Enhanced Non-Viral Gene Delivery to Human Embryonic Stem Cells via Small Molecule-Mediated Transient Alteration of Cell Structure

Abstract

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 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 its 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.

Fig. 1

Y-27632 enhances the transfection efficiencies of various nanocomplexes in hESCs. a) Size and zeta potential of various nanocomplexes (DNA:polymer, w/w): PLR (1:10), PLL (1:10), PEI (1:5), LPF (1:2), and FHD (1:3.5). b) Transfection efficiencies of various reagents in hESCs in the presence (50 µM)or absence of Y-27632as measured by flow cytometry. Cells were pre-treated with Y-27632 cells for 4 h before removal of Y-27632 and addition of various nanocomplexes. (n=3) (* p<0.05, ** p<0.01)

Fig. 2

Y-27632 promotes transfection in hESCs cells by extending the cell surface area. a) Transfection efficiencies of FHD/DNA nanocomplexes in H1 hESCs in the presence of Y-27632 or blebbistatin at various concentrations. b) Fluorescence images of H1 hESCs48 h post transfection with FHD/pEGFP nanocomplexes. Cells were pre-treated with 0 µM, 10 µM, 30 µM, or 50 µM Y-27632 for 4 h before transfection and during the 4-h transfection process (scale bar = 250 µm). c) Bright-field imaging showing the morphological change of hESCs after 4-h treatment with Y-27632 at various concentrations (scale bar = 250 µm). d) Alteration of the cytoplasm area (µm²) per nucleus of hESCs following treatment with Y-27632 of various concentrations. Images were taken and analysed with the GE In Cell Analyzer. (n=5) (* p<0.05, ** p<0.01)

Fig. 3

Y-27632 promotes cellular internalization of the gene cargo as a result of increased surface area. a) Transfection efficiency of FHD in hESCs in the presence of Y-27632. Y-27632 was applied to the cells for 4 h before transfection, during the 48-h transfection period, or a combination thereof. b) Cell uptake level of FHD/YOYO-1-DNA nanocomplexes in hESCs in the presence of various concentrations of Y-27632 (n =3). (* p<0.05, ** p<0.01)

Fig. 4

Y-27632 does not compromise the pluripotency of H1 hESCs. a) DAPI and SSEA-4 staining patterns of H1 hESCs without Y-27632 treatment (control group) or transfected with FHD in the presence of 50 µM Y-27632 (FHD group, scale bar = 250 µm). b) Western blot analysis on the OCT4 expression in hESCs 4 days post FHD transfection and treatment of Y-27632 at various concentrations. (- demonstrates the cells without transfection and + indicates cells with FHD transfection)

Scheme 1

Y-27632 enhances the transfection efficiencies of various polyplexes or lipoplexes in hESCs via increased membrane exposure through transient spreading of the cells.