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被编码，并在成年期保留。脊髓运动神经元是一类表现出实质异质性的细胞，它们根据发育过程中收到的信号对特定肌肉靶点进行神经支配，在肌萎缩侧索硬化症（ALS）和平滑肌萎缩症（SMA）等疾病中受到严重损害。虽然人类多能干细胞（hPSCs）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明确位置身份所必需的发育线索。此外，像ALS这样的疾病是非细胞自主的，涉及复杂的细胞间相互作用，脊髓运动神经元的分离分化可能不能恰当地代表疾病在培养皿中的进展和表型。因此，该建议广泛寻求将hPSCs分化为具有明确位置身份的脊髓运动神经元，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。本研究的目的1是通过Wnt3a、成纤维细胞生长因子8、视黄酸和生长分化因子11等模式因子的联合治疗，将hPSCs分化为沿喙侧/尾侧脊髓轴具有明确位置的神经祖细胞。目标2将结合微接触印刷、表面化学和重组蛋白工程来创建确定大小和形状的神经组织结构。目的3将利用两种微流体装置优化脊柱运动神经元分化。第一个微流体装置将采用一个有源梯度发生器，它将在每个细胞的基础上识别出高产量运动神经元分化所需的超音hedgehog基因浓度。第二微流控装置将采用无源梯度发生器将工程神经组织结构朝向神经管的腹侧部分进行图案化。成功完成Aim 3的前半部分将产生具有明确位置标识的脊髓运动神经元，这将在再生治疗中有潜在的应用，而成功完成Aim 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，可用于治疗ALS的高产量筛选。

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.