Research On The Neural Stem Cells Derived Fom ADSCs Efficiently Engaft In Animal Models Of Presbycusis

Presbycusis which is also called age-related hearing loss（ARHL） refers to aphysiological phenomenon that the slow aging process of the hearing system occur due tothe degeneration of the auditory organ along with the other tissues and organs with agingand the emergence of a hearing loss. It belongs to the areas of multi-gene diseases andcaused by genetic and environmental factors. With the growing population aging trends inthe world, the incidence of presbycusis increased year by year. According to WHO statistics,by2025, the proportion of elderly population over the age of60years old will increase asmuch as1.2billion rapidly. However, presbycusis incidence rate for65-75years old is70%-80%. The occurrence and development of presbycusis play a serious role on quality of lifefor elderly patients. As to the sustained study on the pathogenesis of presbycusis in recentyears, Welsh suggested a concept of central presbycusis based on the classification ofpresbycusis by Schuknecht. It is mainly mentioned that the apoptosis and degeneration ofall levels of central neveous system, especially the primary auditory cortex（Au1） neuronsis the main pathogenesis of presbycusis. In recent years, in order to save the hearingfunction of patients with presbycusis, a variety of methods such as stem cell transplantation,pharmacological intervention therapy and gene therapy were tried, but stem celltransplantation has great potential clinical value of treatment.Kesser and the other people not only succeed in replacing or repairing the apoptoticand degenerated cells in peripheral nervous system by neural stem cells(NSCs) transplantedinto the cochlea，that is to say，NSCs differentiated in to hair cells or spiral ganglion cells,but also pointed out that seed cells impact the recipient by the way of releasing someneurotrophic factors such as GDNF, BDNF and so on, suggesting that the NSCs played animportant role in stem cell transplantation for presbycusis and other neurodegenerativediseases. Owing to the transplanted cells for presbycusis deriving from the striatum, hippocampus, cerebral cortex, the lateral ventricle of the embryonic ventricular zone,human embryonic tissue and human embryonic stem cells are always restricted by ethicsand inadequate sources. Nevertheless, allogeneic transplantation will exist more or lessimmune rejection problems. Adipose-derived stem cells (ADSCs) with characteristics ofovercoming these shortcomings is considered to be an ideal source of seed cells. ADSCscan be obtained through liposuction, with the advantages that can be more easily isolatedand cultured, less rejection, non-tumorigenic characteristics. It has become one of the mostpotential source of adult stem cells.Stem cell transplantation for peripheral nervous system of the animals withpresbycusis was able to confirm the seed cells can replace lost cells, but the restoration ofthe auditory function is not yet clear. Currently, large number of studies show that theincidence of presbycusis is based on the degeneration of the peripheral nervous system andcentral nervous system, espically the central nervous system. The study induced ADSCsinto NSCs and then transplanted into Au1of C57BL/6J mouse, a typical animal model ofpresbycusis, to observe the effect of inducing ADSCs into NSCs. Transmission electronmicroscopy techniques, HE staining and Tunel-POD method were used to compare themorphology differences before tansplantation and varity of apoptotic rates in the Au1between the young group aged at2months and the aged group aged at10months bothbefore and after transplantation. After that, immunofluorescence was applied to detect thesurvival, migration and differentiation of the seed cells transplanted into the recipient.Furthermore, the recovery of auditory function was detected. The whole study is dividedinto three parts:Part one: Dissection, culture and identification of adipose-derived stem cells【Objective】To isolate epididymal fat pads from C57BL/6J mouse under strictlysterile condition. ADSCs were cultured in vitro and then identificated to search for a newsource of stem cells for transplantation.【Methods】 C57BL/6J mouse weight about15g~16g were choosed. After dissectionthe skin of abdoman, it is possible to obtain epididymal fat pads where ADSCs wereisolated by the way of collagenase digestion and then passaged by the trypsin digestion. Atthe third passage, flow cytometry was used to identificate the cell surface antigens (CD45,CD90, CD105) while the fibronectin marker of ADSCs was evaluated by immunofluorescence.【Results】 A great deal of spindle-shaped ADSCs possessed potential ofself-proliferation can be generated from epididymal fat pads if cultured in vitro. Flowcytometry showed that the isolated ADSCs make positive expression of the CD90, CD105,fibronectin，but the expression of CD45on ADSCs is negative.【Conclusion】Generous ADSCs isolated from epididymal fat pads can easily becultured, passaged and amplified in vitro. ADSCs can be seen as a new source of stem cellsfor transplantation.Part two: Induction and differentiation of ADSCs into NSCs in vitro【Objective】To explore a method to induce ADSCs differentiate into NSCs in orderto provide an origin of stem cells for transplantation and repair of the central nervoussystem.【Methods】After the cell surface antigens and the fibronectin marker of ADSCs wereidentificated by the flow cytometry and immunofluorescence respectively, the neurosphereswere induced from mouse ADSCs with basic fibroblast growth factor(bFGF), epidermalgrowth factor(EGF) and2%B27in Neurobasel medium. Immunofluorescence was carriedout to examine the expression of NSCs marker(Nestin), self-proliferation(Brdu), and themultipotential of differentiation.【Results】The neurospheres induced from ADSCs examined by Nestin. They expressBrdu showing their potential of self-proliferation. They can also differentiated into β-TubulinⅢ positive neurons, GFAP positive astrocytes.【Conclusion】A large number of NSCs could be harvested from ADSCs underappropriate growth factors and made a foundation for the transplantation and repair of thecentral nervous system.Part three: NSCs derived from ADSCs efficiently engaft in animal models ofpresbycusis in vivo【Objective】To observe the survival, migration and differentiation of NSCs derivedfrom ADSCs and evaluated the recovery of the auditoy function in C57BL/6J mouse beforeand after tansplantation.【Methods】 With the assistance of stereotaxis system, NSCs derived from ADSCswere injected into the Au1of C57BL/6J mouse including the young goup and the aged group. The efficiency of the therapy was detected by immunofluorescence method,pathology and electophysiology, respectively, such as the suvival, migration anddifferentiation of NSCs was detected by immunofluorescence method，the neuron apoptoticrate was examined by Tunel-POD method. Meanwhile, the Auditoy Brain StemResponse(ABR) could be applied to test the diversity of auditory function before and afterthe therapy.【Results】After the NSCs derived from ADSCs transplantation, immunofluorescencedemonstrates that achieved NSCs differentiated into neurons that engraft and migratedinto the brain even to lesioned sites. Immunofluorescence revealed that the neuronapoptotic rate lower after the transplantation in the aged group but not in the young group.Meanwhile, the electrophysiological data suggests that ABR thresholds significantly decreasedin the aged group after transplantation for2weeks and3weeks.In contrast, there is nodifference neither in the aged group at the4th week nor in the young group at any timepostoperation.【Conclusion】Our results demonstrate that NSCs derived from ADSCs improve theauditory of C57BL/6J mouse with presbycusis through the way of replacing and restoringthe apoptotic neurons in the Au1. In the current research, we present a novel strategy totreat the C57BL/6J mouse with presbycusis.