Acoustic Tweezing Cytometry for Efficient Neural Differentiation

项目摘要 声钳细胞计数法用于有效的神经分化 人类多能干细胞(HPSCs)被誉为治疗退行性变、 恶性和遗传性疾病，或因炎症、感染和创伤造成的损伤。HPSC也一直是 事实证明，这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hPSCs的巨大潜力，第一步，也可能是最关键的一步是 高效率、高纯度的hPSCs定向分化为特定功能细胞类型。 运动神经元是一类特殊的神经元，存在于脊髓和投射轴突中。 以控制肌肉的活动。MN在脊髓损伤、肌营养不良等疾病中受损 侧索硬化症(ALS)和脊髓性肌萎缩症(SMA)。虽然有很大的兴趣 将hPSCs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低效的MN从hPSCs分化的方案显著 阻碍了它们的广泛使用。鉴于胚胎发育是一个动态的过程，需要不断地 不断变化的物理环境，这项拟议研究的中心假设是hPSCs，这是 相当于围着床期人类胚胎中的外胚层，本质上是机械敏感的，并且 细胞微环境中的生物物理线索可以提供强有力的调节信号来控制它们的 对特定神经元亚型的分化和功能成熟，如MNS。这项建议是 我们令人兴奋的初步数据显示，一种新的基于超声波的技术，声学 镊子细胞术(ATC)可以对hPSCs施加受控的动态亚细胞机械力，可以 事实上，与对照组相比，hPSCs更快地诱导神经上皮细胞甚至MN分化 仅依赖于可溶性因子的常规方案。因此，我们在这项研究中建议充分开发 ATC技术不仅阐明了hPSCs的内在机械敏感特性，而且还利用 提高功能性锰的规模化生产的技术。 在本研究中，我们建议(目标1)开发具有改进的高通量ATC技术 机械刺激hPSCs的能力；(目标2)阐明调控网络的作用，包括 机械敏感途径(BMP/YAP活性、RhoA/ROCK/细胞骨架收缩和河马/LATS) 调控ATC促进的hPSCs神经上皮细胞分化；(目标3)应用ATC实现高效 从hPSCs产生功能的MN。这项研究的成功完成将建立一种新的、新颖的 HPSC神经分化和MN生成的方法，潜在地使大型- 规模化生产用于细胞疗法和药物筛选的临床级MN。我们提议的研究将 也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 将绘制一条道路，为未来的再生医学应用揭开它们的全部复杂性。

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.