This application represents an exploratory and development research proposal to develop new lines of engineered adult adipose-derived stem cells ASCs, harvested from chinchillas (resulting in chASCs), that can be used for autologous transplantation for the delivery of trophins directly to the inner ear. There is considerable body of evidence demonstrating the limited intrinsic rehabilitative capabilities of the adult inner ear, yet there are numerous conditions in which such capabilities would be extremely valuable. These include cases of otoprotection, in which other systemic life-threatening conditions warrant the use of aggressive therapies that are also ototoxic. Still other conditions abound that would benefit from regenerative capabilities of Scarpa's or spiral ganglion neurons. At the same time, it is clear that inner ear protection and neurorehabilitation is enhanced through the application of neurotrophins, of which brain-derived neurotrophic factor (BDNF) is an important member. Therefore, the development of a strategy to provide BDNF for prolonged periods would be of significant benefit in providing otoprotection and stimulating inner ear rehabilitation. The present application presents a research plan in which we will test the efficacy of using chASCs as a cell-based delivery system to provide BDNF to the inner ear. This work takes advantage of a chronically-prepared chinchilla model in which direct access to the perilymphatic space has been developed. Experiments will be conducted to harvest, characterize, and engineer chASCs via lentiviral transfection to express enhanced green fluorescent protein (GFP) and GFP-tagged BDNF. Strategies will be implemented to produce stable cell lines. These two cell lines will then be transplanted directly to the perilymphatic space in chinchillas. The use of these lines enable the capability to monitor the intrinsic capabilities of transplanted chASCs to secrete BDNF, as well as the enhanced secretion of BDNF resulting from transgene expression. This paracrine function of chASCs will be evaluated through ELISA methodologies. The GFP expression in both transgene systems will enable us to critically evaluate the integration and survival of these cells in vivo through histologic analysis of the recipient temporal bones. Inner ear function of both the peripheral auditory and vestibular systems will be assessed by recording the auditory brainstem response and through single neuron electrophysiology to identify the potential influence of the transplantation on inner ear function. In summary, these experiments represent a direct test of paracrine function of adult engineered stem cells in the inner ear. While the model system incorporates the secretion of neurotrophin, it will serve as a proof-of- concept of a myriad of other otoactive agents. The successful outcome of these experiments can be immediately tested in a model of inner ear neurorehabilitation in the chinchilla developed in the laboratory of the PI.
The research to be conducted under this exploratory and development proposal will test the efficacy of utilizing adult adipose-derived stem cells as a means to deliver trophin therapies directly to the inner ear. We will test the intrinsic paracrine capabilities of these cells as a source of brain-derived neurotrophic factor (BDNF), as well as BDNF resulting from cells that have been engineered via viral transfection. Such a cell-based therapy system takes advantage of the intrinsic and engineered capabilities of stem cells, and if successful will be extremely advantageous in cases requiring neurorehabilitation or neuroprotection of the inner ear.
