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过程中被编码并在成年期间保留。一类表现出很大异质性的细胞是脊柱运动神经元，该细胞基于发育过程中获得的线索支配特定的肌肉靶标，并且在诸如肌萎缩性侧面硬化症（ALS）和平滑肌萎缩（SMA）等疾病中受到严重损害。尽管与脊柱运动神经元区分的人多能干细胞（HPSC）可以为研究和/或治疗这些疾病提供出色的资源，但当前的分化方法无法概括获得确定的位置身份所需的发育线索。此外，尽管ALS之类的疾病是非细胞自主的，并且涉及复杂的细胞相互作用，但脊柱运动神经元的孤立分化可能不能适当地代表菜肴中的疾病进展和表型。因此，该提案广泛地旨在将HPSC区分开为具有定义的位置身份的脊柱运动神经元，无论是孤立的种群和有组织的多细胞组织结构。该提案的目的1着重于将HPSC与神经祖细胞区分开，该神经祖细胞通过与wnt3a，成纤维细胞生长因子8，视黄酸和生长分化因子和生长分化因子等模式因子结合使用，沿鼻骨/尾部脊髓轴沿鼻/尾部脊髓轴的定义位置区分。定义的尺寸和形状。 AIM 3将利用两个微流体设备来优化脊柱运动神经元分化。第一个微流体设备将采用活跃的梯度发生器，该发电机将以每个细胞基础的基础确定声音刺猬的浓度，以实现高产量运动神经元分化。第二个微流体设备将采用被动梯度发生器，以对工程神经组织结构进行对朝向神经管的腹侧部分进行模拟。直到AIM 3上半年的成功完成，将产生具有定义的位置身份的脊柱运动神经元，该神经元将在再生疗法中具有潜在的应用，而成功完成AIM 3的后半部分将产生脊柱运动神经元，该神经元将产生属​​于腹侧组织结构中的脊柱运动神经元，该神经元可用于腹侧组织结构，该神经元可以在高吞吐量筛查Als的高吞吐量中使用。