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

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种起病晚的神经退行性疾病，导致大脑、后脑和脊髓运动神经元(MN)选择性丧失，导致瘫痪和死亡，发病约5年。目前还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，有助于阐明疾病的病因，并有助于筛选潜在的小分子疗法。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/基因型中强有力地诱导ALS标志性的MN特异性细胞凋亡仍然是一个挑战。我们假设，这一挑战可以通过设计体外疾病模型来克服，该模型最佳地概括了MNS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来建立体外ALS-IPSC衍生的疾病模型，该模型包含了所发现的细胞多样性、空间组织和区域化。 在内源性脊髓组织内。一旦开发出来，我们多功能的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，甚至可能有助于提高对有早期ALS症状的患者的诊断。在R21阶段，我们将设计一个高通量的微阵列平台，用于产生胚胎神经管的体外模拟切片，称为神经管微阵列(NTM)。在目标1中，我们将测试微接触印刷底物诱导人类多能干细胞来源的神经上皮细胞形成玫瑰花环结构的能力。在目标2中，我们将这些底物与显微镜舞台顶端的微流控平台集成在一起，该平台既可以支持长期细胞培养期间的高通量活细胞成像，又可以产生稳定的跨玫瑰花环梯度的可溶性分子。在目标3中，我们将测试相反梯度的Sonic Hedgehog和骨形态发生蛋白-4是否可以诱导阵列花环内神经前体细胞的多样性和空间组织，类似于在发育中的人类神经管中观察到的背腹模式，从而产生NTMS。在R33阶段的目标1中，我们将测试Wnt信号激动剂CT99021、成纤维细胞生长因子-8、生长/分化因子-11和维甲酸的组合是否可以根据HOX转录因子的表达将花环区域化到脊髓的不同部分。最后，在R33阶段的目标2中，包含不同脊髓利基的模拟的NTM将从一组ALS-IPSC系中产生，与类似模式的星形胶质细胞共同培养，并用于筛选NTM平台中独特提供的模拟微环境是否能够有力地诱导MN特异性的细胞凋亡。

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.