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

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种迟发性神经退行性疾病，可导致大脑、后脑和脊髓的运动神经元（MNs）选择性丧失，在症状发作后约5年内导致瘫痪和死亡。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，可以帮助阐明该疾病的病因，并促进筛选潜在的小分子治疗方法。然而，这些细胞的进展是有限的，因为它仍然是一个挑战，在体外从大多数ALS- ipsc系/基因型中强有力地引发ALS的mn特异性凋亡的标志病理学。我们假设这一挑战可以通过设计体外疾病模型来克服，这些模型可以最佳地概括MNs在体内所经历的组织微环境。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，该模型包含所发现的细胞多样性、空间组织和区域化

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.