| Deafness is a major global public health problem. Sensorineural deafness as a result of inner ear hair cells impairment is one of the most common deafness. As a kind of terminal differentiation cells, inner ear hair cells turn sound into electrical signal that is transmitted into brain. Hair cells are prone to be damaged by noise, age and ototoxic drug such as aminoglycoside. Mammalian hair cells loss is irreversible which leads to permanent hearing injury. During last decade, stem cell treatment technology brings revolution in treatment of many human critical diseases. Stem cells have totipotency or pluripotency and highly proliferative capacity. The maintenance and differentiation of stem cells are driving forces of tissue development and regeneration. Adoption of stem cells based technology will make restoring auditory system especially hair cells regeneration become possible. Therefore it is helpful to cure deafness in essence.Inner ear stem cells, which are the important seed cells for inner ear hair cells regeneration, distribute in vestibular utricle, saccule, cochlear basilar membrane and crista ampullaris of semicircular canals. It has been shown that inner ear stem cells can self-renew and have pluripotency that can be induced to differentiate into hair cells. However, there are still problems in studies about inner ear stem cells and hair cells as follows:1. The number of inner ear stem cells is limited, the distribution in inner ear is unclear and the in vitro cultivation is difficult.2. The mechanism of in vitro differentiation of inner ear stem cells into hair cells is remained unclear. The newly generated hair cells are not typical hair cells without normal function.3. It is difficult to identify and sort out inner ear stem cells, progenitor cells and hair cells due to the lack of specific marker. Therefore, the first two parts of this study (Chapter2and Chapter3) mainly investigated isolation, cultivation, identification and in vitro differentiation of inner ear stem cells into mature hair cells with typical stereocilium and electrophysiological response. The last part mainly investigated the gene expression during differentiation of inner ear stem cells into hair cells and the selection of specific marker of stem cells, progenitor cells and hair cells.Part1:We mainly studied the isolation and cultivation of inner ear stem cells distributed in cochlear basilar membrane of newborn ICR mouse (PO). Subsequently, specific markers of stem cells, progenitor cells were examined. Results showed that spheres generated from cells of cochlear basilar membrane expressed markers of inner ear stem cells and progenitor cells which indicated that inner ear stem cells were obtained with partial progenitor cells. The vast majority of spheres were BrdU positive which indicated that most of cells were generated by proliferation. Immunocytochemistry showed that spheres expressed proteins such as Nestin.Part2:In order to further induce inner ear stem cells to differentiate into functional hair cells, we inoculated inner ear stem cells to polylysine treated wells and induced them to adhere to wells and then differentiation. Gene expression was investigated with inner ear stem cells, cells differentiated for7days and14days respectively. Results showed that the expression of markers specific for inner ear stem cells decreased, the expression of inner ear development related genes sustained and the expression of markers specific for inner ear hair cells increased. Immunofluorescence of cells differentiated for14days showed that some cells expressed hair cell-specific markers MyosinVIIA, Brn3c and Espin. Scanning electric microscopy showed that there were some cells differentiated for14days with disordered stereocilia while inner ear stem cells showed no stereocilium, which indicated that some inner ear stem cells differentiated into immature hair cells. In order to obtain mature hair cells, we developed a two step-induction method of generating functional hair cells from inner-ear stem cells:Firstly, inner ear stem cells were induced into progenitor cells on the poly-L-lysine substratum. Then, progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells with stereociliary bundles. Results showed that there were typical stereocilia bundles on the surface of newly generated hair cells with abundant F-actin and kinocilia marker Tubulin. The newly generated hair cells could absorb FM-143with electrophysiological function as examined by patch clamp. The results indicated that inner ear stem cells could be induced to differentiate into the functional mature hair cells under the ideal cultivating condition.Part3:The development of inner ear hair cells is controlled by a series of transcription factor and signal pathway, so the identification and regulation of crucial genes during the development of inner ear hair cells is helpful to control differentiation of hair cells. We selected the inner ear stem cells, the progenitor cells differentiated for7days and the hair cells differentiated for14days to investigate specific markers of these cells. Hair cells were sorted through marker FM1-43. We investigated Phalanx mouse gene expression profiles chip using RNA extracted from three types of cells mentioned above respectively. We sorted out30,38, and31genes expressed specifically in inner ear stem cells, progenitor cells and hair cells, and the specification of the expression of these genes were examined with different mouse tissue. Gdf10+Ccdc121were determined as markers specific for inner ear stem cells, Tmprss9+Orml were determined as markers specific for progenitor cells and Chrna9+Espnl were determined as markers specific for inner ear hair cells.Here, we mainly focused on identification of new markers of hair cells, progenitor cells and stem cells which paved the way for identification and sorting of inner ear stem cells. |
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