Cellular therapy for the inner ear

描述(申请人提供)：治疗神经损伤的一种方法是移植外源性祖细胞。我们使用开发的内耳神经替代模型系统来研究重建神经回路的要求：在当前的工作中，我们研究了干细胞来源的移植神经元是否可以在失去初级传入神经后恢复耳朵的功能。我们以前的工作表明，在化学去传入后，来自移植干细胞的神经元可以在体外和体内沙土鼠模型中重新支配Corti器官的毛细胞，这表明如果引入适当的前体细胞，这种神经回路可以在成年神经系统中重建。在这个方案中，我们使用这个系统来研究供体细胞的哪些特征允许它们与毛细胞形成功能性突触，并询问新的突触是否会导致患有初级神经元变性的动物的听力恢复。了解成人耳朵毛细胞神经连接重建的机制对任何治疗感音神经性听力损失的方法都很重要。具体目标包括3个相互关联的实验，以探索可能影响神经前体细胞移植后毛细胞再神经再生成功的关键变量。在目的1中，在去神经的Corti体外器官中评估干细胞分化阶段对其重新支配毛细胞能力的影响。评估基于免疫组织化学：与内外毛细胞形成的突触的形态和数量；电生理学：刺激毛细胞后神经元激发动作电位和兴奋性突触后电流，以及药物阻滞剂抑制这一突触功能，从而抑制自然耳蜗区传入谷氨酸突触的功能活动。在目标2中，我们使用毒素去传入的耳蜗外植体来研究参与突触发生的分子的表达调控作用。根据我们的证据证明它们在Corti器官中的表达，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。排斥导向分子信号素3a和轴突引导和突触发生的负性调节因子信号素4b被抑制，突触发生的正调控因子神经营养素-3和钙粘附素11过度表达。我们还抑制了再生神经元生长锥中表达的轴突引导受体--神经粘连蛋白1和新生蛋白1的表达。在目标3中，我们建立了体内移植到去传入沙土鼠耳中的体外结果，在这种情况下，感觉细胞完好，但螺旋神经节细胞被破坏。我们测试了我们在这个模型中观察到的毛细胞的再神经支配是否会导致听力功能指标的改善，并使用小鼠模型来研究轴突引导分子对再神经支配和功能改善的影响。这项拨款中描述的工作将试图更好地了解干细胞来源的神经元与其靶点之间的寻路和突触形成所涉及的基因。这些信息在任何替换神经元可能是有益的治疗中都是有用的。由于耳蜗细胞的丧失与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.