Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers

Mouse embryonic fibroblasts are loaded onto the vertical/3D Nep device where they come in contact with the nanochannels. A cocktail of Brn2a, Mytl1, and Ascl1 is transfected into the cells by nanochannel electroporation. Although this achieves deterministic plasmid delivery, several stochastic barriers to induced neuron production exist. Reprogramming requires CCNA2 expression early during the transfection process. Furthermore, several asymmetric divisions during the reprogramming process result in decreased quantities of reprogramming factors. Finally, at least some induced neurons proceed through a Nestin-positive progenitor cell stage prior to becoming terminal differentiation into a neuron.189