Synthetic microfluidic synthesis of spinal cord tissues from human pluripotent stem cells

项目摘要 在脊椎动物神经系统的发育过程中，大量神经元将以离散的方式发育。 解剖位置，获得不同的形态形式，并与特定人群建立联系 目标细胞的数量。神经发育过程中细胞命运和分化的这种空间组织 系统由化学信号的浓度梯度指示，称为形态成因。即使是在 分级形态信号在发育模式形成中的重要性已经得到了很好的认识，它 仍然是生物学中的一个重要问题，即胚胎前体细胞如何在 发育信号转化为基因表达的空间模式和细胞分化的可靠和 强健的时尚。NIH R21项目的长期功能目标是专门解决重要的 在理解细胞内信号级联解释形态原梯度方面的挑战 胚胎前体细胞在发育过程中经历多细胞自组织。 具体地说，我们建议利用神经上皮细胞固有的致瘤和自组织特性。 (NE)细胞，神经管中的胚胎前体细胞，与创新的微流控技术相结合 胚胎学装置，以实现可控和可重复代的腔内NE囊肿，以模拟非... 有图案的脊髓组织。高纯度的NE细胞将从人类多能干细胞(HPSCs)中获得 使用已建立的2D定向分化方案。然后，腔内NE囊将被无缝地用于 用于下游非对称图案化的相同微流控装置，使用形态发生声刺猬 (Shh)实现脊髓腹侧神经元亚型的渐进性获得。成功 这项拟议研究的完成将导致建立一种创新的基于微流体的 可控制、可复制和可伸缩的(自体)人脊髓组织的生成方法 从hPSCs出发，与现有的3D有机培养系统相比，这是一个巨大的飞跃 可控性和重复性。此外，我们的合成图案人类脊髓模型将提供 非常有用的实验平台，提供卓越的关键参数和量化实验控制 允许对新出现的自组织原理和 为胚胎图案提供健壮性和可靠性的图案机制是一个长期存在的问题 在生物学上。

Project Summary During development of the vertebrate nervous system, a vast array of neurons will develop in discrete anatomical positions, acquire varied morphological forms, and establish connections with specific populations of target cells. Such spatial organization of cell fates and differentiation during the development of the nervous system are directed by concentration gradients of chemical signals, termed morphogens. Even though the importance of graded morphogen signaling in developmental pattern formation has been well recognized, it remains a significant question in biology about how embryonic progenitor cells transform dynamic changes in developmental signaling into spatial patterns of gene expression and cellular differentiation in a reliable and robust fashion. The long-term functional goal of this NIH R21 project is to specifically address the significant challenge in understanding the interpretation of morphogen gradients by intracellular signaling cascades while embryonic precursor cells are undergoing multicellular self-organization during developmental patterning. Specifically, we propose to leverage the intrinsic lumenogenic and self-organizing properties of neuroepithelial (NE) cells, the embryonic precursor cells in the neural tube, in conjunction with an innovative microfluidic embryological device, to achieve controllable and reproducible generations of lumenal NE cysts to mimic un- patterned spinal cord tissues. High-purity NE cells will be derived from human pluripotent stem cells (hPSCs) using established 2D directed differentiation protocols. Lumenal NE cysts will then be utilized seamlessly in the same microfluidic device for downstream asymmetrical patterning using the morphogen Sonic hedgehog (Shh) to achieve progressive acquisition of ventral neuronal subtypes in the spinal cord. Successful accomplishment of this proposed research will lead to the establishment of an innovative microfluidics-based methodology for controllable, reproducible, and scalable generation of (autologous) human spinal cord tissues from hPSCs, a quantum leap compared with existing 3D organoid culture systems that are known to lack controllability and reproducibility. Furthermore, our synthetic patterned human spinal cord model will provide a very useful experimental platform that offers superior experimental controls of key parameters and quantitative measurements to allow in-depth mechanistic investigations on the emergent self-organizing principles and pattering mechanisms that provide robustness and reliability to embryonic patterning, a long-standing question in biology.