Enhanced non-viral gene delivery to human embryonic stem cells via small molecule-mediated transient alteration of the cell structure

Non-viral gene delivery into human embryonic stem cells (hESCs) is an important tool for controlling cell fate. However, the delivery efficiency remains low due in part to the tight colony structure of the cells which prevents effective exposure towards delivery vectors. We herein report a novel approach to enhance non-viral gene delivery to hESCs by transiently altering the cell and colony structure. (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632), a small molecule that inhibits the rho-associated protein kinase pathway, is utilized to induce transient colony spreading which leads to an increased transfection efficiency by 1.5 to 2 folds in a spectrum of non-viral transfection reagents including Lipofectamine 2000 and Fugene HD. After removal of Y-27632 post-transfection, cells can revert back to their normal state and do not show alteration of pluripotency. This approach provides a simple, effective tool to enhance non-viral gene delivery into adherent hESCs for genetic manipulation.