Basic Research On Treatment To Drug Deafness With Bone Marrow Mesenchymal Stem Cells

Sensorineural hearing loss represents dysfunction in the chchlea or auditory nerve. The most common site of lesion is the hair cell, a specialized mechanoreceptor located in the organ of Corti in mammals. For patients with sensorinueral hearing loss, the causes are dysfunction, injury, or death of the hair cell.In contrast to non-mammals, hair cc(?)s do not regenerate following hair cell death in mammals, so that it is difficult to restore hearing after hair cell injury or loss. Cell replacement therapy with stem cells represents a major strategy for restoring hearing following hair cell loss. Stem cells are self-renewing pluripotent cells that can give rise to highly specialized cells in the organism depending on the microenvironment in which the stem cells reside, to replace lost, damaged, or defective human cell.Bone marrow mesenchymal stem cells(MSCs), easy to collect and propagate, be used in autologous transplantation, few ethical and immunologic barriers to clinical applications, be multipotent, represent a promising stem cell source for stem cell replacement therapy. It has been demonstrated that MSCs have the competence of differentiating into neureons. However, it is not clear whether MSCs differentiate into hair cells or progenitors in vitro and MSCs transplanted into inner ear survive and migrate to the sites hair cells reside to replace the lost inner ear hair cells.Our experiment carried out a series of researches, including differentiating into cochlear sensory hair cells of bone marrow MSCs in vitro and the survival, migration and differentiation of MSCs transplantation into cochlea in normal and deafened rats. The present study can be divided into following two parts: Part one:Bone marrow MSCs differentiate into hair cell-like cells in vitroObjective:To explore the feasibility of directionally inducing bone marrow MSCs to cochlear sensory hair cells in vitro.Methods:1. Rat bone marrow cells were separated and expanded by adherence culture. The surface molecule expressions of cells were examined by RT-PCR. Multilineage differentiation capability of cells was examined by culturing cells under conditions favorable for adipogenic and osteogenic differentiation in vitro. 2. By using special cell growth factors, bone marrow MSCs were induced to cochlear sensory hair cells, the characteristic marks of which were detected by immunohistochemisty and scanning electron microscope.Results:1. The medium was firstly half changed after primary culture for 24 hours, which made the isolated cells expand fast and cover with cell culture dishes in 7 days. Cultured cells expressed SH2, CD31, CD44, but lacked expression of CD44.The cells were successfully induced to adipocytes and osteogenesis.2. Bone marrow MSCs could be induced to differentiate into neural stem cells, and expressed specific neural marker, such as Nestin. Following that, the cells expressed specific markers of cochlear inner ear hair cell in the other growth factors, such as MyosinⅦa. Scanning electron microscopy showed microvillus on the surface of cells, silimar to the stereocilia of inner ear hair cells.Conclusions:1. The method that medium was firstly half changed after primary culture for 24 hours is benefit for isolation and purity of rat bone marrow MSCs.The cells isolated in this experiment have biological characteristics of bone marrow MSCs. 2. Bone marrow MSCs can be induced to convert into inner ear hair cell-like cells.Part two:The treatment to drug deafness with inner ear implantation of bone marrow MSCsObjective:1. To explore the methods of the sensorineural hearing loss animal model for stem cells replacement therapy to regenerate hair cells.2. To investigate the impact on normal cochlea after bone marrow MSCs transplantation into cochlea through scala tympani (ST).3. To explore survival and differentiation of rat MSCs after transplantation into drug deafened rat cochlea.Methods:1. Rats were treated hypodermically with different dose of amikacin for one week. Auditory brainstem response (ABR), light microscope and scanning electron microscope were used to evaluate to establish a sensorineural hearing loss animal model for bone marrow MSCs transplantation.2. Bone marrow MSCs were transplated into cochlea of normal hearing rats via ST. The cochlear structures and auditory function were measured by ABR, immunohistochemisty and scanning electron microscope.3. The impact on auditory function was evaluated by ABR and the survival, migration and differentiation of engrafted MSCs were examined by immnofluoresence method and scanning electron microscopy after bone marrow MSCs were transplated into cochlea of drug deafened rats.Results:1. There were permanent threshold shift after rats were treated hypodermically with amikacin 500mg-kg-1·d-1 for one week. Three weeks after treatment, loss of cells in the organ of Corti from sensory hair cells to supporting cells and the cuboidal epithelium were observed.2. There were no significantly impact on the auditory function and cochlear structures of normal rats after MSCs transplantation into cochlea via ST. MSCs could survive at least 4 wekks, locating in ST and scala vestibular, adherence or dissociation. 3. After transplantation, Grafted MSCs were visualized in the basilar membrane of every turn of the cochlea, having the characteristics of inner ear hair cells. There were no significant hearing improvements in 8 weeks.Conclusions:1. This amikacin deafness animal model is promise of an ideal animal model for the further study for acoustic hair cell regeneration through bone marrow MSCs replacement therapy. 2. Bone marrow MSCs transplantation is a potential strategy for cochlear lesion. 3. Rat bone marrow MSCs can survive in the sites of the outer hair cells died and have the characteristics of the inner ear hair cells.