Explore The Method Of Differentiation Of HUC-MSCs Combined With DAPT Into Hair-cell Like Cells In The Inner Ear In Vitro

Sensorineural hearing loss(SNHL)is a common form of sensory dysfunction in otorhinolaryngology clinical work and study,which is caused by the degeneration and irreversible injure of inner ear hair cells and the spiral ganglion neurone.People who suffered of hearing loss may have varying degrees of difficulty with speech comprehension.As the limmited regenerative capacity of mammalian inner hair cells,traditional treatments can not restore the physiological function of the damaged inner ear such as hearing aids and cochlear implant(CI).As the further study of stem cells,it maybe possible to repair the damaged hair cells and spiral ganglion neurone.Human umbilical cord mesenchymal stem cells(hUC-MSCs)have the ability of self-proliferation and multiple differentiation potential and plentiful sources.Moreover,they have low immunogenicity as well as low ethical and legal controversies.HUC-MSCs are the ideal seed cells because of their practical clinical value,which are already widely used in the research of cell and tissue engineering.But the inefficiency and low differentiation of stem cells seriously hampers their development.Therefore,it is particularly important to explore a safe and efficient way to induce hUC-MSCs into hair cells.Notch signaling pathway is widely dispersed and highly conserved in evolution,which is crucial to the development of the auditory organ.The lateral inhibition mechanism of Notch signaling can regulate the development of hair cells in the inner ear.The study found that DAPT can inhibit the activity of ?-secretase through the effect on presenilin to reduce the production of NICD.Further more,it can regulate the expression of downstream target genes to break the lateral inhibition of Notch,what can result in the number of hair cells increased.And a test of adult guinea pigs found that it can induce ectopic hair cells with the use of DAPT.However,combinedwith DAPT to differentiate hUC-MSCs into hair-cell like cells in the inner ear in vitro has not been reported.ObjectiveWe use DAPT as the target and human umbilical cord mesenchymal stem cells(hUC-MSCs)as the object to find out the function of DAPT on the differentiation of hUC-MSCs into hair-cell like cells in the inner ear in vitro.And we also want to explore the possible molecular mechanism in order to enhance the efficiency of their differentiation.Thus it would provide a new theoretical basis for the treatment of SNHL.Methods1?Isolate hUC-MSCs from Wharton's jelly by the existing methods of our lab and cultured.The first step of the experiment is divided into protocol group(DMEM/F12+N2+B27+EGF+b FGF)and control group(DMEM/F12+N2+B27)to generate neural stem cells(NSC),which were non-adherent cultured about 3-5days.Then we use CCK-8 assay to detect the proliferation of hUC-MSCs and immunofluorescence to detect the expression of Nestin in induced cells.Meanwhile,Western Blot and q RT-PCR were also used to check the expression of Nestin.2 ? To further induce the NSCs derived from hUC-MSCs,the NSCs were allocated three groups randomly: DAPT group(DFNB + EGF+ATRA +DAPT)?trial(DFNB + EGF+ATRA)and control group(DFNB),which were cultured in gelatin coated flask to acquire inner ear hair cells for 2-4 weeks.Then we used CCK-8 assay to detect the proliferation of NSCs.With the use of immunofluorescence to detect the expression of Math1 ? Myosin VIIa ? Brn3 c in the induced hair cells.Meanwhile,Western Blot and q RT-PCR were also used to detect the expression of Math1 ?Myosin VIIa?Brn3c?Hes1 and Hes5.Results1? The control group have little Nestin after differentiated while the protocol group presented a neurobiological structure and demonstrated a higher Nestin positive ratio after 3-5 days' neural stem cells induction(p< 0.05).This suggested that EGF and b FGF could promote the proliferation of hUC-MSCs significantly.2? Compared to the control group,the m RNA and protein level of Math1, Myosin VIIa and Brn3 c exhibited a significant increase in DAPT and trial group,which induced for 4 weeks in the hair-cell like cells in the inner ear's induced phase(p < 0.05).With use of DAPT in DAPT group,the expressioin of Math1,Myosin VIIa and Brn3 c increased while Hes 1 and Hes 5 decreased.Conclusion1 ? hUC-MSCs can trans-differentiate into neural stem cells through cross embryonic germ layers,then subsequently to generate hair-cell like cells,which may provide a new theoretical opinion on the therapy of SNHL.2?DAPT can down-regulate the expression of Hes1 and Hes5 to promote the differentiation of hUC-MSCs into inner ear hair cells,thus may introduce a new idea for the treatment of sensorineural deafness.