Engineered Stem Cells for Inner Ear Pharmacotherapy

用于内耳药物治疗的工程干细胞

• 批准号: 8048931
• 负责人: LARRY F HOFFMAN
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-13 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8048931
• 关键词: Adipose tissue; Adult; Afferent Neurons; Aminoglycosides; Animal Model; Anterior semicircular canal (body structure); Auditory; Auditory Brainstem Responses; Auditory system; Autologous Transplantation; Biological Models; Brain-Derived Neurotrophic Factor; Cell Line; Cell Survival; Cell Therapy; Cells; Chinchilla (genus); Chronic; Complement; Culture Media; Cytoprotection; Development; Effectiveness; Electrophysiology (science); Engineering; Engraftment; Environment; Enzyme-Linked Immunosorbent Assay; Epithelium; Evaluation; Fatty acid glycerol esters; Green Fluorescent Proteins; Hair Cells; Harvest; Histologic; Human; Immune; Implant; In Vitro; Infiltration; Laboratories; Labyrinth; Lesion; Life; Measurable; Methodology; Modeling; Monitor; Neurons; Neurorehabilitation; Outcome; Oxidative Stress; Pathologic; Perilymph; Peripheral; Pharmacotherapy; Preparation; Protein Secretion; Rehabilitation therapy; Research; Research Proposals; Sampling; Secondary to; Sensory; Shipping; Ships; Source; Stem cells; Subfamily lentivirinae; System; Temporal bone structure; Testing; Time; Tissues; Transfection; Transgenes; Transplantation; Vestibular ganglion; Viral; Work; aggressive therapy; base; chemotherapy; density; efficacy testing; enhanced green fluorescent protein; in vivo; member; neuroprotection; neurotrophic factor; ototoxicity; paracrine; protein expression; regenerative; research and development; research study; spiral ganglion; stable cell line; stem cell therapy; transgene expression

DESCRIPTION (provided by applicant): This application represents an exploratory and development research proposal to develop new lines of engineered adult adipose-derived stem cells ASCs, harvested from chinchillas (resulting in chASCs), that can be used for autologous transplantation for the delivery of trophins directly to the inner ear. There is considerable body of evidence demonstrating the limited intrinsic rehabilitative capabilities of the adult inner ear, yet there are numerous conditions in which such capabilities would be extremely valuable. These include cases of otoprotection, in which other systemic life-threatening conditions warrant the use of aggressive therapies that are also ototoxic. Still other conditions abound that would benefit from regenerative capabilities of Scarpa's or spiral ganglion neurons. At the same time, it is clear that inner ear protection and neurorehabilitation is enhanced through the application of neurotrophins, of which brain-derived neurotrophic factor (BDNF) is an important member. Therefore, the development of a strategy to provide BDNF for prolonged periods would be of significant benefit in providing otoprotection and stimulating inner ear rehabilitation. The present application presents a research plan in which we will test the efficacy of using chASCs as a cell-based delivery system to provide BDNF to the inner ear. This work takes advantage of a chronically-prepared chinchilla model in which direct access to the perilymphatic space has been developed. Experiments will be conducted to harvest, characterize, and engineer chASCs via lentiviral transfection to express enhanced green fluorescent protein (GFP) and GFP-tagged BDNF. Strategies will be implemented to produce stable cell lines. These two cell lines will then be transplanted directly to the perilymphatic space in chinchillas. The use of these lines enable the capability to monitor the intrinsic capabilities of transplanted chASCs to secrete BDNF, as well as the enhanced secretion of BDNF resulting from transgene expression. This paracrine function of chASCs will be evaluated through ELISA methodologies. The GFP expression in both transgene systems will enable us to critically evaluate the integration and survival of these cells in vivo through histologic analysis of the recipient temporal bones. Inner ear function of both the peripheral auditory and vestibular systems will be assessed by recording the auditory brainstem response and through single neuron electrophysiology to identify the potential influence of the transplantation on inner ear function. In summary, these experiments represent a direct test of paracrine function of adult engineered stem cells in the inner ear. While the model system incorporates the secretion of neurotrophin, it will serve as a proof-of- concept of a myriad of other otoactive agents. The successful outcome of these experiments can be immediately tested in a model of inner ear neurorehabilitation in the chinchilla developed in the laboratory of the PI. PUBLIC HEALTH RELEVANCE: The research to be conducted under this exploratory and development proposal will test the efficacy of utilizing adult adipose-derived stem cells as a means to deliver trophin therapies directly to the inner ear. We will test the intrinsic paracrine capabilities of these cells as a source of brain-derived neurotrophic factor (BDNF), as well as BDNF resulting from cells that have been engineered via viral transfection. Such a cell-based therapy system takes advantage of the intrinsic and engineered capabilities of stem cells, and if successful will be extremely advantageous in cases requiring neurorehabilitation or neuroprotection of the inner ear.

描述（由申请人提供）：本申请代表了一项探索性和开发性研究提案，旨在开发新的工程化成人脂肪源性干细胞ASC系，从龙猫中收获（产生chASC），可用于自体移植，将营养因子直接递送至内耳。有大量证据表明成人内耳的内在康复能力有限，但在许多情况下，这种能力将极其有价值。这些包括耳保护病例，其中其他全身性危及生命的疾病需要使用也具有耳毒性的积极治疗。还有很多其他的条件，将受益于斯卡帕或螺旋神经节神经元的再生能力。同时，很明显，内耳保护和神经康复是通过应用神经营养素，其中脑源性神经营养因子（BDNF）是一个重要的成员。因此，开发一种策略，以提供BDNF的延长期将是显着的好处，在提供耳保护和刺激内耳康复。本申请提出了一项研究计划，其中我们将测试使用chASC作为基于细胞的递送系统向内耳提供BDNF的功效。这项工作利用了一个长期准备的龙猫模型，其中直接进入外淋巴空间已经开发。将进行实验以通过慢病毒转染收获、表征和工程化chASC以表达增强的绿色荧光蛋白（GFP）和GFP标记的BDNF。将实施策略以产生稳定的细胞系。然后将这两种细胞系直接移植到龙猫的外淋巴间隙。这些细胞系的使用使得能够监测移植的chASC分泌BDNF的内在能力，以及由转基因表达引起的BDNF分泌增强。将通过ELISA方法评价chASC的这种旁分泌功能。两种转基因系统中的GFP表达将使我们能够通过受体颞骨的组织学分析来严格评估这些细胞在体内的整合和存活。通过记录听觉脑干反应和单神经元电生理学来评估外周听觉和前庭系统的内耳功能，以确定移植对内耳功能的潜在影响。总之，这些实验代表了内耳中成体工程干细胞的旁分泌功能的直接测试。虽然模型系统包含神经营养因子的分泌，但它将作为无数其他耳活性剂的概念验证。这些实验的成功结果可以立即在PI实验室开发的栗鼠内耳神经康复模型中进行测试。 公共卫生关系：根据这一探索性和开发提案进行的研究将测试利用成人脂肪源性干细胞作为直接向内耳提供营养因子治疗的手段的功效。我们将测试这些细胞作为脑源性神经营养因子（BDNF）来源的内在旁分泌能力，以及通过病毒转染工程化细胞产生的BDNF。这种基于细胞的治疗系统利用了干细胞的内在和工程化能力，并且如果成功，在需要内耳的神经康复或神经保护的情况下将是非常有利的。