Light-Patterned RNA Interference of 3D-Cultured Human Embryonic Stem Cells

A novel approach to regulate genes in human embryonic stem cells in a 3D matrix was developed with unprecedented spatiotemporal control. HGN-transfected human embryonic stem cells (hESCs) were switched into a synthetic thermoreversible hydrogel for 3D culture and laser-selected gene silencing. However, only a small fraction of the cells survived. Shortening the hollow gold nanoshells (HGN) incubation time resulted in decreased internalization. Te addition of Rho-associated proten kinase (ROCK) inhibitor was tested during the HGN incubation and this treatment found significantly increased cell viability in the 3D matrix without detectable changes in HGN internalization. It was also observed that dissociated single cells aggregate into cell clusters with ROCK inhibitor treatment in suspension forming cellular spheroids within 3 d in the 3D matrix. 2D-cultured HeLa cells transduced with green fluorescent protein (GFP) gene were used to optimize the laser irradiation treatment, and it was found that laser at 3% of the maximum power releases insufficient siRNA, whereas a power of 8% causes some immediate changes in cell morphology.