| Deafness is the most common human disease caused by sensorineural damage,which affects more than 6%of the global population and has serious social and economic implications.The main cause of deafness is the damage of hair cells,and the secondary cause is the damage of sensory neurons.As terminally differentiated cells,hair cells and sensory neurons in the inner ear cannot regenerate after injury,which makes it difficult to effectively treat deafness.At present,hearing improvement is often achieved by wearing hearing aids and cochlear implants,but the basis of these approaches is that there are still a certain amount of hair cells and sensory neurons in the cochlea of deaf patients.Stem cell therapy provides an effective way to cure deafness.Embryonic stem cells have the potential of infinite proliferation and differentiation into a variety of cell types.The embryonic stem cells can be induced to differentiate into a variety of inner ear cells in vitro,and these new cells can be used to replace the innate loss or injury of inner ear cells in mammals,so as to achieve the restoration of inner ear function.Therefore,in the process of using stem cell therapy to treat deafness,it is very important to induce a large number of stable inner ear cells in vitro.In this study,the MC3T3 induction system was successfully constructed using mouse embryonic stem cells(mESCs)as experimental material to induce the efficient differentiation of mESCs into otic progenitors.Oepithelial progenitors(OEPs)and otic neural progenitors(ONPs)were isolated and enriched from otic progenitors,and a system was constructed to effectively induce OEPs to differentiate into inner ear hair cells.First of all,we prepared the mESCs into the embryonic body of 10~4cells,and induced embryonic bodies with MC3T3 conditioned medium for 7 days.The differentiated cells were determined to be otic progenitors with high expression of Pax2,Pax8 and Nestin by immunofluorescence and other experiments.Next,by setting different inoculation densities and cultivating otic progenitors with the otic stem cell full media,we determined that 6×10~4cells/cm~2 inoculated cells were the ONPs with high expression of Nestin gene,and 10×10~4cells/cm~2 inoculated cells were the OEPs with high expression of Pax2 and Pax8 genes.We also amplified OEPs and ONPs in the otic stem cell full media,and determined that OEPs could still express its signature genes and proteins stably during the amplification process,and found that the second-generation OEPs were most suitable for subsequent experiments to induce differentiation into hair cell-like cells.Finally,OEPs were induced by MC3T3conditioned medium for 6 days,and the differentiated cells were identified as inner ear hair cell-like cells with high expression of Brn3c,Math1 and MYO7A by immunofluorescence and other experiments.At the same time,the double staining experiments of FM1-43 and MYO7A confirmed that the hair cells differentiated in this study had the preliminary electrophysiological function,and the scanning electron microscopy experiments showed that the hair cells differentiated in this study had abundant immature and disorganized ciliary structures on the surface.In conclusion,this study successfully constructed the MC3T3 induction system that can effectively induce and differentiate mESCs into otic progenitors and hair cell-like cells,and determined the specific method of isolating OEPs and ONPs from otic progenitors.In this study,the induction time is short,the induction efficiency is high and the induction method is simple,which can provide a large and stable cell source for stem cell therapy in the treatment of deafness disease. |
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