Personalized In Vitro Nerve Model

由遗传疾病、损伤/感染和衰老相关变性引起的600多种神经系统疾病对生活质量提出了独特的挑战，因为它们通常会损害认知、感觉和运动功能。虽然部分由于人口老龄化，这类疾病的发病率呈指数级增长，但几乎没有有效的治疗方法。虽然新技术使新疗法的快速发展成为可能，但这些疗法在临床试验中往往失败，主要原因是在人类中翻译/验证基于动物的结果方面效率低下。因此，人们越来越需要一种用于健康和患病神经的人源化体外（体外）模型，以促进对神经科学的基本理解并开发针对神经异常的潜在干预措施。该项目通过开发和验证由神经元和来自神经干细胞的支持细胞组成的个性化神经组织模型来满足这一需求，该模型旨在在结构和功能上模仿天然神经。这一成果具有广泛的社会影响潜力，包括：1）在当前老龄化社会对长期疗效的需求快速增长的情况下，为实现个性化再生医学提供科学依据; 2）提供个性化药物试验床，从而可能导致开发患者定制的治疗方法; 3）导致开发新的植入物，使目前无法实现的受损脊神经再生和4）为许多基础神经科学研究提供理想的平台。与该项目相关的学生将在材料科学，纳米技术，神经成像和神经科学的接口进行培训。教育和外展计划以STEM中代表性不足的群体为中心，包括：1）加州大学河滨分校与（一所西班牙裔服务机构））和加州浸会大学（主要是本科院校），2）各种K-12外展计划，和3）将研究成果整合到本科和研究生课程中。该项目的目标是开发组织形态发生策略，以产生结构和功能上的组织与天然神经组织相当，并通过纵向观察的非破坏性成像来验证结构/功能。这项工作建立在实验室最近开发的增强神经细胞活动的技术基础上;同时进行机械和电（机械-电）刺激a）促进神经突伸长和B）激活神经胶质细胞表达促神经生长因子。研究计划分为两个任务。任务1是开发一种策略，通过使用可以声学振动的电纺压电PVDF-TrFE支架，通过机械电刺激，在结构上和功能上与天然组织相媲美。初始步骤是通过调整接种有细胞成分（每种细胞类型分别接种）的压电支架的厚度来确定用于源自人神经干细胞（NSC）的神经的细胞成分（神经元、少突胶质细胞和星形胶质细胞）的功能增强的最佳机械电刺激方案。下一步是通过将H9衍生的NSC接种在支架上来确定机械电刺激对人NSC的多表型分化的影响，所述支架然后将经历每天2小时/天的刺激持续6天，然后通过计数每种分化类型的细胞的数量来评估。在优化分化过程后，在适当的刺激持续时间后，将比较自下而上（允许神经元、少突胶质细胞和星形胶质细胞自组装神经结构）和自上而下（同时诱导NSC向三种细胞表型和结构组装的分化）方法的神经生成，即，在髓鞘形成和组织形态发生之后。然后将细胞/支架结构卷起以形成圆柱形，并进行标准神经传导测试和组织学检查。任务2是通过非破坏性偏振敏感光学相干断层扫描（PS-OCT）验证工程化神经组织的形态和功能特征。将通过比较多发性硬化症（MS）小鼠模型的切除脊髓与年龄匹配对照的脊髓来鉴定中枢神经系统中神经活力的基于PS-OCT的生物标志物。PS-OCT能够在支架材料内和通过支架材料成像，然后将用于定量监测工程化神经结构的形态发生和结构，这将通过与从对照脊髓样品获得的光学生物标志物进行比较来优化细胞密度和髓鞘形成程度的期望终点。 最后，PS-OCT将用于量化工程神经结构中化学诱导（溶血素）变性导致的结构和功能损失。在退化过程中光学测量的变化预计将导致参数，可以优化，以模拟在病理/MS脊髓samples.This奖项反映了NSF的法定使命中观察到的退化，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

The more than 600 neurological diseases caused by genetic disorders, injuries/infections and aging-related degeneration uniquely challenge the quality of life as they typically impair cognitive, sensory and motor functions. Although there is an exponential increase in the prevalence of such diseases, partly due to the aging population, few effective treatments are available. Though new technologies have enabled the rapid development of new therapeutics, these often fail in clinical trials, largely due to inefficiency in translating/validating animal-based results in humans. Thus, there is a growing need for a humanized in vitro (outside the body) model for healthy and diseased nerves in order to further the fundamental understanding of neuroscience and develop potential interventions for neuro-abnormalities. This project addresses this need by developing and validating a personalized nerve tissue model composed of neurons and supporting cells derived from neural stem cells and designed to structurally and functionally mimic native nerves. This outcome has potential for broad society impact, including: 1) providing a scientific basis to realize personalized regenerative medicine amid fast rising demand for long-term efficacy in the current ageing society; 2) providing a personalized drug test bed which could lead to the development of patient-tailored therapeutics; 3) leading to development of novel implants that enable currently unachievable regeneration of damaged spinal nerves and 4) providing ideal platforms for many fundamental neuroscience studies. Students associated with the project will be trained at the interface of materials science, nanotechnology, neuroimaging and neuroscience. The education and outreach initiatives center on under-represented groups in STEM, including: 1) an undergraduate research partnership between the University of California-Riverside (a Hispanic serving institution)) and