Modeling spinal cord axis patterning with human pluripotent stem cells

描述(由申请人提供)：研究人员越来越意识到，中枢神经系统(CNS)的异质性在胚胎发育期间编码，并在成年后保留。一类表现出显著异质性的细胞是脊髓运动神经元，它根据发育过程中收到的提示来支配特定的肌肉靶点，在肌萎缩侧索硬化症(ALS)和平滑肌萎缩(SMA)等疾病中严重受损。虽然人类多能干细胞分化为脊髓运动神经元可以为研究和/或治疗这些疾病提供极好的资源，但目前的分化方法无法概括实现特定位置认同所需的发育线索。此外，尽管像肌萎缩侧索硬化症这样的疾病是非细胞自主的，涉及复杂的细胞间相互作用，但脊髓运动神经元的单独分化可能不能恰当地代表疾病的进展和表型。因此，这一建议广泛地寻求区分hPSCs和具有明确位置同一性的脊髓运动神经元，无论是作为一个孤立的群体还是在一个有组织的多细胞组织结构中。本方案的目的1主要是通过与WNT3a、成纤维细胞生长因子8、维甲酸和生长分化因子11等构图因子的组合，将hPSCs分化为沿脊髓吻侧/尾轴方向具有特定位置的神经前体细胞。目的2将结合微接触打印、表面化学和重组蛋白质工程来创建确定大小和形状的神经组织结构。Aim 3将利用两个微流控设备来优化脊髓运动神经元的分化。第一个微流体设备将采用主动梯度发生器，该发生器将在每个细胞的基础上识别实现高产量运动神经元分化所必需的声波刺猬浓度。第二个微流控装置将使用被动梯度发生器，将工程化的神经组织结构朝着神经管的腹侧部分形成图案。成功完成目标3的前半部分将产生具有特定位置标识的脊髓运动神经元，这将在再生治疗中具有潜在应用，而成功完成目标3的后半部分将产生驻留在腹侧组织结构中的脊髓运动神经元，该结构可用于高通量筛选治疗ALS的药物。

DESCRIPTION (provided by applicant): Researchers are becoming increasingly aware that heterogeneity in the central nervous system (CNS) is encoded during embryonic development and retained during adulthood. One class of cells exhibiting substantial heterogeneity is spinal motor neurons, which innervate specific muscle targets based on cues received during development and are critically impaired in diseases such as Amyotrophic Lateral Sclerosis (ALS) and smooth muscle atrophy (SMA). While human pluripotent stem cell (hPSCs) differentiated to spinal motor neurons could provide an excellent resource for studying and/or treating these diseases, current differentiation methods are unable to recapitulate the developmental cues necessary to achieve defined positional identity. Furthermore, whereas diseases like ALS are non-cell-autonomous and involve complex cell- cell interactions, isolated differentiation of spinal motor neurons may not appropriately represent the disease progression and phenotype in a dish. As such, this proposal broadly seeks to differentiate hPSCs to spinal motor neurons possessing defined positional identity, both as an isolated population and within an organized multicellular tissue structure. Aim 1 of this proposal focuses on differentiation of hPSCs to neural progenitors possessing a defined position along the rostral/caudal spinal cord axis by combinatorial treatment with patterning factors such as Wnt3a, fibroblast growth factor 8, retinoic acid, and growth differentiation factor 11. Aim 2 will use a combination of micro-contact printing, surface chemistry, and recombinant protein engineering to create neural tissue structures of defined size and shape. Aim 3 will utilize two microfluidic devices to optimize spina motor neuron differentiation. The first microfluidic device will employ an active gradient generator that will identify the concentration of sonic hedgehog on a per cell basis necessary to achieve high yield motor neuron differentiation. The second microfluidic device will employ a passive gradient generator to pattern the engineered neural tissue structure towards the ventral portion of the neural tube. Successful completion up to the first half of Aim 3 will yield spinal motor neurons with defined positional identity that will have potential applications in regenerative therapy, while successful completion of the second half of Aim 3 will yield spinal motor neurons that reside in a ventral tissue structure that can be used in high throughput screening of therapeutics to treat ALS.