Cellular therapy for the inner ear

内耳细胞疗法

• 批准号: 8233927
• 负责人: Albert Edge
• 金额: $ 41.06万
• 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/8233927
• 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 治疗神经丧失的一种方法是外源祖细胞的移植。我们 使用为内耳中神经替换开发的模型系统，调查 重建神经回路：在当前的工作中，我们研究是否衍生自干细胞的移植神经元 一级传入神经损失后，可以将功能恢复到耳朵。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后加剧皮质器官的毛细胞 在体外外植体和体内香水模型中的传入，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构。在这个建议中，我们使用 该系统研究供体细胞的哪些特征使它们可以与头发形成功能突触 细胞，我们询问新的突触是否导致原发性神经元动物的听力恢复 退化。了解神经连接与毛细胞的重新形成的机制 成年耳朵对于任何治疗性听力损失的治疗方法都很重要。 具体目的包括3个相互关联的实验，以探测可能影响的关键变量 通过移植的神经祖细胞重新开始毛细胞的成功。在AIM 1中 干细胞在重新染色毛细胞的能力上的分化在体外被剥离。 Corti器官。评估基于免疫组织化学：突触的形态和数量 由内毛细胞形成；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的突触后电流，并抑制这种突触功能 药理阻滞剂抑制天然人工耳蜗传入的谷氨酸能突触的功能活性。在 目的2我们使用毒素去征服的人工耳蜗外植体研究调节表达的效果 参与突触发生的分子。基于我们的证据表明它们在Corti器官中的表达和 它们增加了其他神经再生系统中突触发生的能力，我们选择了一组 研究分子。 Semaphorin 3a，排斥性引导分子和Semaphorin 4B，一个负调节剂 轴突引导和突触发生被抑制，神经营养蛋白3和钙粘蛋白11，阳性调节剂 突触发生过表达。我们还抑制Neuropilin1和neogenin1的表达 在再生神经元的生长锥中表达的引导受体。在AIM 3中，我们建立在体外 体内移植到脱落的沙鼠耳朵中的结果，其中感觉细胞完整但螺旋形 神经节细胞被破坏。我们测试我们在此观察到的毛细胞是否重新连接 模型导致听力功能度量的改善，我们使用小鼠模型来研究 轴突引导分子对重新支配和功能改善的影响。