Acoustic Tweezing Cytometry for Efficient Neural Differentiation

项目摘要 声推流式细胞术用于有效的神经分化 人多能干细胞（hPSC）已被誉为治疗退行性疾病的有希望的细胞来源， 恶性和遗传性疾病，或由于炎症、感染和创伤引起的损伤。hPSC还被 被证明是研究人类发育以及开发和测试新药的宝贵发现工具。 然而，为了充分实现hPSC的巨大潜力，第一步也可能是最关键的一步是 以高效率和高纯度将hPSC定向分化为特定的功能细胞类型。 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 来控制它们的活动。MN在诸如脊髓损伤、肌萎缩性侧索硬化症、脊髓损伤和脊髓损伤等疾病中受损。 脊髓侧索硬化症（ALS）和脊髓性肌萎缩症（SMA）。虽然有重大利益， 将hPSC分化为功能性MN用于细胞治疗和了解MN退行性疾病， 不明确的培养条件和从hPSC分化MN的低效方案显著影响了细胞的分化。 阻碍了它的广泛应用。鉴于胚胎发育是一个动态过程， 不断变化的物理环境，这项拟议研究的中心假设是hPSC，即 相当于植入期人类胚胎中的外胚层，本质上是机械敏感的， 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的功能。 分化和功能成熟的神经元亚型，如MN。这项建议是 我们令人兴奋的初步数据表明，一种新的基于超声的技术，声学 镊子式细胞术（ATC）可以向hPSC施加受控的动态亚细胞机械力， 事实上，诱导hPSC神经上皮甚至MN分化的速度要快得多， 传统的协议，仅依赖于可溶性因子。因此，我们建议在这项研究中充分开发 ATC技术不仅阐明了hPSC的内在机械敏感性，而且还利用了hPSC的机械敏感性。 改进功能MN的大规模生产的技术。 在这项研究中，我们提出（目标1）开发高通量ATC技术， （目的2）阐明调控网络的作用，所述调控网络包括： 机械敏感性途径（BMP/雅普活性、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;（目的3）应用ATC高效地 从hPSC产生功能性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产生的方法，可能使大规模的研究取得巨大进展。 用于细胞治疗和药物筛选的临床级MN的规模生产。我们的研究计划将 也有助于建立一个新的机制框架，以了解机械敏感的hPSC性质， 将绘制一条路径，以解开他们的全部复杂性，为他们未来的再生医学应用。

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.