PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.
PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.
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DOI：
10.1038/ijos.2015.18
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发表时间：
2015-09-14
影响因子：
14.9
通讯作者：
Sato M
中科院分区：
文献类型：
--
作者：
Inada E;Saitoh I;Watanabe S;Aoki R;Miura H;Ohtsuka M;Murakami T;Sawami T;Yamasaki Y;Sato M
人乳牙牙髓细胞（HDDPC）分化为生成矿化组织的成牙本质细胞的能力在牙齿再生医学领域的治疗应用中具有巨大的潜力。这种潜力的实现取决于高效和优化的HDDPC遗传操作方案。在这项研究中，我们证明了使用PiggyBac（PB）为基础的基因转移系统作为一种方法引入非病毒转座子DNA到HDDPC和HDDPC衍生的诱导性多能干细胞。基于PB的系统的转染效率显著大于先前报道的基于电穿孔的质粒DNA转染。使用新霉素抗性基因作为选择标记，HDDPC以比使用常规方法高近40倍的速率稳定转染。使用该系统，还可以将两种构建体同时引入单个细胞中。得到的表达tdTomato和增强的绿色荧光蛋白的稳定转染子表现出红色和绿色荧光。建立的细胞系在培养三个月后没有失去获得的表型。基于我们的结果，我们得出结论，PB是上级目前可用的方法引入质粒DNA到HDDPC。由于安全风险和伦理问题，这种方法用于人类牙齿组织工程的直接临床应用可能存在重大挑战。然而，用PB实现的高水平转染可能在牙科组织工程应用的基础科研中具有显著优势，例如基因和蛋白质的功能研究。此外，它是一个有用的工具，用于分离基因工程HDDPC衍生的干细胞，用于牙齿再生医学的研究。
The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.