iPS细胞定向分化为耳蜗神经元及其治疗感音神经性耳聋的实验研究

Loss of inner ear spiral ganglion neurons and hair cells is a major cause of sensorineural hearing loss. The long-term goal of this research is to develop an autologous cell-based therapy in the treatment of sensorineural hearing loss using induced pluripotent stem cells (iPS cells) and by providing essential preclinical studies of efficacy and safety. ..First, iPS cells will be sequentially inducted and differentiated to otic placode, otocyst cells and spiral ganglion precursor cells and spiral ganglion neurons by examing the effects of such morphogens as Wnts, Wnt antagonists, FGFs and Shh, individually, in combination or sequentially. We propose to investigate the complex signals and mechanisms that control iPS cells differentiation into spiral ganglion neurons, in order to establish an efficient chemical defined system to abtaining a large number of fuctional spiral ganglion neurons for cell transplantation. Based on the results, we will transplant the iPS cell-derived spiral ganglion-like progenitors or neurons into a chemically de-afferented mouse ear, in which the sensory cells are intact but spiral ganglion neurons are destroyed. In this model system, success of reinnervation and integration will be assayed both functionally and histologically...Finally, a specific iPS cell line will be generated from a specific mouse with normal hearing, then the spiral ganglion neurons in this mouse will be chemically destroyed. This mouse's specific iPS cells will be directly differentiated into spiral ganglion-like progenitors or neurons and will be transplanted into this mouse's inner ear to achieve an autologous transplantation. The extent of cell survival, reinnervation and integration with the host inner ear and hearing improvement will be evaluated. ..Overall, these studies would provide critical experimental basis and new insights into an antologous cell replacement therapy for sensorineural hearing loss, and thus bring approaches using human iPS cells closer to future clinical application.

利用内耳螺旋神经元缺失的小鼠耳聋模型，探讨诱导性多潜能干细胞（iPS细胞）对感音神经性耳聋的治疗潜能。将iPS细胞序贯诱导到听泡细胞，内耳螺旋神经元前体细胞及螺旋神经元细胞，研究iPS细胞向内耳细胞定向分化的规律和机制，并为细胞移植建立高效获取内耳神经元细胞的限定化学成分的体外培养及鉴定系统。取听力正常小鼠成纤维细胞建立该小鼠自体iPS细胞系后，药物致聋该小鼠，将该小鼠自体iPS细胞诱导分化为内耳螺旋神经元样前体细胞和神经元细胞并移植入该小鼠内耳，动态检测小鼠听力改善，利用小鼠iPS细胞实现感音神经性耳聋小鼠的自身个体化细胞移植治疗，原则性验证iPS细胞对感音神经性耳聋的治疗作用，为未来临床应用iPS细胞治疗人类感音神经性耳聋提供关键实验依据，为进一步探索新的有效的感音神经性耳聋治疗手段奠定基础。

本研究成功建立了精准的内耳螺旋神经元缺失的小鼠耳聋模型，发现高浓度(3mM)哇吧因可损伤I 型和II型耳蜗螺旋神经元，哇吧因对耳蜗螺旋神经元和胶质细胞的损伤与给药浓度和时间相关，给药浓度越高，给药后时间越长，损伤越严重，为确定细胞移植的时机以提高被移植细胞的存活率提供了重要依据。本研究初步建立了小鼠胚胎干细胞及小鼠iPS细胞定向诱导分化为耳蜗神经元的技术方法和培养体系并成功获取特定小鼠自体iPS细胞，鉴定并建系，已开始小鼠iPS细胞分化而来的耳蜗神经元细胞移植入耳聋小鼠。本实验结果为未来人iPS细胞自体移植治疗感音神经性耳聋提供了关键的实验依据，奠定了坚实的工作基础。