therapies and regenerative medicine. Nevertheless, implantation of ES cell derived differentiated cells holds the risk of teratoma formation due to residual undifferentiated cells. In order to tackle this problem, we used pluripotent stem cells consisting of ES and iPS cells of mouse genetically modified by lentiviral vectors (LVs) carrying herpes simplex virus thymidine kinase (HSV-TK) under the control of different promoters of pluripotency genes. Cells expressing TK in turn are eliminated upon administration of the prodrug ganciclovir (GCV). Our aim was to study the conditions required for a safe mechanism to clear residual undifferentiated cells but using low MOIs of lentiviruses to reduce the risk of insertional mutagenesis. Our in vitro data demonstrated that TK expression in pluripotent stem cells upon treatment with GCV led to elimination of undifferentiated cells. However, introduction of hygromycin resistance in the LV transduced ES cells followed by pre-selection with hygromycin and GCV treatment was required to abolish undifferentiated cells. Most importantly, transplantation of pre-selected ES cells that had been transduced with low MOI LV in mice resulted in no teratoma development after GCV treatment in vivo. Taken together, our data show that pre-selection of ES cells prior to in vivo application is necessary if vector integration events are minimized. The study presented here gives rise to safer use of pluripotent stem cells as promising cell sources in regenerative medicine in the future.
胚胎干细胞（ES）和诱导多能干细胞（iPS）细胞代表了基于细胞治疗和再生医学的有前途的工具。然而，由于残留的未分化细胞，植入 ES 细胞衍生的分化细胞存在形成畸胎瘤的风险。为了解决这个问题，我们使用了由 ES 和 iPS 细胞组成的多能干细胞，这些细胞是通过携带单纯疱疹病毒胸苷激酶（HSV-TK）的慢病毒载体（LV）遗传修饰的小鼠产生的，该基因受多能基因不同启动子的控制。表达 TK 的细胞在给予前药更昔洛韦（GCV）后被消除。我们的目的是研究清除残留未分化细胞的安全机制所需的条件，但使用低 MOI 的慢病毒来降低插入突变的风险。我们的体外数据表明，GCV 处理后多能干细胞中 TK 的表达导致未分化细胞的消除。然而，需要在 LV 转导的 ES 细胞中引入潮霉素抗性，然后用潮霉素和 GCV 处理进行预选择，以消除未分化细胞。最重要的是，在体内给予 GCV 后，移植预先用低 MOI LV 转导的经过预选择的 ES 细胞不会导致畸胎瘤的发展。总之，我们的数据表明，如果最小化载体整合事件，则在体内应用之前对 ES 细胞进行预选择是必要的。本研究为将来在再生医学中更安全地使用多能干细胞作为有前途的细胞来源提供了依据。
