The Experimential Study On Transplanting The Induced Pluripotent Stem Cells Into The Cochlea Of Mature Mice

Objective：In this study，we transplanted iPSCs into mature mice cochlea through the roundwindow to observe differentiation outcome of the situation by microinjectionmethod.The purpose of the study is to provide a theoretical and experimental basis forthe treatment of deafness by iPSCs.Methods：1.Establishing a stable culture system of mouse iPSCs and then identificatingstable passage of the mouse iPSCs in vitro.2. Labeling and transplantating the mouse iPSCs: the iPSCs was labeled withCM-Dil and identified by flow cytometry.20healthy6-8weeks old CD1/ICR micewere selected and randomly divided into two groups: A group was the experimentalgroup, B group was the control group. all the implant surgery was operated on the leftear;10mice were microjected the cell suspension of-miPSCs labeled by CM-Dil intothe cochlea,10mice were implanted with DMEM basic medium of equal volume.the thresholds of all mice were tested by Auditory brainstem response(ABR) at oneweek before surgery and one month after surgery.3. Identification of the differentiated cells by iPSCs in cochlea All mice weresacrificed at one month after transplantation. we identify the differentiated cellswhether expression of the hair cell markers (Myosin Ⅶ a, Espin) and neuronal cellmarkers (Nestin, Neurofilament-M) by Immunohistoche-mical staining.4. HE staining was used to determine whether there are teratoma formation afteriPSCs were transplanted into the cochlea.Results:1. We successfully established a stable culture system of mouse iPSCs. MouseiPSCs by AP staining were positive and can form EBs.2.96.42%of the mouse iPS cells were labeled by Fluorescent dye (CM-Dil).One week before surgery, The ABR response threshold in group A and B was24.50±5.50dB SPL,and26.00±6.15dB SPL,respectively; The difference of ABR threshold between group A and B was no statistically significance at one week before surgery (p=0.572>0.05). ABR response threshold at1month postoperatively was70.50±4.97dB SPL in group A and68.00±5.37dB SPL in group B, There is no statisticallysignificant between two groups (p=0.295>0.05). ABR response thresholds beforesurgery is higher than that one month later in two groups, the difference wasstatistically significant(p <0.05).3. In the group A, CM-Dil-labeled iPSCs would be seen in the inside and outsidelymph and the modiolus of the cochlea, and some red fluorescence-labeled cellsexpressed auditory hair cell markers (Myosin Ⅶ a, Espin) and neural cell markers(Nestin, Neurofilament-M). No red fluorescence was observed in the cochlear ofgroup B.4. Teratoma was observed in some cochlea.Conclusion:1. Mouse iPSCs could differentiate into cells with hair cell markers after theywere transplanted into the cochlear of mice one month later;.2. Mouse iPSCs could differentiate into cells with neural cell markers afterthey were transplanted into the cochlear of mice one month later;3. Mouse iPSCs cochlear transplantation may form teratomas；4. The hearing loss of mice caused by cell transplantation through Roundwindow pathways can not be improved in the short term by iPSCs cochleartransplantation.