California Baptist University (a primarily undergraduate institution), 2) various K-12 outreach programs, and 3) integration of research outcomes into both undergraduate and graduate courses.The objective of this project is to develop a tissue morphogenesis strategy to produce a tissue structurally and functionally comparable to native nerve tissues and to validate the structure/functionality by non-destructive imaging for longitudinal observations. The work builds on the lab's recently developed technology to enhance neural cell activities; simultaneous mechanical and electrical (mechano-electrical) stimulation a) facilitated neurite elongation and b) activated glial cells to express neourotropic factors. The research plan is organized under two tasks. Task 1 is to develop a strategy to engineer spinal nerves structurally and functionally comparable to the native tissues via mechano-electrical stimulation by using electrospun piezoelectric PVDF-TrFE scaffolds that can be vibrated acoustically. The initial step is to determine the optimal mechano-electrical stimulation regimen for the functional enhancement of cellular constituents of the nerve (neurons, oligodendrocytes and astrocytes) derived from human neural stem cells (NSCs) by adjusting the thickness of piezoelectric scaffolds seeded with the cellular constituents (each cell type separately seeded). The next step is to determine the effects of mechano-electrical stimulation on the multi-phenotype differentiation of human NSCs by seeding H9-derived NSCs on the scaffolds that will then undergo daily stimulations for 2 hrs/day for 6 days before being assessed by counting the number of cells of each differentiated type. After optimization of the differentiation process, nerve generation from bottom-up (neurons, oligodendrocytes and astrocytes allowed to self-assemble the nerve structure) and top-down (simultaneously induce the differentiation of NSCs towards the three cell phenotypes and structural assembly) approaches will be compared after an appropriate stimulation duration, i.e., after myelination and tissue morphogenesis has occurred. The cell/scaffold constructs will then be rolled to form a cylindrical shape and subjected to standard nerve conduction testing and histological examination. Task 2 is to validate the morphological and functional characteristics of the engineered nerve tissue via non-destructive polarization-sensitive optical coherence tomography (PS-OCT). PS-OCT based biomarkers for nerve viability in the central nervous system will be identified by comparing excised spinal cords from a mouse model of multiple sclerosis (MS) with spinal cords from age matched controls. PS-OCT, which is capable of imaging within and through the scaffolding material, will then be used to quantitatively monitor the morphogenesis and structure of the engineered nerve structures, which will enable optimization of the desired end points for cell density and degree of myelination through comparison to optical biomarkers obtained from the control spinal cord samples. Finally, PS-OCT will be used to quantify the loss of structure and functionality resulting from chemically induced (lysolecithin) degeneration in engineered nerve structures. Changes in optical measures during degeneration are expected to lead to parameters that can be optimized to model the degeneration observed in the pathologic/MS spinal cord samples.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.