Cellular therapy for the inner ear

内耳细胞疗法

• 批准号: 7901281
• 负责人: Albert Edge
• 金额: $ 9.71万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-14 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7901281
• 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; Ear; Electrophysiology (science); Environment; Epithelium; Excision; Gene Expression; Gene Silencing; Genes; Gerbils; Glutamates; Goals; Grant; Growth; Growth Cones; Hair Cells; 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; Public Health; Quantitative Evaluations; Recovery; Recovery of Function; Semaphorin-3A; Sensorineural Hearing Loss; Sensory; Signal Transduction; Staging; Stem cell transplant; Stem cells; Synapses; System; Testing; Therapeutic; Tissues; Toxin; Transgenic Organisms; Transplantation; Undifferentiated; Upper arm; Work; 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

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.

描述（由申请人提供）：治疗神经丧失的一种方法是外源祖细胞的移植。我们使用用于内耳神经替代的模型系统来研究重建神经回路的需求：在当前的工作中，我们研究了在失去一级传入神经支配后，从干细胞中得出的移植神经元是否可以恢复到耳朵的功能。我们先前的工作表明，来自移植的干细胞的神经元可以在化学脱发后和体外epplatiant和体内gerbil模型中重新连接皮质器官的毛细胞。在此提案中，我们使用该系统来研究供体细胞的哪些特征使它们可以与毛细胞形成功能突触，我们询问新的突触是否导致原发性神经元变性的动物的听力恢复。了解神经连接与成年耳朵中毛细胞的重新形成的机制对于任何治疗性听力损失的方法都很重要。该特定目的包括3个相互关联的实验，以探测可能影响毛细胞通过移植神经祖细胞加重毛细胞的成功的关键变量。在AIM 1中，在干燥的Corti体外器官中评估了干细胞分化阶段对重新染色细胞能力的影响。评估基于免疫组织化学：由内部和外毛细胞形成的突触的形态和突触数量；和电生理学：刺激毛细胞后神经元的动作电位和兴奋性突触后电流的发射，并通过药理学阻滞剂抑制这种突触功能，从而抑制天然耳蜗传入谷氨酸胶质氨基氨基氨基氨基氨基氨基甲基的功能活性。在AIM 2中，我们使用毒素去征服的耳蜗外植体来研究调节突触发生的分子表达的效果。基于我们证明它们在Corti器官中表达的证据及其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。信号蛋白3a，排斥性引导分子和Semaphorin 4b（轴突引导和突触发生的负调节剂）被抑制，神经营养蛋白3和钙粘蛋白11，突触发生的阳性调节剂过表达。我们还抑制了神经蛋白1和Neogenin1的表达，在再生神经元的生长锥中表达的轴突引导受体。在AIM 3中，我们建立在体外的体外结果基础上，用于体内移植到脱落的Gerbil耳朵中，其中感觉细胞完好无损，但螺旋神经节细胞被破坏。我们测试在此模型中观察到的毛细胞的重新连接是否会改善听力度量，并使用小鼠模型研究轴突引导分子对重新连接和功能改善的影响。该赠款中描述的工作将试图更好地理解干细胞衍生的神经元与其靶标之间的探路和突触形成的基因。该信息在任何替代神经元可能是有益的治疗方法中都是有用的。由于耳蜗细胞的丧失与感官性听力丧失之间存在明显的相关性，因此我们再生这些神经元的能力以及它们与毛细胞的传入突触的改善将直接导致新的治疗方法，以造成听力丧失。