CAREER: Engineering Human CNS Morphogenesis Ex Vivo: Spinal Cord

最近观察到的人类多能干细胞（hPSCs）组合在体外自发转化为含有多种脑组织的“脑类器官”的能力表明，类器官可以在体外被改造，以产生具有结构、组成和功能（包括神经回路）的大脑和脊髓组织，这些组织可以模仿人类中枢神经系统（CNS）的许多特征。可控的离体中枢神经系统模型的可用性可以为以下方面提供平台：1)研究人类发育生物学和生理学；2)研究退行性疾病和创伤性损伤；3)加强药物筛选和个性化医疗；4)消除对相关动物研究的需要。该项目的目标是开发人类脊髓类器官的工程方法，显示脊髓灰质结构和类似中枢神经系统的神经回路。成功可能导致脊髓损伤研究和药物筛选的范式转变。所提出的方法可以用于设计各种神经、心脏和肠道类器官。教育和推广计划包括：1)开发一个全面的“基于干细胞的组织工程和形态发生（Stem）”网站，作为BME课程和初中和高中教师的教育资源，并作为分享研究成果的网站；2)旨在接触未被充分代表的少数民族（URM）学生（包括小学和初中）的外展计划；3)举办“建造人类脊髓”现场展览，让公众参与干细胞和组织工程研究。该项目将使用创新的培养平台来开发标准化的方法，用于可重复工程的人类脊髓类器官，选择脊髓的组织形态相对简化。研究计划分为三个目标和相关假设：1)具有后神经管（PNT）组织结构的工程人类神经类器官——在发育长度尺度上对类器官形态的空间控制可以诱导仿生组织结构的假设将通过一种新的成型技术进行测试，以开发一种标准化的方法来模拟早期后神经管结构的极化、hpsc衍生的神经干细胞（hNSCs）的工程环形管。2)用脊髓灰质组织的背腹侧（D/V）细胞结构模拟工程人类神经类器官——外源性形态梯度可以应用于指导类器官内的仿生细胞组成和组织细胞结构的假设将使用新型“Stamp-On”微流体来测试，以产生类似的形态梯度，并研究它们是否可以再现仿生细胞组成和细胞结构的D/V模式3)模拟脊髓灰质组织的rostroaudal （R/C）细胞结构的工程人类神经类器官——定向组装神经类器官组织可以诱导形成仿生神经的假设将通过使用来自颈、胸、和腰椎hNSCs，并测试它们的定向组装是否产生具有仿生R/C组织细胞结构和功能性神经回路的复合脊髓类器官。第四个目标是通过整合多方面的方法来激励和教育公众和学生，特别是代表性不足的少数民族，促进未来以hpsc为基础的组织工程行业多元化劳动力的发展。尽管人类psc衍生的类器官具有模拟人类生物学和生理学的巨大可能性，但由于缺乏对其自发形态发生过程的控制，它们的效用受到限制，这些过程产生随机的类器官结构和组成。利用脊髓类器官样本，该项目将开发新的工具和方法，用于在微到毫米尺度上设计人类类器官形态发生。在这种规模上指导可复制的离体形态发生对于开发先进的生物制造工艺来生产功能性、仿生组织和器官至关重要。工程参数被认为是指导仿生3-D类脊髓器官出现的关键，可以阐明广泛适用的形态发生工程原理，并为中枢神经系统的发育和疾病提供新的见解。所产生的工具和知识将显著提高科学家设计仿生人体组织的能力，特别是为研究退行性脊髓疾病和创伤性损伤提供迄今为止最具人形的实验平台。所提出的工具和方法设计简单，因此可以广泛传播和实施工程人类类器官。人类类器官形态发生的迷人数据和解释将作为在线教育资源进行策划，并介绍给公众和学生，以促进政治支持，兴趣和劳动力发展。

PI: Ashton, Randolph S.Proposal #: 1651645The recently observed ability of combinations of human pluripotent stem cells (hPSCs) to spontaneously transform in vitro into "cerebral organoids" containing diverse brain tissues suggests the possibility that organoids can be engineered ex vivo to generate brain and spinal cord tissues with structure, composition and function (including neural circuits) that can mimic many features of the human Central Nervous System (CNS). The availability of controlled ex vivo CNS models can lead to platforms for 1) investigating human developmental biology and physiology, 2) investigating degenerative diseases and traumatic injury, 3) enhanced drug screening and personalized medicine and 4) eliminating the need for questionably relevant animal studies. The goal of this project is to develop methods for engineering human cord organoids displaying spinal cord grey matter architecture and CNS-like neuronal circuitry. Success could lead to a paradigm shift relative in spinal cord injury research and drug screening. The proposed methods can be implemented to engineer diverse neural, heart, and gut organoids. The education and outreach plan includes: 1) development of a comprehensive "Stem cell-based Tissue Engineering & Morphogenesis (STEM)" website to serve as an educational resource for BME courses and middle and high school teachers and as a site for sharing research findings, 2) outreach programs designed to expose underrepresented minority (URM) students, including at elementary and middle school levels, to STEM experiences and 3) creation of a live "Building a Human Spinal Cord" exhibit to engage the public in stem cell and tissue engineering research.This project will use innovative culture platforms to develop standardized methodologies for reproducibly engineering human spinal cord organoids, chosen