iPSC for Neurodegenerative Diseases

iPSC 治疗神经退行性疾病

• 批准号: 8737569
• 负责人: Weiming Xia
• 金额: --
• 依托单位: EDITH NOURSE ROGERS MEMORIAL VETERANS HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-10-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8737569
• 关键词: Address; Alzheimer' s Disease; Amyloid Proteins; Animals; Automobile Driving; Autopsy; Biochemical; Biological Assay; Biological Markers; Blood Cells; Blood donor; Brain; Brain Pathology; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell model; Cells; Cerebrospinal Fluid; Characteristics; Clinical Trials; Collection; Consent; Cultured Cells; Data; Diagnosis; Disease; Disease Progression; Elderly; Enzyme-Linked Immunosorbent Assay; Evaluation; Generations; Goals; Human; Human Cell Line; In Vitro; Individual; Inpatients; Institutional Review Boards; Laboratories; Life; Life Expectancy; Mammalian Cell; Measures; Metabolism; Methods; Modeling; Neuraxis; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Participant; Pathogenesis; Pathologic; Pathology; Patient Selection; Patients; Pharmaceutical Preparations; Pilot Projects; Positioning Attribute; Process; Property; Protein Isoforms; Proteins; Protocols documentation; Proxy; Publishing; Research; Resources; Role; Sampling; Senile Plaques; Techniques; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Tissues; Treatment Efficacy; Ursidae Family; Variant; Ventricular; Veterans; Work; amyloid imaging; amyloid precursor protein processing; brain cell; brain tissue; cell preparation; clinical Diagnosis; experience; hospice environment; human tissue; in vivo Model; induced pluripotent stem cell; inhibitor/antagonist; kinase inhibitor; monomer; patient population; presenilin; protein oligomer; public health relevance; response; secretase; tau Proteins; tau phosphorylation; tau-1; therapeutic development

DESCRIPTION (provided by applicant): We propose to establish cellular biomarkers in human neurons differentiated from induced pluripotent stem cells (iPSC) of Alzheimer's disease (AD) patient origin to predict specific drug responses. These human cell lines will be used as an ex vivo cellular model to characterize key proteins driving AD pathogenesis, amyloid ¿ protein (A¿) and Tau, and to correlate to postmortem brain pathology from the same donor. We will also collect postmortem brain and CSF samples from the same donor. We will quantify A¿ monomers and oligomers, pTau and Tau levels and validate intrinsic properties of these biomarkers in cells and brain tissues. We will test the hypotheses that neurons having higher A¿ or pTau proteins respond to therapeutic agents differently. By testing this hypothesis, we will address a critical question in AD therapeutic development, whether characteristic A¿/Tau biomarkers can be used to predict therapeutic efficacy in sporadic AD (SAD) patients. Currently, biomarkers consisting of a combination of brain amyloid imaging and cerebrospinal fluid (CSF) A¿ and pTau/Tau proteins have been developed and are used in patient selection for clinical trials. There is no in vitro method of testing AD biomarkers to predict individual's brain pathology and CSF profile without performing brain amyloid imaging and CSF collection. We are uniquely positioned to carry out these studies. We have almost two-decades of experience in cellular modeling for AD research and a well-characterized patient population contributing blood cells and postmortem tissue for ex vivo modeling and in vivo confirmation. Work in my laboratory has used the mammalian cell culture model to study the functional role of presenilin in amyloid precursor protein (APP) processing and A¿ generation. Using extensive biochemical assays and highly sensitive ELISA, we have quantified various isoforms of A¿ and Tau proteins in cultured cells, animals, and human tissues. In bringing this expertise to bear on human iPSC, we will directly assess the A¿ and Tau protein signatures in AD-derived human neurons and use this signature to predict the responses to therapeutics. Our Institutional Review Board (IRB) application has been approved, and 29 subjects have been consented for participation. All participants have agreed, by proxy, to brain autopsy to confirm the diagnosis of AD. We will derive iPSC from patients living at an inpatient AD hospice unit, where life expectancy is 6 months or less. Blood cells will be used for iPSC preparation. Ventricular CSF and postmortem brain tissues will be collected from the same individuals who have donated blood cells for iPSC preparation. Clinical diagnosis will be confirmed by detailed neuropathological evaluation and characterization. By creating iPSC originating from AD patients, this work will generate iPSC-derived human neuronal cells that carry different signatures of A¿/Tau proteins. To avoid the variation introduced in the course of iPSC conversion and differentiation, we will compare the changes of A¿/Tau in same neuronal cell lines with or without pharmacologic treatment and predict responses to different therapeutic agents. Specifically, we will achieve the following aims. Aim 1. To determine whether the A¿/Tau protein signature in iPSC-derived human neurons is consistent with that in postmortem brain tissue from the same donor. Established methods will be applied to convert blood cells to iPSC, followed by differentiation into neuronal cells. We will quantify the A¿ monomer and oligomer proteins, total and pTau proteins. We will determine whether levels of A¿ and Tau/pTau in cultured neurons are inherently reflected in postmortem CSF and brain tissue from the original cell donors, validating cellular biomarkers for therapeutic tests. Aim 2. o determine whether levels of A¿ and pTau predict cellular responses to secretase or kinase inhibitors. Human neurons with different levels of A¿/Tau will be treated with a ¿-secretase inhibitor, and the effect on A¿42 and A¿ oligomers will be quantified. GSK3¿ inhibitor will be used to reduce Tau phosphorylation. We will determine whether high levels of A¿ or pTau in cultured neurons predict better responses to secretase and kinase inhibitors, respectively.

