Cellular therapy for the inner ear

内耳细胞疗法

• 批准号: 8432824
• 负责人: Albert Edge
• 金额: $ 39.01万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-12 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432824
• 关键词: Action Potentials; Adult; Affect; Afferent Neurons; Animals; Axon; Biological Assay; Biological Models; Cell Differentiation process; Cell Maturation; Cell Therapy; Cells; Characteristics; Chemicals; Cochlea; Deafferentation procedure; Ear; Electrophysiology (science); Environment; Epithelium; Excision; Gene Expression; Gene Silencing; Genes; Gerbils; Glutamates; Goals; Grant; Growth; Growth Cones; Hair Cells; Health; Hearing; Immunohistochemistry; In Vitro; Knock-out; Knowledge; Labyrinth; Lead; Ligands; Measures; Mediating; Modeling; Morphology; Mus; Natural regeneration; Nerve Degeneration; Nerve Growth Factor Receptors; Nerve Regeneration; Nervous system structure; Neurons; Neurotrophin 3; Organ of Corti; Outer Hair Cells; Pattern; Process; Progress Reports; Protocols documentation; Quantitative Evaluations; Recovery; Recovery of Function; Semaphorin-3A; Semaphorins; Sensorineural Hearing Loss; Sensory; Signal Transduction; Staging; Stem cell transplant; Stem cells; Synapses; System; Testing; Therapeutic; Tissues; Toxin; Transgenic Organisms; Transplantation; Undifferentiated; Work; arm; axon growth; axon guidance; axonal guidance; base; cadherin-11; experience; functional improvement; functional restoration; ganglion cell; hearing impairment; human embryonic stem cell; in vivo; inhibitor/antagonist; mouse model; nerve stem cell; nerve supply; neural circuit; neurotrophic factor; overexpression; patch clamp; postsynaptic; progenitor; receptor; reconstitution; reinnervation; relating to nervous system; research study; spiral ganglion; stem cell differentiation; success; synaptic function; synaptogenesis

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.

抽象的 治疗神经损失的一种方法是移植外源祖细胞。我们 使用为内耳神经替代而开发的模型系统来研究以下要求： 重建神经回路：在当前的工作中，我们研究移植的神经元是否源自干细胞 可以在失去初级传入神经支配后恢复耳朵的功能。我们之前的工作表明 移植干细胞的神经元在化学脱除后可以重新支配柯蒂氏器的毛细胞 在体外外植体和体内沙鼠模型中的传入，表明该神经回路可以是 如果引入适当的祖细胞，则在成人神经系统中重建。在这个提案中我们使用 该系统研究供体细胞的哪些特征使它们能够与头发形成功能性突触 细胞，我们想知道新的突触是否会导致具有初级神经元的动物的听力恢复 退化。了解神经与毛细胞连接重建的机制 成人耳朵对于感音神经性听力损失的任何治疗方法都很重要。 具体目标包括 3 个相互关联的实验，以探讨可能影响 通过移植神经祖细胞成功实现毛细胞神经再支配。在目标 1 阶段的效果 在体外去神经状态下评估干细胞分化对其毛细胞重新神经支配能力的影响 柯蒂氏器。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组成；和电生理学：动作电位和兴奋性的发射 刺激毛细胞后神经元产生突触后电流，并通过抑制这种突触功能 抑制天然耳蜗传入谷氨酸突触功能活动的药理学阻断剂。在 目标 2 我们使用被毒素去传入的耳蜗外植体来研究调节表达的效果 参与突触发生的分子。基于我们在柯蒂氏器官中表达的证据和 他们有能力增加其他神经再生系统的突触发生，我们选择了一组 用于研究的分子。信号蛋白 3a（排斥性引导分子）和信号蛋白 4b（负调节分子） 轴突引导和突触发生受到抑制，神经营养素 3 和钙粘蛋白 11（正调节因子） 突触发生过度表达。我们还抑制神经毡蛋白1和neogenin1、轴突的表达 引导受体在再生神经元的生长锥中表达。在目标 3 中，我们建立在体外 体内移植到去传入沙鼠耳中的结果，其中感觉细胞完整但呈螺旋状 神经节细胞被破坏。我们测试了我们在此观察到的毛细胞的神经支配是否 模型导致听力功能测量的改善，我们使用小鼠模型来研究 轴突引导分子对神经支配和功能改善的影响。