Migration And Differentiation Of Embryonic Stem Cells Transplanted Into Rat Cochlea

Embryonic stem cells (ESCs) have the capacities of proliferating infinitely and maintaining the undifferentiated state in vitro. They can be induced to differentiate into any kind of tissue cell types under particular conditions, which made them ideal candidates for replacement therapies after losing the human organ function. At present, there is still no effective treatment for permanent sensorineural hearing loss caused by inner hair cell damage. Many scholars both at home and abroad started exploring the feasibility of treating deafness by directing ESCs to differentiate into inner ear cells. This study aims to observe the survival, distribution and differentti- ation in the cochlea of undifferentiated ESCs, which were transplanted into scala tympani of the rat with normal hearing and aminoglycoside-induced hearing loss.Part I:Preparation of rat model with aminoglycoside-induced hearing lossObjective:To observe the injury of newborn rat cochlea by amikacin sulfate, and to establish an animal model with complete hair cells loss.Methods: Thirty newborn rats aged 9 days were divided into control group (n=10) and amikacin group (n=20). The latter group received subcutaneous injection of an amikacin sulfate solution with a dose of 500 mg/kg/day for seven consecutive days. 1, 6 and 12 weeks after injection, auditory brainstem response (ABR) was tested, frozen section observation and scanning electron microscopy of the inner ear were conducted.Results: The 20 rats in amikacin group were all survived. After injection, ABR thresholds were raised to over 95dB SPL at 6 weeks for each rat, which didn't show any recovery when retested at 12 weeks. Scanning electron microscopy and HE staining showed all the inner and outer hair cells of the cochlea were lost 6 weeks after injection. Conclusions: Continuous administration of medium dose of amikacin sulfate to neonatal rat leads to a complete loss of cochlea hear cells and resultant permanent sensorineural hearing loss, which made it an ideal model for embryonic stem cells transplantation.Part II: Methodological study for transplantation of embryonic stem cells into inner earObjective:To explore effective methods of transplanting exogenous embryonic stem cells (ESCs) into the inner ear.Methods: 10 wistar rats aged 5 to 6 weeks were used. The acoustic capsule of the right ear was opened under anesthesia, in which the ESCs carrying the green fluorescent protein (EGFP-ESCs) were infused into the scala tympani through a thin soft tube connected to a micro-syringe. As control, the left ears didn't receive surgery. Auditory brainstem response (ABR) thresholds were determined 1 week before and immediately after surgery. 36 hours after transplantation, the animals were sacrificed for morphological observation by frozen section.Results: Transplanted cells were mostly aggregated into clumps and suspended in the perilymph of the scala tympani, a few of them attached to the outer bony wall of the scala tympani. No grafted EGFP-ESCs were found in other compartments of the cochlea and vestibular. ABR tests showed an approximately 10 dB decrease of hearing immediately after surgery.Conclusions: ESCs could survive in the cochlea when transplanted into the scala tympani, and the operation itself causes little damage to inner ear. As a result, it might be an effective method for cell transplantation into the inner ear.Part III:Migration and differentiation of embryonic stem cells transplanted into the rat cochlea with aminoglycoside-induced hearing lossObjective:To observe the survival, migration and differentiation of embryonic stem cells in deafened rat cochlea.Methods: 18 wistar rats were randomly divided into two groups, 8 in the control group (without receiving drug) and 10 in the experimental group. The embryonic stem cells were transplanted into the scala tympani of the right ears, while the left ears received no surgery. Histological observation of the cochlea was conducted by frozen section fluorescence 2 and 6 weeks respectively after transplantation.Results: In the experimental group, the grafted EGFP-ESCs migrated into the bony wall of the scala tympani in clusters as well as into the endolymph compartment of cochlea two weeks after the transplantation.At six weeks after the transplantation, the grafted EGFP-expressing embryonic cells could localize in the osseous spiral lamina, the spiral ligament, the inner sulcus cell, the organ of Corti, the outer sulcus cell, and the inside wall of the scala tympani. Some grafted stem cells even expressed myosin VIIa, the molecular marker of hair cells. While in the control group, the transplanted cells stayed only in the scala tympani at two weeks, three were no survival EGFP-ESCs were found either in the scala tympani or in the other compartments of the cochlea.Conclusions: Embryonic stem cells can survive longer in the microenvironment of damaged cochlea, some of them may migrate into the scala media in which a few even differentiated into precursor hair cells.