描述(由申请人提供)： 我们建议在阿尔茨海默病(AD)患者来源的诱导多能干细胞(IPSC)分化的人类神经元中建立细胞生物标记物，以预测特定的药物反应。这些人类细胞系将被用作体外细胞模型，以表征推动AD发病的关键蛋白--淀粉样蛋白(A？)和Tau，并与同一供体的死后脑病理相关。我们还将收集同一捐赠者的死后脑和脑脊液样本。我们将量化A？单体和低聚物、ptau和tau水平，并验证这些生物标记物在细胞和脑组织中的内在属性。我们将测试这些假设，即具有较高A‘或ptau蛋白的神经元对治疗剂的反应是不同的。通过检验这一假设，我们将解决AD治疗发展中的一个关键问题，即特征A？/Tau生物标志物是否可以用于预测散发性AD(SAD)患者的疗效。目前，由脑淀粉样蛋白成像和脑脊液(CSF)A？和ptau/tau蛋白组成的生物标记物已经被开发出来，并用于临床试验的患者选择。如果不进行脑淀粉样蛋白成像和脑脊液收集，就没有体外方法来测试AD生物标志物来预测个体的脑病理和脑脊液特征。我们处于开展这些研究的独特地位。我们在AD研究的细胞建模方面拥有近20年的经验，并拥有一支为体外建模和体内确认贡献血细胞和死后组织的具有良好特征的患者群体。我在实验室的工作使用哺乳动物细胞培养模型来研究早老素在淀粉样前体蛋白(APP)加工和A？代中的功能作用。利用广泛的生化分析和高灵敏度的ELISA法，我们对培养的细胞、动物和人类组织中的A和Tau蛋白的各种亚型进行了定量。在将这一专业知识应用于人类IPSC时，我们将直接评估AD来源的人类神经元中的A？和Tau蛋白签名，并使用该签名来预测对治疗的反应。我们的院校评审委员会(IRB)的申请已获批准，已有29名受试者获准参加。所有参与者都同意通过代理进行脑部尸检，以确认AD的诊断。我们将从居住在住院AD临终关怀病房的患者那里获得IPSC，那里的预期寿命不超过6个月。血细胞会 用于IPSC的制备。脑室脑脊液和死后脑组织将从为IPSC准备捐献血细胞的同一人身上收集。临床诊断将通过详细的神经病理评估和特征确定。通过创造源自AD患者的IPSC，这项工作将产生IPSC来源的人类神经细胞，这些细胞携带不同签名的A？/Tau蛋白。以避免在课程中引入的变化 在IPSC转化和分化方面，我们将比较相同神经细胞系在药物治疗前后A？/Tau的变化，并预测不同治疗药物的反应。具体来说，我们将实现以下目标。目的1.确定IPSC来源的人类神经元中的A？/Tau蛋白特征是否与同一供体的死后脑组织中的一致。已建立的方法将用于将血细胞转化为IPSC，然后分化为神经细胞。我们将对A？单体和寡聚蛋白、总蛋白和ptau蛋白进行定量。我们将确定培养神经元中的A？和Tau/ptau水平是否固有地反映在原始细胞捐赠者的死后脑脊液和脑组织中，从而验证用于治疗测试的细胞生物标记物。目的2.确定Aβ和ptau水平是否能预测细胞对分泌酶或激酶抑制剂的反应。具有不同水平的A？/Tau的人神经元将被？分泌酶抑制剂处理，其对A？42和A？寡聚体的影响将被量化。GSK3？抑制剂将被用来减少Tau的磷酸化。我们将确定培养神经元中高水平的A‘或ptau是否分别预示着对分泌酶和激酶抑制剂的更好反应。