Feasibility Study Of Human Pluripotent Stem Cells-derived Peripheral Sensory Neurons For Cochlear Auditory Nerve Connection

Hearing impairment is a common clinical disease.Common causes include damage to the hair cells responsible for perceiving sound or damage to the cochlear auditory nerve responsible for transmitting sound.If the auditory nerve is damaged,even if the cochlear implant is implanted,the signals generated by the cochlear implant cannot be transmitted to the auditory center.Human embryonic stem cells(h ESCs)and human induced pluripotent stem cells(hi PSCs)are both human pluripotent stem cells(h PSCs).Inducing h PSCs into specific types of cells for cell therapy has gradually brought hope for the treatment of many diseases.In recent years,there have been many studies attempting to repair auditory nerve damage by stem cells,but there is very few exploration on the structural basis of auditory nerve connection by stem cells,especially on the structural connection with cochlear nucleus.In this study,h PSCs such as h ESCs and hi PSCs were induced into peripheral sensory neurons with almost the same characteristics in vitro,and then,under the co-culture and in vivo condition,h ESCs were mainly used to study the differentiation,nerve fiber extension and the terminal junction structure of peripheral sensory neurons.In the first part,using retrovirus,we induced urine cells into stable passaged hi PSCs cell lines.Alkaline phosphatase staining,pluripotent gene expression,the expression of pluripotent protein markers and capacity of differentiation into 3 germ layers showed that the hi PSCs cell lines we established had same characteristics as h ESCs.Furthermore,we introduced GFP tag into h ESCs cells and hi PSCs cells,and established stable h ESCs-GFP and hi PSCs-GFP cell lines for co-culture and in vivo traceability.In the second part,we induced h PSCs into peripheral sensory neurons in vitro and identified them at each induction and differentiation stage,which provided a cell source for co-culture and in vivo studies.In the third part,we confirmed that there was a terminal dendritic bifurcation structure formed,similar to endbulbs of Held,between cochlear nucleus and peripheral sensory neurons under co-culture condition.We also detected the signal transmission between cells by optogenetics preliminarily.In the fourth part,the peripheral sensory neural progenitor cells derived from h ESCs were transplanted into Mongolian gerbils,and the survival,differentiation,nerve fiber extension and fiber-end structure formation after transplantation were detected.In summary,this study demonstrated that peripheral sensory neurons derived from h PSCs were capable of forming terminal structures similar to those of endogenous nerve,and to some extent showed the feasibility of peripheral sensory neurons derived from human pluripotent stem cells for auditory nerve connection.