due the spinal cord's comparatively simplified tissue morphology. The Research Plan is organized under 3 objectives with related hypotheses: 1) Engineer human neural organoids with posterior neural tube (PNT) tissue structure--The hypothesis that spatial control of organoid morphology at developmental length scales can induce biomimetic tissue structure will be tested using a novel molding technology to develop a standardized methodology for engineer annular tubes of polarized, hPSC-derived neural stem cells (hNSCs), which is mimetic of early PNT structure, 2) Engineer human neural organoids with dorsoventral (D/V) cytoarchitecture mimetic of spinal cord grey matter tissue--The hypothesis that exogenous morphogen gradients can be applied to instruct biomimetic cellular composition and tissue cytoarchitecture within organoids will be tested using novel "Stamp-On" microfluidics to generate analogous morphogen gradients and investigate whether they can recapitulate D/V patterning of biomimetic cellular composition and cytoarchitecture within cylindrical hNSC organoids and 3) Engineer human neural organoids with rostrocaudal (R/C) cytoarchitecture mimetic of spinal cord grey matter tissue--The hypothesis that directed assembly of neural organoid tissues can induce formation of biomimetic neural will be tested by using organoids generated from cervical, thoracic, and lumbar hNSCs and testing whether their directed assembly yields a composite spinal cord organoid with biomimetic R/C tissue cytoarchitecture and functional neuronal circuitry. The 4th objective is to promote development of a diverse workforce for future hPSC-based tissue engineering industries by integrating a multifaceted approach for inspiring and educating the general public and students, especially underrepresented minorities. Though Human PSC-derived organoids possess tremendous possibilities for modeling human biology and physiology, their utility is limited by a lack of control over their spontaneous morphogenesis processes, which produces random organoid structure and composition. Using a spinal cord organoid exemplar, this project will develop novel tools and methodologies for engineering human organoid morphogenesis at the micro-to-millimeter scale. Instructing reproducible ex vivo morphogenesis at this scale is critical for developing advanced biomanufacturing processes to produce functional, biomimetic tissues and organs. Engineering parameters identified as critical to instructing emergence of biomimetic 3-D spinal organoids could elucidate broadly applicable morphogenesis engineering principles and provide new insights into CNS development and disease. The tools and knowledge generated will significantly advance scientists' ability to engineer biomimetic human tissues, specifically providing the most humanoid experimental platform to date for investigating degenerative spinal cord diseases and traumatic injury. The proposed tools and methods are designed for simplicity, thus can be broadly disseminated and implemented for engineer human organoids. Captivating data and explanations of human organoid morphogenesis will be curated as an online educational resource and introduced to the general public and students to promote political support, interests, and workforce development.