Cellular therapy for the inner ear

描述（由申请人提供）：治疗神经丧失的一种方法是移植外源性祖细胞。我们使用一个模型系统开发的内耳神经替代重建神经回路的要求：在目前的工作中，我们研究是否移植的神经元来源于干细胞可以恢复功能的主要传入神经支配的损失后的耳朵。我们以前的工作已经表明，来自移植干细胞的神经元可以在化学去传入后重新支配Corti器官的毛细胞，在体外外植体和体内沙鼠模型中，这表明如果引入适当的祖细胞，这种神经回路可以在成人神经系统中重建。在这个提议中，我们使用这个系统来研究供体细胞的什么特征使它们能够与毛细胞形成功能性突触，并且我们询问新的突触是否会导致患有原发性神经元变性的动物的听力恢复。了解成年人耳毛细胞神经连接重建的机制对任何感音神经性听力损失的治疗方法都很重要。具体目标包括3个相互关联的实验，以探索可能影响移植神经祖细胞的毛细胞神经再支配成功的关键变量。在目的1中，在去神经支配的体外Corti器官中评估干细胞的分化阶段对其再神经支配毛细胞的能力的影响。评估基于免疫组织化学：与内毛细胞和外毛细胞形成的突触的形态和数量;以及电生理学：刺激毛细胞后神经元的动作电位和兴奋性突触后电流的激发，以及抑制天然耳蜗传入神经元突触功能活动的药理学阻断剂对该突触功能的抑制。在目的2中，我们使用耳蜗外植体去传入毒素研究参与突触发生的分子的调节表达的影响。基于我们在Corti器官中表达的证据以及它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。排斥性导向分子Semaphorin 3a和轴突导向和突触发生的负调节因子semaphorin 4 b被抑制，而突触发生的正调节因子神经营养素-3和cadherin 11过表达。我们还抑制了再生神经元生长锥中表达的轴突导向受体neuropilin 1和neogenin 1的表达。在目标3中，我们建立在体内移植到去传入沙鼠耳，其中感觉细胞是完整的，但螺旋神经节细胞被破坏的体外结果。我们测试我们在该模型中观察到的毛细胞的神经再支配是否会导致听力功能指标的改善，并且我们使用小鼠模型来研究轴突引导分子对神经再支配和功能改善的影响。这项资助中描述的工作将试图更好地了解干细胞衍生的神经元及其靶点之间的寻路和突触形成所涉及的基因。这一信息在神经元替代可能有益的任何治疗中都是有用的。由于耳蜗细胞的损失和感觉神经性听力损失之间存在明显的相关性，我们再生这些神经元及其与毛细胞的传入突触的能力的提高将直接导致听力损失的新治疗方法。

DESCRIPTION (provided by applicant): 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. The work described in this grant will attempt to better understand the genes involved in pathfinding and synapse formation between stem cell-derived neurons and their targets. This information is useful in any treatment in which replacement of neurons could be beneficial. Because of the clear correlation between loss of cochlear cells and sensorineural hearing loss, improvements in our ability to regenerate these neurons and their afferent synapse with hair cells will lead directly to new treatments for loss of hearing.