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

• 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
• 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

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（由申请方提供）：肌萎缩侧索硬化症（ALS）是一种迟发性神经退行性疾病，可导致大脑、后脑和脊髓中运动神经元（MN）的选择性丧失，导致症状发作后约5年内瘫痪和死亡。没有治愈或阻止疾病进展的方法，但来自ALS患者的诱导多能干细胞（iPSC）具有巨大的潜力，可以帮助阐明疾病的病因，并有助于筛选潜在的小分子治疗剂。然而，这些细胞的进展是有限的，因为在体外从大多数ALS-iPSC系/基因型中稳健地引发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，这一挑战可以克服工程在体外疾病模型，最佳概括的MN在体内经历的组织微环境。因此，我们提出了一种高通量的组织工程方法，用于创建体外ALS-iPSC衍生的疾病模型，该模型包含所发现的细胞多样性，空间组织和区域化。 在内源性脊髓组织中。一旦开发出来，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切面的体外模拟物，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底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，我们将这些基板与显微镜载物台顶部的微流体平台相结合，该微流体平台既可以在长期细胞培养期间支持高通量活细胞成像，又可以产生可溶性分子的稳定trans-rosette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式神经元内神经祖细胞的细胞多样性和空间组织，这类似于在发育中的人类神经管中观察到的背腹图案，从而产生NTM。在R33阶段的目标1中，我们将测试Wnt信号传导激动剂CT 99021、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roximal区域化到脊髓的不同部分，如Hox转录因子的表达所示。最后，在R33阶段的目标2中，将从一组ALS-iPSC系产生含有不同脊髓小生境的模拟物的NTM，与类似图案化的星形胶质细胞共培养，并用于筛选NTM平台中唯一提供的模拟微环境是否可以稳健地诱导MN特异性细胞凋亡。

项目成果

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

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)
• 作者: Knight GT;Sha J;Ashton RS
• 通讯作者: Ashton RS

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

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