High-Throughput Modeling of ALS Using iPSC-Derived Neural Tube Microarrays

使用 iPSC 衍生的神经管微阵列对 ALS 进行高通量建模

基本信息

批准号：
9548846
负责人：
Randolph S Ashton
金额：
$ 39.44万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9548846
关键词：
Agonist Amyotrophic Lateral Sclerosis Anatomy Anterior Apoptosis Astrocytes Biomimetics Brain Cell Culture Techniques Cell Line Cell physiology Cells Central Nervous System Diseases Cervical Cessation of life Chest Coculture Techniques Complex DNA Sequence Alteration Derivation procedure Diagnosis Disease Disease Progression Disease model Embryo Engineering Etiology Fibroblast Growth Factor Fibroblast Growth Factor 8 GDF11 gene Genetic Genotype Horns Human In Vitro Incubators Investigation Link Methodology Methods Microfabrication Microfluidics Microscope Modeling Morphology Motor Cortex Motor Neurons Neural tube Neuraxis Neurodegenerative Disorders Neuroepithelial Cells Neuronal Differentiation Neurons Paralysed Pathologic Pathology Patients Pattern Pattern Formation Phase Population Population Heterogeneity Probability Rodent SHH gene Spinal Spinal Cord Stem cells Structure Surface Symptoms Techniques Testing Therapeutic Tissue Engineering Tissues Tretinoin Vertebral column WNT Signaling Pathway bone morphogenetic protein 4 bone morphogenic protein density drug discovery experience experimental study high throughput screening hindbrain human pluripotent stem cell improved in vivo induced pluripotent stem cell mimetics morphogens nerve stem cell nervous system disorder progenitor public health relevance relating to nervous system screening small molecule therapeutics tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): Amyotrophic Lateral Sclerosis (ALS) is a late-onset neurodegenerative disease that causes selective loss of motor neurons (MNs) in the brain, hindbrain, and spinal cord leading to paralysis and death within ~5 years of symptomatic onset. There is no cure or means to halt disease progression, but induced pluripotent stem cells (iPSC) derived from ALS patients have enormous potential to aid elucidation of the disease's etiological factors and facilitate screening for potential small molecule therapeutics. However, progress with these cells is limited because it remains a challenge to robustly elicit ALS' hallmark pathology of MN-specific apoptosis from the majority of ALS-iPSC lines/genotypes in vitro. We hypothesize that this challenge can be overcome by engineering in vitro disease models that optimally recapitulate the tissue microenvironments experienced by MNs in vivo. Thus, we propose a high-throughput tissue engineering approach for creating in vitro ALS-iPSC-derived disease models that contain the cellular diversity, spatial organization, and regionalization found within endogenous spinal cord tissues. Once developed, our versatile high-throughput platform would facilitate investigating ALS' pathological mechanisms, screening for potential therapeutics, and even possibly aid in improving the diagnosis of patients with early ALS symptoms. In the R21 phase, we will engineer a high-throughput microarray platform for generating in vitro mimics of transverse sections of the embryonic neural tube, called Neural Tube Microarrays (NTM). In Aim 1, we will test the ability of micro-contact printed substrates to induced formation of rosette structures from human pluripotent stem cell-derived neuroepithelial cells. In Aim 2, we will integrate these substrates with a microscope stage-top microfluidic platform that can both support high-throughput live-cell imagining during long-term cell culture and produce stable trans-rosette gradients of soluble molecules. In Aim 3, we will test whether opposing gradients of Sonic hedgehog and Bone morphogenic protein-4 can induce the cellular diversity and spatial organization of neural progenitors within arrayed rosettes that is analogous to the dorsoventral patterning observed in the developing human neural tube, thus creating NTMs. In Aim 1 of the R33 phase, we will test whether combinations of Wnt signaling agonist CT99021, Fibroblast growth factor-8, Growth/differentiation factor-11, and Retinoic Acid can regionalize the patterned rosettes to diverse sections of the spinal cord as indicated by expression of Hox transcription factors. Finally in Aim 2 of the R33 phase, NTMs containing mimics of diverse spinal cord niches will be generated from a panel of ALS-iPSC lines, co- cultured with similarly patterned astrocytes, and used to screen whether the mimetic microenvironments uniquely provided in the NTM platform can robustly induce MN-specific apoptosis.

描述（由申请人提供）：肌萎缩性侧性硬化症（ALS）是一种晚期神经退行性疾病，会导致大脑，后脑和脊髓中运动神经元（MNS）的选择性丧失，导致症状发作5年内麻痹和死亡。没有阻止疾病进展的治疗或手段，但是诱导的多能干细胞（IPSC）衍生自ALS患者具有巨大的潜力，可以帮助阐明该疾病的病因学因素，并促进筛查潜在的小分子疗法。但是，这些细胞的进展是有限的，因为在体外大多数ALS-IPSC系/基因型中，强有力地引起ALS的MN特异性细胞凋亡的标志性病理仍然是一个挑战。我们假设可以通过工程化体外疾病模型来克服这一挑战，这些模型可以最佳地概括MNS In Vivo经历的组织微环境。因此，我们提出了一种高通量组织工程方法，用于创建包含细胞多样性，空间组织和区域化的体外ALS-IPSC衍生疾病模型在内源性脊髓组织中。一旦开发，我们多功能的高通量平台将有助于研究ALS的病理机制，筛查潜在的治疗疗法，甚至可能有助于改善早期ALS症状患者的诊断。在R21阶段，我们将设计一个高通量微阵列平台，用于生成胚胎神经管的横向截面的体外模仿，称为神经管微阵列（NTM）。在AIM 1中，我们将测试微接触印刷底物从人多能干细胞衍生的神经上皮细胞中诱导玫瑰花结构形成的能力。在AIM 2中，我们将将这些底物与显微镜阶段的微流体平台整合在一起，该平台都可以支持长期细胞培养过程中的高通量活细胞想象，并产生可溶性分子的稳定的反式镜梯度。在AIM 3中，我们将测试Sonic刺猬和骨形态形态蛋白-4的相对梯度是否可以诱导阵列的玫瑰花结构内神经祖细胞的细胞多样性和空间组织，该玫瑰花结中与在发育中的人神经管中观察到的背腹图相似，从而产生NTMS。在R33阶段的AIM 1中，我们将测试Wnt信号传导激动剂CT99021，成纤维细胞生长因子-8，生长/分化因子-11和视黄酸是否可以将图案化的玫瑰花质分化到脊髓的各个部分，如hox转录因子的表达所示。最后，在R33阶段的AIM 2中，将通过一系列ALS-IPSC系列产生包含不同脊髓壁ni的模仿的NTM，并与类似图案的星形胶质细胞共同培养，并用于筛选模拟微型微环境是否独特地提供在NTM平台中提供的Mn-apoptosis。