| Hearing loss is one of the most frequent diseases that disable people. Options for improvement of hearing of patient with sensorineural deafness are limited to hearing aid and cochlear implants. The effect of aid and cochlear implants depends on the number of live hair cell and spiral ganglion neuron. It's new strategy for restoring hearing to improve inner ear hair cell and spiral ganglion neuron regeneration and to prevent necrosis of spiral ganglion neuron after hair cell injury. Cell therapy may replace damaged and lost hair cell and spiral ganglion neuron and hence restore hearing pathway. Stem cells are undifferentiated cells, which have the ability to undergo numerous divisions and self-renewal in culture, as well as to differentiate into multilineage, functionally specialized cells. The replacement of lost or damaged neurons by neural stem cells (NSCs) or neural progenitor cells(NPCs) represents a great promise for clinical treatment for hearing loss. NSCs, with the capacity for unlimited self renewal and the production of none-restricted lineage committed progenitors in contrast to neural'progenitor'or'precursor'cells, have long been thought of as central to the repair and regeneration processes to replacing cells lost in hearing loss diseases. Although hair cell replacement occurs spontaneously following injury in birds and lower vertebrates, the mature mammalian cochlea is unable to regenerate new hair cells. As a matter of fact, cochlear hair cell loss is one of the major causes of hearing impairment. Hair cell replacement, either by stimulation of regeneration or by transplantation of progenitor cells capable of differentiating into hair cells, remains the ultimate goal in the development of treatment applications to reconstruct damaged inner ears. An alternative way to replace lost hair cells is by introducing exogenous stem cells into the inner ear. Recent work by Heller and co-workers has demonstrated the existence of progenitor/stem cells in the sensory epithelium of a mature mouse vestibular end organ, which is responsible for balance. These cells are not only able to differentiate into cells that express hair cell markers in vitro, but also differentiate into hair cell-like cells in developing chick ears. The recent discovery of stem cells in the adult inner ear that is capable of differentiating into hair cells, as well as the finding that embryonic stem cells can be converted into hair cells, raise hope for the future development of stem-cell-based treatment regimens. While stem cells/progenitors may exist in adult vestibular end organs, stem cells/progenitors from the cochlear nucleus have not been isolated.We have for the first time isolated neural precursor cells from the newborn rat cochlear nucleus. Under our optimized conditions, precursor cells isolated from the cochlear nucleus proliferate in culture in the same way as neural stem cells after epigenetic stimulation and retain all of the typical characteristics of neural stem cells: they proliferate in response to mitotic factors (basic fibroblast growth factor, bFGF, and epidermal growth factor, EGF) and have the ability to give rise to neurons, astrocytes, and oligodendrocytes. Part I Isolation and culture of NPCs from the newborn rat cochlear nucleus.NPCs were isolated and cultured from P3, P5, P7, P9, P11 and adult rat cochlear nucleus using DMEM/F12 (1:1) containing 10% heat-inactivated fetal bovine serum. Cell clones were formed 8 days after culture. Cell clones and most single round cells were semi-suspending. Cell clones can be formed again after dilution, purification and passage. Observe the growth of NPCs every day in inverted microscope, 24h later, in experiment group, we can observe many living cell homogeneous distribution and many collagenoblast sink in the base of culture flask, part of cells grow floating in culture fluid, cell appear conjugation usually and some cells begin to division. 72h later, cell aggregation and the cell colony consist of about 4-6 cells; 5 days later, cell colony is consisted of about ten to hundred cells; we can observe lots of cell colony. The cell sphere growth floating and arrange tight among cells, some cell colony appear cellular necrosis because of alimentary deficiency. In control group, we can not observe cell proliferate. Cell clones were nestin immuno-positive and Musashi1 immuno- positive. The Cell viability of P5 and P7 rat cochlear nucleus was higher than the others. The viability of NPCs had significant difference between P5,P7 and the others (P>0.05). Made cell sphere into mono-cell suspension, add dispase during dispersive process. The method is to obtain neural cell spheres that growth good in culture flask first\digest them for 2 hours\then