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：
8900372
负责人：
Randolph S Ashton
金额：
$ 17.42万
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 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8900372
关键词：
Agonist Amyotrophic Lateral Sclerosis 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 Differentiation and Growth Disease Disease Progression Disease model Embryo Engineering Erinaceidae Fibroblast Growth Factor Fibroblast Growth Factor 8 Genetic Genotype Health Horns Human In Vitro Incubators Investigation Life Link Methodology Methods Microfabrication Microfluidics Microscope Modeling Motor Cortex Motor Neurons Neural tube Neuraxis Neurodegenerative Disorders Neuroepithelial Cells Neuronal Differentiation Neurons Paralysed Pathology Patients Pattern Pattern Formation Phase Pluripotent Stem Cells Population Population Heterogeneity Printing Probability Rodent Signal Transduction Spinal Spinal Cord Staging Stem cells Structure Surface Symptoms Techniques Testing Therapeutic Tissue Engineering Tissues Tretinoin Vertebral column bone morphogenetic protein 4 bone morphogenic protein density drug discovery experience high throughput screening hindbrain improved in vivo induced pluripotent stem cell mimetics morphogens nerve stem cell nervous system disorder progenitor relating to nervous system research study screening small molecule 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）：肌萎缩侧索硬化症 (ALS) 是一种迟发性神经退行性疾病，会导致大脑、后脑和脊髓中的运动神经元 (MN) 选择性丧失，导致出现症状后约 5 年内瘫痪和死亡发病。目前尚无治愈方法或方法来阻止疾病进展，但源自 ALS 患者的诱导多能干细胞 (iPSC) 具有巨大的潜力，有助于阐明该疾病的病因因素，并有助于筛选潜在的小分子疗法。然而，这些细胞的进展有限，因为在体外从大多数 ALS-iPSC 系/基因型中强烈引发 MN 特异性细胞凋亡的 ALS 标志性病理学仍然是一个挑战。我们假设可以通过设计体外疾病模型来克服这一挑战，该模型可以最佳地重现 MN 在体内经历的组织微环境。因此，我们提出了一种高通量组织工程方法，用于创建体外 ALS-iPSC 衍生的疾病模型，其中包含发现的细胞多样性、空间组织和区域化内源性脊髓组织内。一旦开发完成，我们的多功能高通量平台将有助于研究 ALS 的病理机制、筛选潜在的治疗方法，甚至可能有助于改善对早期 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横截面的体外模拟物，称为神经管微阵列（NTM）。在目标 1 中，我们将测试微接触印刷基材诱导人多能干细胞衍生的神经上皮细胞形成玫瑰花结结构的能力。在目标 2 中，我们将这些基底与显微镜载物台顶部的微流体平台集成，该平台既可以支持长期细胞培养过程中的高通量活细胞成像，又可以产生稳定的可溶性分子的反玫瑰花梯度。在目标 3 中，我们将测试 Sonic Hedgehog 和骨形态发生蛋白 4 的相反梯度是否可以诱导阵列花结内神经祖细胞的细胞多样性和空间组织，类似于在发育中的人类神经管中观察到的背腹模式，从而创建非关税措施。在 R33 阶段的目标 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 和视黄酸的组合是否可以将图案化的玫瑰花结区域化到脊髓的不同部分，如下所示Hox 转录因子的表达。最后，在 R33 阶段的目标 2 中，将从一组 ALS-iPSC 系中生成包含不同脊髓生态位模拟物的 NTM，与类似图案的星形胶质细胞共培养，并用于筛选 NTM 中是否唯一提供的模拟微环境平台可以强烈诱导 MN 特异性细胞凋亡。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The case for applying tissue engineering methodologies to instruct human organoid morphogenesis.

DOI：
10.1016/j.actbio.2017.03.023
发表时间：
2017-05
期刊：
Acta biomaterialia
影响因子：
9.7
作者：
Marti-Figueroa CR;Ashton RS
通讯作者：
Ashton RS

Deriving, regenerating, and engineering CNS tissues using human pluripotent stem cells.

使用人类多能干细胞衍生、再生和改造中枢神经系统组织。

DOI：
10.1016/j.copbio.2017.05.010
发表时间：
2017
期刊：
Current opinion in biotechnology
影响因子：
7.7
作者：
Lemke,KristenA;Aghayee,Alireza;Ashton,RandolphS
通讯作者：
Ashton,RandolphS

Tracking and Predicting Human Somatic Cell Reprogramming Using Nuclear Characteristics.

DOI：
10.1016/j.bpj.2019.10.014
发表时间：
2019-10
期刊：
Biophysical journal
影响因子：
3.4
作者：
Kaivalya Molugu;Ty Harkness;Jared Carlson-Stevermer;Ryan Prestil;Nicole J. Piscopo;Stephanie K Seymour;G. Knight;R. Ashton;Krishanu Saha
通讯作者：
Kaivalya Molugu;Ty Harkness;Jared Carlson-Stevermer;Ryan Prestil;Nicole J. Piscopo;Stephanie K Seymour;G. Knight;R. Ashton;Krishanu Saha

Micropatterned, clickable culture substrates enable in situ spatiotemporal control of human PSC-derived neural tissue morphology.

DOI：
10.1039/c4cc08665a
发表时间：
2015-03-28
期刊：
Chemical communications (Cambridge, England)
影响因子：
0
作者：
Knight GT;Sha J;Ashton RS
通讯作者：
Ashton RS

Single-injection ex ovo transplantation method for broad spinal cord engraftment of human pluripotent stem cell-derived motor neurons.

单次注射卵外移植方法，用于人类多能干细胞衍生的运动神经元的广泛脊髓移植。

DOI：
10.1016/j.jneumeth.2018.01.006
发表时间：
2018
期刊：
Journal of neuroscience methods
影响因子：
3
作者：
Estevez-Silva,MariaC;Sreeram,Akshitha;Cuskey,Stephanie;Fedorchak,Nikolai;Iyer,Nisha;Ashton,RandolphS
通讯作者：
Ashton,RandolphS