Improving the Gene Transfection in Human Embryonic Stem Cells: Balancing with Cytotoxicity and Pluripotent Maintenance.
Improving the Gene Transfection in Human Embryonic Stem Cells: Balancing with Cytotoxicity and Pluripotent Maintenance.
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DOI：
10.1021/acsami.6b00353
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发表时间：
2016-03
影响因子：
9.5
通讯作者：
Chunhua Luo;Dongyuan Lü;Jun Pan;M. Long
中科院分区：
文献类型：
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作者：
Chunhua Luo;Dongyuan Lü;Jun Pan;M. Long
人类胚胎干细胞（hESC）中的基因操作对于其高度潜在的应用是必不可少的;然而，转导效率仍然非常低。虽然已有证据表明载体的类型、大小和zeta电位影响基因在细胞中的转染效率，但在hESCs中的系统研究很少。本研究以末端为胺、羟基或羧基的聚酰胺-胺（PAMAM）树枝状大分子为模型，考察了树枝状大分子的大小、表面基团以及细胞毒性和内吞作用对hESC基因转染的影响。我们发现，在mTeSR的培养基中，G5，G7，G4.5COOH和G5 OH的颗粒尺寸约为5 nm，G1的颗粒尺寸较小，为3.14 nm。G5和G7分别具有轻微和显著的正zeta电位，而G1为轻微负zeta电位，G4.5COOH和G5 OH为显著负zeta电位。我们证明，只有胺封端的树枝状聚合物完成基因转染hESC，这是大于从Lipofectamine 2000转染。10微摩尔G5的效率最高，优于1000 μM G1。只有低浓度（0.5和1 μM）的G7实现了基因递送。胺末端的树枝状聚合物，特别是具有较高的代数，是有害的生长和多能性的维持的hESC。相比之下，类似大小的羟基和羧基封端的树枝状聚合物表现出低得多的细胞毒性，其中羧基封端的树枝状聚合物保持了hESC的多能性。我们还使用FITC标记的G5树枝状聚合物证实了hESC的内吞和显著的胞吐。这些结果表明，在设计用于hESC基因转导的载体时，仔细考虑大小、浓度和zeta电位，特别是基团的身份和位置，以及最小化胞吐是必要的，这有助于更好地设计用于hESC基因转导的有效载体。
Manipulation of genes in human embryonic stem cells (hESCs) is imperative for their highly potential applications; however, the transduction efficiency remains very low. Although existing evidence revealed the type, size, and zeta potential of vector affect gene transfection efficiency in cells, the systematic study in hESCs is scarce. In this study, using poly(amidoamine) (PAMAM) dendrimers ended with amine, hydroxyl, or carboxyl as model, we tested the influences of size and surface group as well as cytotoxicity and endocytosis on hESC gene transfection. We found that in culture medium of mTeSR the particle sizes of G5, G7, G4.5COOH, and G5OH were around 5 nm and G1 had a smaller size of 3.14 nm. G5 and G7 had a slight and significant positive zeta potential, respectively, whereas G1 was slightly negative, and G4.5COOH and G5OH were significantly negative. We demonstrated that only amine-terminated dendrimers accomplished gene transfection in hESCs, which is greater than that from Lipofectamine 2000 transfection. Ten micromolar G5 had the greatest efficiency and was better than 1000 μM G1. Only a low concentration (0.5 and 1 μM) of G7 realized gene delivery. Amine-ended dendrimers, especially with higher generations, were detrimental to the growth and pluripotent maintenance of hESCs. In contrast, similarly sized hydroxyl- and carboxyl-terminated dendrimers exerted much lower cytotoxicity, in which carboxyl-terminated dendrimer maintained pluripotency of hESCs. We also confirmed the endocytosis into and significant exocytosis from hESCs using FITC-labeled G5 dendrimer. These results suggested that careful considerations of size, concentration, and zeta potential, particularly the identity and position of groups, as well as minimized exocytosis in the design of a vector for hESC gene delivery are necessary, which helps to better design an effective vector in hESC gene transduction.