The Experimental Study Of Sensorineural Hearing Loss Treatment By Induced Pluripotnet Stem Cells Transplantation

Background:sensorineural hearing loss is one of the most common diseases, which is mainly related to the degeneration, death, or damage of auditory hair cells or/and associated spiral ganglion neurons. However, in mammals, because the hair cells and spiral ganglion can not regenerate, the two damage is impossible to be repaired. Thus, stem cell transplantation to replace the damaged hair cell and/or spiral ganglion neuro which result in SNHL would become the focus and hot issues in scientific research of ear. Objective:1. Establishing a stable culture system of mouse i PSCs in vitro.2. Making sensorineural hearing loss(SNHL) mice model by use of ototoxic drugs.3. The i PSCs labeled with CM-Di1 were microinjected into the SNHL model cochlea through the round window to observe the survival, migration, differentiation in cochlea, which would provide a theoretical and experimental basis for the treatmentof SNHL.Methods:1. Culture and label of the i PSCs: The mouse i PSCs were passaged through adherent culture method and labeled by fluorescent dye CM-Di1.2. The establishment of SNHL mice model: we evaluated the hearing loss in mice through auditory brainstem response(ABR) after continuous subcutaneous injection with neomycin.3. The transplantation of mouse i PSCs: 40 SNHL mice were randomly divided into two groups :A was the experimental group, B was the control group. Beside, 20 normal contemporary mice act as the normal group C. The experimental groups were microinjected with the mouse i PSCs and the control group were transplanted with equal volume DMEM basic medium. Sacrificed 10 mice 4 weeks postoperation of each group, and evaluated the survival, migration, differentiation of the transplanted i PSCs in cochlea by immunofluorescence staining and PT-PCR. The others were use to observe the ABR threshold in different time point.4. Cochlear HE staining was used to identify whether there are teratoma formation after i PSCs transplantation. Results:1. We successfully established a stable culture system of mouse i PSCs and formed embryoid body(EB) in vitro. And we also identified the pluripotent with genes Nanog, Oct4, Sox2.2. After 12 days subcutaneous injection with neomycin, the ABR hearing threshold was increased to 70-80 d Bn HL.3. The two group showed no improvement in ABR hearing threshold postoperation. In the group A, CM-Di1-labeled i PSCs would be found in the the modiolus of the cochlear and Rosenthal’s canal, and some red fluorescence-labeled cells could expressed auditory hair cell markers or Spiral ganglion makers. No red fluorescence was observed in the cochlea of group B and C.4. The ABR threshold difference between the two groups of mice with preoperative and postoperative has no statistical significance.5. No teratoma was observed in the cochlea after i PSCs transplantation by HE staining. Conclusion:1. This method can establish a stable culture system of mouse i PSCs.2. Fluorescent dye CM-Di1 can label cells in vitro and in vivo.3. Neomycin can induce SNHL and establish a stable deafness model.4. Mouse i PSCs could differentiate into hair cell-like cells with hair cell markers after transplanted into the cochlear of mice one month later.5. Mouse i PSCs could differentiate into spiral ganglion neuron-like cells with neural cell markers after transplanted into the cochlear of mice one month later.6. i PSCs transplatation through Rosenthal’s canal cannot improve the ABR thresholds in short-term.7. Mouse i PSCs transplantation into cochlear could not form teratomas in short-term.8. The transplanted i PSCs were mainly distributed in the Rosenthal’s canal and the modiolus of cochlear.