Kim C-Yoon, Hwang In-Kyu, Kang Changhee, Chung Eun-Bin, Jung Cho-Rok, Oh Hanseul, Jeong Young-Hoon, Moon Sung-Hwan, Kim Jong Soo, Hong Ki-Sung, Park Jae-Hak, Chung Hyung-Min
Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Korea.
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
Int J Stem Cells. 2018 Nov 30;11(2):149-156. doi: 10.15283/ijsc18037.
Human embryonic stem cells (hESCs) are pluripotent cells widely used in conventional and regenerative medicine due to their ability to self-renew, proliferate and differentiate. Recently, genetic modification of stem cells using genome editing is the most advanced technique for treating hereditary diseases. Nevertheless, the low transfection efficiency of hESCs using enzymatic methods is still limited in preclinical research. To overcome these limitations, we have developed transfection methods using non-enzymatic treatments on hESCs. In this study, hESCs were transfected following enzymatic (TrypLE and trypsin) and non-enzymatic treatment ethylenediaminetetraacetic acid (EDTA) to increase transfection efficiency. Flow cytometric analysis using an enhanced green fluorescent protein vector showed a significantly increased transfection efficiency of EDTA method compared to standard enzyme method. In addition, the EDTA approach maintained stable cell viability and recovery rate of hESCs after transfection. Also, metabolic activity by using Extracellular Flux Analyzer revealed that EDTA method maintained as similar levels of cell functionality as normal group comparing with enzymatic groups. These results suggest that transfection using EDTA is a more efficient and safe substitute for transfection than the use of standard enzymatic methods.
人胚胎干细胞（hESCs）是多能干细胞，因其自我更新、增殖和分化的能力而广泛应用于传统医学和再生医学。近来，利用基因组编辑对干细胞进行基因改造是治疗遗传性疾病的最先进技术。然而，在临床前研究中，使用酶法对hESCs进行转染的效率较低，仍然存在局限性。为克服这些局限性，我们开发了对hESCs采用非酶处理的转染方法。在本研究中，对hESCs进行酶处理（胰蛋白酶和胰蛋白酶）和非酶处理乙二胺四乙酸（EDTA）后进行转染，以提高转染效率。使用增强型绿色荧光蛋白载体进行的流式细胞术分析显示，与标准酶法相比，EDTA法的转染效率显著提高。此外，EDTA方法在转染后维持了hESCs稳定的细胞活力和回收率。而且，使用细胞外通量分析仪进行的代谢活性分析表明，与酶处理组相比，EDTA法维持的细胞功能水平与正常组相似。这些结果表明，与使用标准酶法相比，使用EDTA进行转染是一种更高效、更安全的转染替代方法。
