Basal Study On Regeneration Of Inner Ear Hair Cells

Most hearing loss results from the incapacity of the cochlear sensory epithelium to replace lost hear cells, deafness due to hair cells loss is normally irreversible. In mammalian cochlea, the terminal mitosis of hair cells (HCs) and supporting cells (SCs) occurs during middle embryonic development (E13-E15) . Because of lack of treatments to restore auditory function or alter the course of progressive hearing loss, experimental strategies have been developed to approach the treatment of sensory deafness. Cell therapy is a rapidly growing area of research and has potential applications in the treatment of inner ear disorders. At present, many researchers have focused on this reaearch field, especially using stem cells as cell resources. Given this potential of isolation stem cells from mammalian inner ear, we have tried to isolate and culture stem cells from cochlea of postnatal 7d rats, as a first step in a possible replacement therapy of the loss HCs. The proliferation and differentiation of these cells were tested. Second, to evaluate the degeneration and regeneration of hair cells in the development mammalian inner ear, we have set up models of the cultured cochleae in vitro. The most definite regeneration results from the combination of insulin with FGF-2 and EGF were found. It suggests the possibility of reconstructing the damaged inner ear by stimulating quiescent stem cells to proliferate and generate new hair cells. One new approach to restore hearing function is to use a biological implant that has the potential to replace the degenerated or absent sensory cells. Here, we used stem cells as cell resources to transplant into the damaged inner ear and tried to explore their survival, differentiation, and possible integration with the host tissue. This work raises the capability for the future development of stem-cell-based treatment regimens for the damaged inner ear.The findings in this study inclused: First part:This study showed acutely dissociated cells from the cochlea of young rats. The inner ear cells, cultured with EGF and FGF-2, the cells developed into otoshpereswhich showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). The subcultured otospheres maintained for up to 10 passages. In addition, the cochlea sphere-derivatives contributed to a variety of cell types. They were found to differentiate into neuron, glia, hair cell and supporting cell phenotypes. The results suggest that the young rat inner ear stem cell has the self-renewal capability and multipotent differentiation potential. In the case of P7 day, the cochlea and the Corti have not yet completed the process of terminal developments. The presence of sphere-forming cells suggests the possibility of reconstructing the damaged inner ear by stimulating quiescent stem cells to proliferate and generate new hair cells. This work also raises the capability that the inner ear stem cells might be sources of regenerated cells for replacement of lost inner cells.Second part:The aim is to set up a new model for examining the course of hair cell degeneration and regeneration in the mammalian cochlea following aminoglucoside treatment. Organotypic cultures were established from postnatal rats and treated 1mmol/L neomycin sulfate for 48h. Hair cell morphology was examined using phalloidin labeling, and analyzed under fluorescence microscopy. Several molecules and growth factors were used to test the possible regenerative potential of the lost hair cells. Slight regeneration effects were tested with insulin or growth factors alone, however, the most definite regeneration results from the combination of insulin with FGF-2 and EGF were found. The tested molecules and growth factors did promote cochlear hair cell regeneration in vitro after neomycin treatment in this organotypic culture condition.Third part:The cochlear sensory epithelium and spiral ganglion neurons (SGNs) in the adult mammalian inner ear do not regenerate following severe injury. To replace the degenerated hair cells and SGNs, stem cell is an attractive alternative for substitution cell therapy. Rat NSC cells and inner ear cells were labled by enhanced green fluorescent protein (EGFP) and then transplanted into the cochlea of adult guinea pigs through the scala tympani in order to explore their survival, differentiation, and possible integration with the host tissue. At 4 weeks following transplantation, implanted cells were found close to the sensory epithelium and adjacent to the SGNsand their peripheral processes. Parts of the surviving cells had surived well and had the possiblibities to integrate with the host tissue. The results illustrate not only the survival of xenografted stem cells in the adult inner ear but also indicate the potential of using inner ear stem cells as graft material in cell therapy for auditory defects, including sensorineural hearing disorders.Taken together, our results suggest that:1. The results in the present studies demonstrate the occurrence of stem cells within the P7 d rat inner ear is restricted to the Corti, including its underlying basal membrane. These cells display the requisite characteristics of stem cells. They possess high proliferative potential and have the ability for self-renewal, which is shown by the ability of forming new spheres after been passaged. In addition, these cells have the potential to differentiate into different cell types. Immunostaining showed these cells could express myosinVIIa and P27^kip1 of the hair cell and supporting cell characteristic marker protein respectively. Cell-type specific antibodies demonstrated their multipotency with MAP-2, NF positive neurons and GFAP positive glia cells.2. There are three major findings of the present studies: (1) Cochlea culture in floating method is a handling method for test hair cell loss and regeneration. (2) The IHCs seem to be most susceptible to aminoglucoside treatment in the basal part of the Corti. (3) Combined insulin, EGF with FGF-2 dramatically increased the regeneration of hair cell loss. The results do have implications for designing controlled methods for inducing hair cell degeneration and regeneration in the mammalian cochlea.3. The survival of xenografted stem cells in the adult inner ear indicates the feasibility of a transplantation strategy in the degenerated auditory system, thereby creating possibilities to replace degenerative or losing hair cells. In addition, it suggests that the lesion microenvironment in the adult may be one of the factors which improve the survival and differentiation of grafted cells in the host auditory system.