| Backround and ObjectiveSensorineural hearing loss is one of the common ENT diseases. According to the 39th World Health Assembly information,there are 420,000,000 deaf and hearing impaired people worldwide. Various factors such as viral or bacterial infections, noise, ototoxic drugs can lead to sensorineural hearing loss. Study confirmed the pathological changes are mainly associated with cochlear hair cells and spiral ganglion neurons loss. So if we can find a way to restore lost hair cells or spiral ganglion neurons, there is the hope for curing sensorineural hearing loss. Cell transplantation is one of treatment methods of sensorineural hearing loss in the future. In other medical fields, it has been confirmed from clinic and experiment stem cell transplantation can cure a disease, such as the transplantation of neural stem cells to treat Parkinson's disease, cardiac stem cell transplantation to treat myocardial infarction. Recent studies have found that the inner ear vestibular organs and the cochlea of mammals such as mice have also stem cells and highly proliferative cells, and in vitro to differentiate into hair cells-like cells and neurons. Stem cell transplantation offers hope for the treatment of sensorineural hearing loss. In addition, Japanese scholars transplanted rat neural stem cells into rat cochlea of the amino acid glycosides antibiotics or noise induced deafness, they found neural stem cells are alive in the cochlea and they can differentiate into a small number of hair cells and neurons. Some scholars also found the transplantation of neural stem cells into the cochlea can improve hearing loss caused by cochlear ischemia. These results show that stem cell transplantation in the treatment of sensorineural deafness is a promising way. As the inner ear stem cells or neural stem cells locate in the inner ear or central nervous system, obtaining them is difficult in clinical applications, which will affect their value. Therefore, we must find other stem cells that are convenient in obtaining material, and can differentiate into inner ear hair cells. The study is designed to extract bone marrow mesenchymal stem cells from the SD rats,then MSCs are co-cultured with the cochlear organ of Corti, we investigate if bone marrow mesenchymal stem cells cultured in vitro can differentiate into inner hair cells, which will offer theoretical basis for the clinical applications of MSCs transplantation in treating sensorineural deafness in the future.Methods1. purification and identification of SD rat MSCs: SD rat MSCs were isolated, cultured and purified by adherence screening method, the changes of cell shape and number were observed under inverted phase contrast microscope,At the same time,The expression of MSCs markers (CD29,CD34,CD45,CD90 and CD117 ) were identified by immunocytochemistry, and the fourth generation MSCs were collected for co-culture experiments.2. The organs of Corti were isolated. we dissected the cochlear organs of Corti from newborn twenty Sprague-Dawley rats, the Spiral ligaments and cochlear axises were carefully removed under Dissecting microscope. The cochlear organs of Corti were treated with 0.25% trypsin in PBS at 37℃for 8 min. The enzymatic digestion was stopped by the addition of serum-free medium. Supernatant was discarded after centrifugation. we plated cells in a 15 cm plastic Petri dish.3. Gene expression analysis of differentiated cells: two groups were divided: the control group and the co-culture group. In control group, we plated the fourth generation of MSCs (5×104cells/ml)in 6-well plates, they were cultured in serum-free DMEM/high glucose and F12 medium (mixed 1:1) supplemented with N2 and B27 solutions and growth factors .Growth factors include epidermal growth factor (EGF) at 20 ng/ml,basic fibroblast growth factor (bFGF) at 10 ng/ml and IGF-1 at 50 ng/ml, the media was replaced every second day, the cultured cells were collected after 14 days. In co-culture group, at the same condition with control group, we co-culture MSCs with the organ of Corti for 14 days, then the differentiated cells in the bottom of 6- well plates of two groups were collected and analyzed by total RNA preparation followed by reverse transcriptase (RT)-polymerase chain reaction (PCR) , electrophoretic analysis, and by immunocytochemistry.4. Identification of differentiated cells:The expression of hair cell markers(Myosin7A,Math1,Calretinin)was examined by immunofluorescent staining. Results:1. The fourth generation of MSCs shows a fibroblast-like shape and most cells expressed CD29,CD90,CD117 instead of CD34,CD45 by immunofluorescent staining.2. After MSCs were cultured with the cochlear organ of Corti cells for 14 days,differentiated cells expressed hair cell markers(Myosin7A,Math1,Calretinin)by immunofluorescent staining. Among them, Myosin7A is one hair cells marker with relatively high specificity,Math1 always expresses from the early stage formation of hair cells to maturity. Calretinin is one early specific molecular marker of vestibular hair cells.3. We did not find mRNA expression of hair cell markers in the control group. The co-culture group can be seen between the electrophoretic bands 500bp and 750bp, there are Myosin7A electrophoresis band with amplified fragment length of 624bp and Math1 electrophoresis band with amplified fragment length 545bp.Conclusion:1. MSCs could be successfully induced to differentiate into inner ear hair cells with molecular characteristics in vitro.2. the cochlear organ of Corti may secrete a variety of nerve growth factors, which can induce Bone marrow-derived mesenchymal stem cells to differentiate into inner ear hair cells. |
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