Modeling spinal cord axis patterning with human pluripotent stem cells

用人类多能干细胞模拟脊髓轴模式

• 批准号: 8852002
• 负责人: Ethan Lippmann
• 金额: $ 4.21万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-08-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8852002
• 关键词: Adult; Amyotrophic Lateral Sclerosis; Astrocytes; Axon; Cell Communication; Cell Culture Techniques; Cells; Characteristics; Chemistry; Clinical; Complex; Cues; Custom; Development; Differentiation and Growth; Disease; Disease Progression; Disease model; Embryo; Embryonic Development; Engineering; Erinaceidae; Exhibits; Fibroblast Growth Factor 8; Future; Generations; Goals; Heterogeneity; Human; Location; Methods; Microfluidic Microchips; Microfluidics; Modeling; Molecular; Motor Neurons; Mus; Muscle; Muscular Atrophy; N-Cadherin; Neural tube; Neuraxis; Neurons; Pattern; Peptides; Phenotype; Pluripotent Stem Cells; Population; Positioning Attribute; Preclinical Drug Evaluation; Printing; Procedures; Process; Protein Engineering; Protocols documentation; Recombinant Proteins; Recombinants; Research; Research Personnel; Resources; Shapes; Smooth Muscle; Somatic Cell; Source; Spinal; Spinal Cord; Spinal Muscular Atrophy; Structure; Surface; Techniques; Therapeutic; Time; Tissue Engineering; Tissues; Translating; Translations; Trauma; Tretinoin; base; cell type; combinatorial; disease phenotype; genetic manipulation; high throughput screening; human disease; improved; in vivo; induced pluripotent stem cell; injured; interest; morphogens; nerve stem cell; neuroepithelium; progenitor; protein expression; public health relevance; regenerative therapy; relating to nervous system; scale up; stem cell differentiation; tool; transcription factor; two-dimensional

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.

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。表现出实质异质性的一类细胞是脊髓运动神经元，其基于发育期间接收的线索来支配特定的肌肉靶标，并且在诸如肌萎缩性侧索硬化症（ALS）和平滑肌萎缩（SMA）的疾病中严重受损。虽然分化为脊髓运动神经元的人多能干细胞（hPSC）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限定的位置同一性所必需的发育线索。此外，尽管像ALS这样的疾病是非细胞自主的并且涉及复杂的细胞-细胞相互作用，但是脊髓运动神经元的孤立分化可能不能适当地代表培养皿中的疾病进展和表型。因此，该提议广泛地寻求将hPSC分化为具有确定的位置特性的脊髓运动神经元，既作为分离的群体又在有组织的多细胞组织结构内。该提议的目的1集中于通过用图案化因子如Wnt 3a、成纤维细胞生长因子8、视黄酸和生长分化因子11的组合处理将hPSC分化为具有沿脊髓头侧/尾侧轴沿着限定位置的神经祖细胞。目标2将使用微接触印刷，表面化学和重组蛋白工程的组合来创建定义大小和形状的神经组织结构。目的3将利用两种微流控装置优化脊髓运动神经元分化。第一个微流体装置将采用主动梯度发生器，其将在每个细胞的基础上鉴定实现高产运动神经元分化所需的音刺猬蛋白的浓度。第二微流体装置将采用被动梯度发生器来使工程化神经组织结构朝向神经管的腹侧部分图案化。成功完成Aim 3的前半部分将产生具有限定的位置同一性的脊髓运动神经元，其将在再生治疗中具有潜在的应用，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，其可用于治疗ALS的治疗剂的高通量筛选。