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，大量的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，并建立与特定人群的联系 的靶细胞。神经系统发育过程中细胞命运和分化的这种空间组织 系统由称为形态发生素的化学信号的浓度梯度指导。即使 分级形态发生蛋白信号在发育模式形成中的重要性已被充分认识， 仍然是生物学中的一个重要问题，即胚胎祖细胞如何将 发育信号转化为基因表达和细胞分化的空间模式， 健壮的时尚该NIH R21项目的长期功能目标是专门解决 理解细胞内信号级联对形态发生梯度的解释的挑战， 胚胎前体细胞在发育模式期间经历多细胞自组织。 具体地说，我们建议利用神经上皮细胞的内在管腔生成和自组织特性， (NE)细胞，神经管中的胚胎前体细胞，结合创新的微流体 胚胎学装置，以实现可控制和可再生的内腔NE囊肿世代，以模拟非- 图案化的脊髓组织高纯度NE细胞将来源于人多能干细胞（hPSC） 使用已建立的2D定向分化方案。然后，管腔NE囊肿将被无缝地用于 使用形态原Sonic hedgehog进行下游不对称图案化的相同微流体装置 (Shh)以实现脊髓中腹侧神经元亚型的渐进性获取。成功 这项研究的完成将导致建立一个创新的基于微流体的 用于可控、可再现和可扩展地产生（自体）人脊髓组织的方法 与现有的3D类器官培养系统相比， 可控性和再现性。此外，我们的合成图案化的人类脊髓模型将提供一个 非常有用的实验平台，提供关键参数和定量的上级实验控制 测量，以允许深入的机制调查的紧急自组织原则， 为胚胎模式提供鲁棒性和可靠性的模式机制，这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.