title: Improving the Gene Transfection in Human Embryonic Stem Cells: Balancing with Cytotoxicity and Pluripotent Maintenance.
abstract: 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.
authors.affiliation: Key Laboratory for Biorheological Science and Technology of Ministry of Education, College of Bioengineering, Chongqing University , Chongqing 400044, China.|Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences , Beijing 100190, China.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Luo
authors.affiliation: Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences , Beijing 100190, China.
authors.forename: Dongyuan
authors.initials: D
authors.lastname: Lü
authors.affiliation: Key Laboratory for Biorheological Science and Technology of Ministry of Education, College of Bioengineering, Chongqing University , Chongqing 400044, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Pan
authors.affiliation: Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory) and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences , Beijing 100190, China.
authors.forename: Mian
authors.initials: M
authors.lastname: Long
mesh_terms: D050091:Dendrimers / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics; D018014:Gene Transfer Techniques; D000066449:Human Embryonic Stem Cells / Q000187:drug effects*; D006801:Humans; D008055:Lipids / Q000737:chemistry / Q000494:pharmacology; D010316:Particle Size; D014162:Transfection / Q000379:methods*
chemical_list: D050091:Dendrimers; D008055:Lipids; C086724:Lipofectamine; C104700:PAMAM Starburst
keywords: dendrimer; embryonic stem cells; end group; endocytosis; gene transfection; size