A combinatory microfluidic and in vivo modeling approach to evaluate collective migration during retinogenesis

一种组合微流体和体内建模方法来评估视网膜发生过程中的集体迁移

• 批准号: 1804411
• 负责人: Maribel Vazquez
• 金额: $ 30万
• 依托单位: CUNY City College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1804411&HistoricalAwards=false
• 关键词: combinatory; microfluidic; vivo; modeling; approach

The coordinated migration of groups of cells is a central element of tissue development as well as of stem cell-based repair in the nervous system. In an idealized model, stem-like cells (STLCs) are introduced into a damaged tissue and migrate collectively, as one unit, toward precise injury sites to reestablish neuronal connectivity. In truth, the effects of cues from a cell's genetic makeup and its external environment on collective migration have been only partially explored. The developing retina provides a unique opportunity for quantitative study of collective migration to support the natural development of vision. As the signaling cues that guide retinal development are surprisingly similar among different species, the common fruit fly (Drosophila melanogaster) provides a simple yet excellent model to study this phenomenon. The study of how STLCs naturally migrate to initiate or re-initiate connectivity of the neurons with the retina will greatly deepen our understanding of retinal development and could greatly advance therapies to restore vision. Educational efforts will develop opportunities for undergraduates to teach and mentor summer high school students in retinal research by establishing integrated course modules that engage student teams in device prototyping, design innovation and cell-based laboratory experiments.This project will evaluate the collective migration of STLCs using in vivo genetics to regulate intracellular Fibroblast Growth Factor Receptor (FGF-R) signaling in Drosophila melanogaster (as a model) and microfluidic systems to control extrinsic FGF environments. The study is motived by knowledge that FGF signaling pathways in the Drosophila model are known to mediate the neural migration needed to initiate vision via the optic stalk, but it is not clear if FGF-R regulation alone is sufficient. The system will facilitate genetically-controlled study of FGF-R-mediated chemotaxis with micrometer resolution in tandem with quantitative study of the intercellular signaling needed to preserve spatial cohesion across motile STLC collectives (cell-cell adhesion via innexin-1). The Research Plan is organized under 3 aims. AIM 1 will develop a microfluidic model of the retinal optic stalk (the portion of the developing retina between the Drosophila Brain Lobe and the Eye Imaginal Disc) to generate controlled, extrinsic FGF fields. Experiments will prototype an in vitro system on the scale of the Drosophila retina and develop an analytical model (Finite Element simulation) to describe concentration gradient fields therein. Characteristics of collective in vitro migration will be evaluated by targeted, quantitative parameters. AIM 2 will examine the collective migration of glial and neuronal cells expressing genetically-modified elements of FGF-R, i.e., cells with and without functional receptors. Genetic FGF-R alteration will be used to experimentally determine contribution of FGF-R to the size of motile collectives as well as their neural composition and cell-cell adhesion within the microfluidic system. AIM 3 will evaluate in vivo retinal formation using genetically modified FGF-R glial and neuronal collectives. The specific role for FGF-R during collective migration in vivo will be studied by examining retinogenesis using neural collectives lacking FGF-R function.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.

细胞群的协调迁移是组织发育以及神经系统中基于干细胞的修复的核心要素。在一个理想的模型中，干细胞样细胞(STLC)被引入受损组织中，并作为一个单位集体迁移到精确的损伤部位，以重建神经元连接。事实上，细胞的遗传组成及其外部环境对集体迁移的影响还只被部分探索过。视网膜的发育为集体迁移的定量研究提供了独特的机会，以支持视力的自然发展。由于引导视网膜发育的信号信号在不同物种之间惊人地相似，常见的果蝇(果蝇)为研究这一现象提供了一个简单但极好的模型。研究STLC如何自然迁移以启动或重新启动神经元与视网膜的连接将极大地加深我们对视网膜发育的理解，并可能极大地促进恢复视力的治疗。教育努力将为本科生提供教授和指导夏季高中生视网膜研究的机会，方法是建立综合课程模块，让学生团队参与设备原型、设计创新和基于细胞的实验室实验。该项目将评估STLC的集体迁移，使用体内遗传学来调节细胞内成纤维细胞生长因子受体(FGF-R)信号在果蝇(作为模型)和微流体系统，以控制外部的FGF环境。这项研究的动机是已知果蝇模型中的成纤维细胞生长因子信号通路介导了通过视柄启动视觉所需的神经迁移，但尚不清楚仅有成纤维细胞生长因子-R调节是否足够。该系统将有助于以微米级分辨率对成纤维细胞生长因子受体介导的趋化作用进行基因控制研究，同时定量研究维持运动的STLC集体之间的空间凝聚力所需的细胞间信号(通过innexin-1进行的细胞间黏附)。研究计划是在三个目标下组织的。目的1将建立视网膜视柄(位于果蝇脑叶和眼睛成像盘之间的发育中的视网膜的一部分)的微流体模型，以产生受控的、外部的成纤维细胞生长因子(FGF场)。实验将在果蝇视网膜的规模上建立一个体外系统的原型，并开发一个分析模型(有限元模拟)来描述其中的浓度梯度场。集体体外迁移的特征将通过有针对性的定量参数进行评估。目的2将研究表达转基因成纤维细胞生长因子受体元件的神经胶质细胞和神经细胞的集体迁移，即具有和不具有功能性受体的细胞。在微流控系统中，将利用遗传的成纤维细胞生长因子受体改变来实验确定成纤维细胞生长因子受体对运动集合体的大小及其神经组成和细胞-细胞黏附的贡献。AIM 3将使用转基因的成纤维细胞生长因子受体神经胶质细胞和神经细胞集体评估体内视网膜的形成。本奖项反映了NSF的法定使命，通过使用基金会的智力优势和更广泛的影响审查标准进行评估，认为值得支持。

项目成果

Re: “Organ-On-A-Chip Technologies for Advanced Blood–Retinal Barrier Models,” by Ragelle et al.

回复：“用于先进血液的器官芯片技术——视网膜屏障模型”，作者：Ragele 等人。

• DOI: 10.1089/jop.2022.0003
• 发表时间: 2022
• 期刊: Journal of Ocular Pharmacology and Therapeutics
• 影响因子: 2.3
• 作者: Castro, Natalia G.;Cohen, Rick;Vazquez, Maribel
• 通讯作者: Vazquez, Maribel