Culture Of Neural Stem Cells From Rat Olfactory Pathway And Cochlear Transplantation

Olfactory dysfunction is a common disease, which has substantial adverse effects on the quality of life. Up to date, effective therapy for neuropathic loss of olfactory remains a major challenge. Because olfactory epithelium and olfactory bulb host stem cells and progenitor cells and remain active neural regeneration through life, it may be an effective way to treat olfactory dysfunction by inducing and promoting the regeneration of neuron in olfactory epithelium and olfactory bulb. NSCs transplantation has achieved promising result in laboratory in the management of traumatic and degenerative diseases of nervous system. As embryonic stem cells and allogenous NSCs transplantation may bring about ethic dilemma and rejective response, autogenous NSCs transplantation may be the way of choice. NSCs from olfactory bulb and olfactory epithelium in peripheral nervous system may be the ideal sources for autogenous NSCs transplantation. NSCs with self-renewal and multipotent ability have been isolated and cultured from olfactory epithelium and olfactory bulb of adult human and mice successfully. However, NSCs from rat olfactory epithelium have not been reported and there is little literature on NSCs from rat olfactory bulb, and the cell biologicalcharacteristics of NSCs from olfactory pathway remain unknown.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 restoringhearing 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. Embryonic and adult NSCs can survive in inner ear microenvironment of mice, guinea pig and newborn rat for up to 4 weeks. Grafted cells differentiate into astrocyte and neuron, migrant to sensory epithelium of cochlear and vestibular and express special markers of hair cell phenotype, and too, migrant spiral ganglion in modiolus. However, the characteristics of survival, migration and differentiation of olfactory bulb NSCs in cochlear of normal adult rat remain unknown.In present study, methods as cell culture, gene transfection and cell transplantation are employed to isolate, culture NSCs from olfactory epithelium of newborn and adult rat and NSCs from olfactory bulb of embryonic, newborn and adult rat respectively, and to investigate the survival, migration and differentiation of olfactory bulb NSCs with reporter gene EGFP from newborn rat in the inner ear of normal adult rat after transplantation. 1. Isolation and culture of NSCs from rat olfactory epithelium NSCs were isolated and cultured from P3 and zinc sulfate in situ injured adult rat olfactory epithelium using DMEM/F 12(1:1) containing 10% heat-inactivated fetal bovine serum. Cell clones were formed 14 days after culture. Cell clones and most single round cells were semi-suspending. Cell clones can be formed again after dilution, purification and passage. These changes were similar in newborn and adult rat. Cell clones were nestin immuno-positive and cytokeratin immuno-negative. The forming rate of clone sphere of P3 and adult rat olfactory epithelium was 0.05%~0.1%. Cultured in MEM, cell clones differentiated into GFAP immuno-positive astrocytes(70%) and NSE immuno-positive neurons(30%). Differentiated cells were GalC and cytokeratin immuno-negative. The viability of NSCs had no significant difference between P3 and adult rat (?>0.05). bFGF can significantly promote proliferation of olfactory epithelium NSCs, while EGF had no effect. The results showed that NSCs with self-renewal capacity and multi-potent could be cultured in vitro from olfactory epithelium of newborn and adult rat. NSCsfrom olfactory epithelium may be ideal source for autogenous NSCs transplantation.2. Culture and cell biologic characteristics of NSCs from rat olfactory bulb2.1 Apoptosis and proliferation in the developing rat olfactory bulb The changing pattern of apoptosis and proliferation in the developing ratolfactory bulb was investigated with TUNEL and PCNA indirect immunofiuorescence staining. Apoptosis cells were widely distributed and PCNA immuo-reactive cells were mainly around ependymal layer in E14 and PI rat olfactory bulb. Apoptosis and PCNA immuo-reactive cells were mainly distributed in glomerular layer and granule layer of P20 and adult rat olfactory bulb. Apoptosis and PCNA immuo-reactive cells reduced significantly as rat getting mature. The results indicated that there are NSCs and progenitor cells in rat olfactory bulb and there exits active apoptosis and neuron regeneration from embryo to adult. Apoptosis and proliferation in rat olfactory bulb reduces gradually during developing.2.2 Culture of NSCs from rat olfactory bulbNSCs were isolated and cultured from El4, PI and adult rat olfactory bulb using serum free media with mitogen and neurosphere forming method. Cultured NSCs were round or oval without process. Cells from El4 and PI olfactory bulb divided 2 days after primary culture, looked like bean sprout and at 4th day formed suspending small neurospheres, which grew gradually. Neurospheres can be formed again after passages and were nestin immunopositive. Cells from adult olfactory bulb formed neurosphere at 2nd passage. The characteristic of appearance and passage of NSCs from adult are much the same as that of NSCs from PL The forming rate of neurosphere of E14, PI and adult rat olfactory bulb was 25%~30%, 20%~30% and 0.1%, respectively. After omitting mitogen and adding fetal bovine serum, most of NSCs from El 4 and PI olfactory bulb differentiated into NSE immunopositive neuron and GFAP immunopositive astrocyte, some of them differentiated into GalC immunopositive oligodendrocyte. The proliferation ofE14 and PI olfactory bulb NSCs depended on EGF and bFGF, in which EGF increased proliferation of cells stronger than bFGF. The results showed that NSCs with self-renewal capacity and potential multi-differentiation could be cultured in vitro from El4, PI and adult rat olfactory bulb. Rat olfactory bulb is rich source of NSCs and thereby an ideal providing organ for the study of NSCs transplantation.2.3 Ultrastructure of NSCs from rat olfactory bulbThe ultrastructures of NSCs from rat olfactory bulb were investigated with scanning and transmission electron microscopes. Cultured NSCs were round or oval, with many tiny processes or microvilli on the surface. 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 NSCs from rat olfactory bulb were primitive cells that can differentiate into nerve cells.3. Label and inner ear transplantation of NSCs from rat olfactory bulbNSCs from PI rat olfactory bulb were transplanted into cochlear of normal adult rat using gene transfection and cell transplantation methods. The transfection efficiency of pIRES-EGPF-C2 into NSCs was 46%, detected with flow cytometry 48 hours after transfection mediated with liposome Lipofectamine 2000. Live cell and neurosphere emitted bright green fluorescence under fluorescence microscope 1 month after G-418 filtration. 4 weeks after transplantation, graft cells were founded in every turn of cochlear in all experimental animals. Grafted cells were single or aggregated, chiefly located in scala vestibuli and scala tympani of the cochlear and most grafted cells were attached to the cochlear architecture, only a few of them were as...