Tissue engineering traditionally has taken an “outside-in” approach to address deformities, injuries, and wear-and-tear of tissues. The current thesis examines an opposite approach through an “inside-out” strategy using non-viral gene delivery with mesenchymal stem cells to regenerate mechanosensory hair cells and supporting cells of the inner ear responsible for hearing and balance. Primary cells, stem cells, and progenitor cells are often difficult to transfect unless using a viral vector, which may have systemic safety concerns. However, non-viral vectors circumvent the safety issues associated with viral vectors, but commonly exhibit low transfection efficiencies. The work of the current thesis identified and enhanced an effective nonviral gene delivery approach that reprogramed human mesenchymal stromal cells, isolated from Wharton’s jelly of human umbilical cords to produce characteristics similar to the hair cell and supporting cell phenotype found in the cochlea and vestibular organs of the inner ear. Studies from the literature highlighted electroporative methods as effective non-viral strategies for difficult-to-transfect cells. In vitro studies demonstrated that human Wharton’s jelly cells (hWJCs) that underwent electroporation and were treated with Y-27632 ROCK Inhibitor outperformed untreated cells in transfection efficiency and cell viability by factors of four and three, respectively. The identification and tracking of positively transfected cells was tremendously improved by use of a photo-converting reporter, which greatly increased signal to noise ratios. The up-regulation of atoh1, and down-regulation of hes1 and hes5, in hWJCs produced a complex phenotype that exhibited over an 11-fold increase in gene expression of the critical hair cell marker, myosin VIIa, with visual morphological changes compared to untreated cells. The current thesis has demonstrated that hWJCs are susceptible to non-viral gene delivery methods, and for the first time non-viral genetic reprogramming of hWJCs induced phenotypic changes characteristic of hair cells and neural epithelium. The current thesis has bridged the gap between non-viral gene delivery, stem cell therapy, and tissue engineering, which now presents new opportunities for further investigation utilizing non-viral gene delivery in concert with stem cell therapies for regenerative medicine applications.