Cellular therapy for the inner ear

摘要 治疗神经缺失的一种方法是移植外源性祖细胞。我们 使用为内耳神经置换开发的模型系统来调查以下要求： 重建神经回路：在目前的工作中，我们研究移植的神经元是否来自干细胞。 可以在失去初级传入神经支配后恢复耳朵的功能。我们以前的工作表明， 来自移植干细胞的神经元可以在化学去神经后重新支配Corti器官的毛细胞， 传入，无论是在体外外植体和在体内沙鼠模型，表明这种神经回路可以 如果引入适当的祖细胞，则在成人神经系统中重建。在本提案中，我们使用 该系统旨在研究供体细胞的哪些特征使其能够与毛发形成功能性突触， 细胞，我们问是否新的突触导致恢复听力的动物与初级神经元 退化了解毛细胞神经连接重建的机制， 成年人的耳朵对于感音神经性听力损失的任何治疗方法都是重要的。 具体目标包括3个相互关联的实验，以探讨可能影响 移植神经前体细胞的毛细胞神经再支配成功。在目标1中， 在去神经支配的体外培养中评估干细胞的分化对它们再神经支配毛细胞的能力的影响。 Corti器官评估基于免疫组织化学：突触的形态和数量 由内毛细胞和外毛细胞形成;电生理学：动作电位和兴奋性 突触后电流的神经元刺激后的毛细胞，并抑制这种突触功能， 抑制天然耳蜗传入突触的功能活性的药理学阻断剂。在 目的2利用毒素去传入的耳蜗植块，研究调节 参与突触发生的分子。根据我们的证据，它们在Corti器官中的表达， 他们在其他神经再生系统中增加突触发生的能力，我们选择了一组 分子进行研究。脑信号蛋白3a，排斥性导向分子，和脑信号蛋白4 b， 轴突导向和突触发生受到抑制，神经营养素3和钙粘蛋白11， 突触发生过度表达。我们还抑制了neuropilin 1和neogenin 1的表达， 在再生神经元的生长锥中表达的引导受体。在目标3中，我们在体外 结果在体内移植到去传入的沙鼠耳，其中感觉细胞是完整的，但螺旋形 神经节细胞被破坏。我们测试我们在这个实验中观察到的毛细胞的神经再支配是否 模型导致听力功能测量的改善，我们使用小鼠模型来研究 轴突导向分子对神经再支配和功能改善的作用。

ABSTRACT One approach to the treatment of neural loss is the transplantation of exogenous progenitor cells. We use a model system developed for neural replacement in the inner ear to investigate requirements for rebuilding a neural circuit: in the current work we study whether transplanted neurons derived from stem cells can restore function to an ear after the loss of primary afferent innervation. Our previous work has shown that neurons from transplanted stem cells can reinnervate hair cells of the organ of Corti after chemical de- afferentation, both in an in vitro explant and in an in vivo gerbil model, suggesting that this neural circuit can be reconstituted in the adult nervous system if appropriate progenitor cells are introduced. In this proposal we use this system to study what characteristics of the donor cells allow them to form functional synapses with hair cells, and we ask whether the new synapses lead to recovery of hearing in animals with primary neuronal degeneration. Understanding the mechanisms underlying re-formation of neural connections to hair cells in the adult ear is important to any therapeutic approach to sensorineural hearing loss. The Specific Aims comprise 3 inter-related experiments to probe key variables likely to influence the success of hair cell reinnervation by transplanted neural progenitor cells. In Aim 1 the effect of the stage of differentiation of the stem cells on their capacity to reinnervate hair cells is assessed in a denervated in vitro organ of Corti. Assessments are based on immunohistochemistry: the morphology and number of synapses formed with inner and outer hair cells; and electrophysiology: firing of action potentials and excitatory postsynaptic currents by the neurons after stimulation of hair cells, and inhibition of this synaptic function by pharmacological blockers that inhibit functional activity of native cochlear afferent glutamatergic synapses. In Aim 2 we use cochlear explants de-afferented by a toxin to study the effect of modulating expression of molecules involved in synaptogenesis. Based on our evidence for their expression in the organ of Corti and their ability to increase synaptogenesis in other systems of neural regeneration, we have selected a group of molecules for study. Semaphorin 3a, repulsive guidance molecule, and semaphorin 4b, a negative regulator of axon guidance and synaptogenesis are inhibited, and neurotrophin-3 and cadherin 11, positive regulators of synaptogenesis are overexpressed. We also inhibit the expression of neuropilin1 and neogenin1, axon guidance receptors expressed in the growth cone of regenerating neurons. In Aim 3 we build on the in vitro results for in vivo transplantation into a de-afferented gerbil ear, in which the sensory cells are intact but spiral ganglion cells are destroyed. We test whether the reinnervation of hair cells that we have observed in this model results in improvements in functional measures of hearing, and we use a mouse model to study the effect of axonal guidance molecules on reinnervation and functional improvement.