blow lightly and get out supernatant after sink nature and obtain mono-cell suspension. Divide into 4 groups, Add EGF in experiment group 1; add bFGF in experiment group 2; add EGF and bFGF together in experiment group 3; add base serum-free medium only in control group. Count cell amount of cell spheres in micro after cultured for 3 days and measure the diameter of cell spheres at 7 days. EGF and bFGF can significantly promote proliferation of cochlear nucleus NPCs. EGF can excite neural stem cells proliferate lonely; bFGF can not excite neural stem cells proliferate lonely but can enhance the proliferation function of EGF. The results showed that NPCs with self-renewal capacity could be cultured in vitro from cochlear nucleus of newborn rat.Part II Cell differentiation biologic characteristics of NPCs from cochlear nucleus of newborn rat.NPCs and NSCs were isolated and cultured from P7 rat cochlear nucleus, E14 rat olfactory bulb and adult rat olfactory epithelium using serum free media with mitogen and neurosphere forming method. Cultured NPCs and NPCs were round or oval without process. Cells from P7 cochlear nucleus, E14 olfactory bulb and adult olfactory epithelium divided 2 days after primary culture, looked like bean sprout and at 4th day formed suspending small neurospheres, which grew gradually. After omitting mitogen and adding fetal bovine serum, most of NSCs/NPCs from P7 rat cochlear nucleus, E14 olfactory bulb and adult rat olfactory epithelium differentiated into Neun and Tuc-4 immunopositive neuron and GFAP immunopositive astrocyte, some of them differentiated into GalC immunopositive oligodendrocyte. RT-PCR and In-cell Western also show the same result. The proliferation of P7 rat cochlear nucleus, E14 olfactory bulb and adult rat olfactory epithelium NSCs/NPCs depended on EGF and bFGF. The results showed that NSCs with self-renewal capacity and potential multi-differentiation could be cultured in vitro from P7 rat cochlear nucleus. The newborn rat cochlear nucleus maybe is rich source of NSCs/NPCs and thereby an ideal providing organ for the study of NSCs/NPCs transplantation.Part III Ultrastructure of NSCs from rat olfactory bulbThe ultrastructures of NPCs from newborn rat cochlear nucleus were investigated with scanning and transmission electron microscopes. Cultured NPCs were round or oval, with many tiny processes or microvilli on the surface. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. The nucleus/cytoplasm ratio was very high. Cells had very little cytoplasm with various numbers of immature mitochondria, ribosome and Golgi complex. Inside neurosphere, dividing cells and apoptosis can be found and the structure of pre-differentiating NSCs remains the same at different time after culture. Differentiated cells with rough endoplasmic reticulum and thick and long processes appeared 1~2 months after culture. Gap junction was found between the processes of adjacent differentiated cells. The results showed that cultured NPCs from newborn rat cochlear nucleus were primitive cells that can differentiate into nerve cells.Part IV Proliferation, Multipotency and Neuronal Differentiation of Cryopreserved Neural Progenitor Cells Derived from Newborn Rat Cochlear NucleusStem cell replacement has emerged as the novel therapeutic strategy for many diseases. These researches not only offers unique opportunities for developing new medical therapies for devastating diseases, but also provides a new mode to explore fundamental questions of biology. The study of newborn rat cochlear nucleus NPCs requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to evaluate different cryopreservation techniques for NPCs derived from newborn rat cochlear nucleus. Initially, we compared the survival rates of cryopreserved NPCs treated with four different cryoprotectants: dimethylsulphoxide (DMSO), glycerol and each with or without 10% FBS, meanwhile with two different storage period at liquid nitrogen (–196℃) , that is 3 days standing for short-term storage and 3 months for long-term storage. We assessed the recovery efficiency of NPCs after freezing and thawing by viability testing, colony-forming assay as well as immunocytochemistry under different conditions. No significant difference in survival rate was observed among these different cryoprotectants. With these protocols, NPCs from newborn rat cochlear nucleus retained their multipotency, differentiated into glial (GFAP-positive), neuronal (NeuN-positive). Collectively, our results imply that, under the optimal conditions, NPCs from newborn rat cochlear nucleus might be cryopreserved for longer storage period than 3 months without losing proliferation and multipotency activity.
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