Acoustic Tweezing Cytometry for Efficient Neural Differentiation

用于高效神经分化的声学镊子细胞术

基本信息

批准号：
10274928
负责人：
CHERI X DENG
金额：
$ 44.12万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-22 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10274928
关键词：
ALS patients Acoustics Adoption Adult Amyotrophic Lateral Sclerosis Axon Biological Assay Biomechanics Biomedical Engineering Biophysics Cell Culture Techniques Cell Differentiation process Cell Therapy Cell physiology Cells Clinical Cues Cytometry Cytoskeleton Data Degenerative Disorder Derivation procedure Development Disease Disease model Drug Screening Embryo Embryonic Development Engineering Epiblast Ethnic Origin Future Generations Genetic Diseases Goals Grant Human Human Development In Vitro Infection Inflammation Injury Integrins Investigation Malignant - descriptor Mechanical Stimulation Mechanics Mediating Microbubbles Morphogenesis Motor Neuron Disease Motor Neurons Muscle Neuroepithelial Neurons Pathway interactions Periodicity Pharmaceutical Preparations Pharmacology Physical environment Pluripotent Stem Cells Process Property Protocols documentation Regenerative Medicine Reporting Research Role Signal Transduction Source Spatial Distribution Spinal Cord Spinal Muscular Atrophy Spinal cord injury System Technology Testing Therapeutic Toxicology Transcription Coactivator Trauma Ultrasonography base blastocyst cell type clinically relevant design differentiation protocol human pluripotent stem cell implantation improved in vivo interest large scale production mechanical force nerve stem cell nervous system disorder neuron loss new technology novel novel strategies novel therapeutics operation pluripotency public health relevance regenerative therapy relating to nervous system self-renewal sex single cell analysis stem cells tool

项目摘要

Project Summary Acoustic Tweezing Cytometry for Efficient Neural Differentiation Human pluripotent stem cells (hPSCs) have been hailed as a promising cell source for treating degenerative, malignant, and genetic diseases, or injuries due to inflammation, infection, and trauma. hPSCs have also been proven as an invaluable discovery tool to study human development and for developing and testing new drugs. However, to fully realize the tremendous potential of hPSCs, the first and perhaps the most critical step is the directed differentiation of hPSCs to specific functional cell types with high efficiency and purity. Motor neurons (MNs) are a specialized class of neurons that reside in the spinal cord and project axons to muscles to control their activity. MNs are damaged in diseases such as spinal cord injury, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). While there are significant interests in differentiating hPSCs into functional MNs for cell therapies and understanding of MN degenerative diseases, poorly defined culture conditions and inefficient protocols of MN differentiation from hPSCs have significantly hindered their broad use. Given that embryonic development is a dynamic process involving constantly changing physical environments, the central hypothesis of this proposed research is that hPSCs, which is equivalent to the epiblast in the peri-implantation human embryo, are intrinsically mechanosensitive, and biophysical cues in the cell microenvironment can provide potent regulatory signals to control their differentiation and functional maturation towards specific neuronal subtypes such as MNs. This proposal is strongly motivated by our exciting preliminary data showing that a novel ultrasound-based technology, acoustic tweezing cytometry (ATC), which can apply controlled dynamic subcellular mechanical forces to hPSCs, can indeed elicit neuroepithelial and even MN differentiation of hPSCs much more rapidly compared to conventional protocols that solely rely on soluble factors. Thus we propose in this research to fully develop the ATC technology to not only elucidate the intrinsic mechanosensitive properties of hPSCs, but also utilize the technology to improve large-scale production of functional MNs. In this research we propose to (Aim 1) develop high-throughput ATC technology with improved capability for mechanical stimulation of hPSCs; (Aim 2) elucidate the role of a regulatory network comprising mechanosensitive pathways (BMP/YAP activity, RhoA/ROCK/cytoskeleton contractility, and Hippo/LATS) in regulating ATC-facilitated neuroepithelial differentiation of hPSCs; (Aim 3) apply ATC for high-efficiency functional MN generation from hPSCs. Successful completion of this research will establish a new, novel approach for hPSC neural differentiation and MN generation, potentially enabling drastic advances in large- scale production of clinical-grade MNs for cell-based therapies and drug screens. Our proposed research will also help establish a novel mechanistic framework for understanding mechanosensitive hPSC properties and will chart a path to unravel their full complexity for their future regenerative medicine applications.

项目概要用于高效神经分化的声学镊子细胞术人类多能干细胞（hPSC）被誉为治疗退行性、恶性和遗传性疾病，或由于炎症、感染和外伤引起的损伤。 hPSC 也已被事实证明，它是研究人类发展以及开发和测试新药的宝贵发现工具。然而，要充分发挥 hPSC 的巨大潜力，第一步，也许也是最关键的一步是以高效率和纯度定向 hPSC 分化为特定功能细胞类型。运动神经元 (MN) 是一类特殊的神经元，位于脊髓中并投射轴突使肌肉控制其活动。 MN 因脊髓损伤、肌萎缩等疾病而受损侧索硬化症（ALS）和脊髓性肌萎缩症（SMA）。虽然人们对以下领域有重大兴趣将 hPSC 分化为功能性 MN，用于细胞治疗和了解 MN 退行性疾病，明确的培养条件和从 hPSC 分化 MN 的低效方案显着阻碍了它们的广泛使用。鉴于胚胎发育是一个不断变化的动态过程改变物理环境，这项研究的中心假设是 hPSC，它是相当于植入期人类胚胎中的外胚层，本质上具有机械敏感性，并且细胞微环境中的生物物理线索可以提供有效的调节信号来控制它们向特定神经元亚型（例如 MN）的分化和功能成熟。这个提议是我们激动人心的初步数据强烈激励了我们，该数据表明一种新颖的基于超声波的技术——声学镊子细胞术 (ATC) 可对 hPSC 施加受控的动态亚细胞机械力，与相比，确实更快地引发 hPSC 的神经上皮甚至 MN 分化仅依赖于可溶性因子的传统方案。因此，我们在这项研究中建议充分开发 ATC 技术不仅可以阐明 hPSC 的固有机械敏感特性，还可以利用技术来提高功能性MN的大规模生产。在这项研究中，我们建议（目标 1）开发高通量 ATC 技术，并改进 hPSC 的机械刺激能力；（目标 2）阐明监管网络的作用，包括机械敏感通路（BMP/YAP 活性、RhoA/ROCK/细胞骨架收缩性和 Hippo/LATS）调节 ATC 促进的 hPSC 神经上皮分化；（目标3）应用ATC实现高效率 hPSC 生成功能性 MN。这项研究的成功完成将建立一个新的、新颖的 hPSC 神经分化和 MN 生成的方法，有可能在大用于细胞疗法和药物筛选的临床级 MN 的规模生产。我们提出的研究将还有助于建立一个新的机制框架来理解机械敏感的 hPSC 特性和将为未来的再生医学应用阐明其全部复杂性。