A Microfluidics-Enabled In Vitro Model for Early Human Placental Development

用于早期人类胎盘发育的微流体体外模型

• 批准号: 1706118
• 负责人: Balaji Rao
• 金额: $ 40万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1706118&HistoricalAwards=false
• 关键词: Microfluidics; Enabled; Vitro; Model; Early

PI: Rao, Balaji M.Proposal: 1706118The placenta is a complex organ at the maternal-fetal interface that sustains fetal development during pregnancy. During early human placental development, a subset of trophoblast (TB) cells of the placenta, called the invasive TB, penetrates the uterine tissue and alters the structure of the uterine arteries. This remodeling of the arteries is critical for enabling efficient perfusion of the placenta with maternal blood and ensuring fetal nutrition. Both insufficient and excessive TB invasion are associated with potentially serious pregnancy disorders; thus TB invasion is highly regulated. The precise regulation is orchestrated by a complex interplay between TB cells and other cell types at the placental interface. Formation of invasive TB and their invasion behavior is also affected by external stimuli such as variations in oxygen concentration or environmental contaminants. The goal of this project is to develop a microfluidics-enabled in vitro model of human TB development for quantitative analysis of TB differentiation and invasion in 3D cultures. A key feature of the proposed approach is to use TB derived from human embryonic stem cells (hESCs) as a bona fide surrogate for human TB. This model system will be used to assess the effect of external stimuli and intercellular communication between other cell types and TB, on TB differentiation and migration. The knowledge gained has the potential to guide therapies for placental disorders and thus impact the health of pregnant women and their babies. Integration of research with education activities include: an interactive lecture-discussion module on "Engineering in Pregnancy" targeted towards high-school students; a high school student workshop designed to create general awareness of the science, technology, ethics and regulation of pluripotent stem cell research; collaboration with the Juntos Program at NC State to increase participation of Latino youth in STEM disciplines; and, active engagement of undergraduate students in targeted research projects that contribute to the overall project goals.The proposal focuses on creating a microfluidic device that will enable in vitro analysis of early human placental development during the time in which a subset of trophoblast (TB) cells of the placenta (invasive TBs) penetrates the uterine tissue, alters the structure of the uterine arteries and establishes remodeling that enables efficient perfusion of the placenta with maternal blood and ensuring fetal nutrition. The precise regulation of TB differentiation to an invasive phenotype and subsequent invasion is orchestrated by dynamic interactions between TB cells and other cell types at the placental interface, specifically cells of the uterine decidua and certain types of immune cells (decidual natural killer (dNK) cells and decidual macrophages). Furthermore, TB differentiation and invasion are affected by external stimuli such as variation in oxygen concentration or environmental contaminants. Experimental platforms to systematically and quantitatively probe the effects of external stimuli and/or intercellular communication on TB differentiation and invasion are largely lacking. The project addresses the two major limitations that impede the development of realistic in vitro models. First, availability of TB from early gestation is very limited. The project will use TB derived from human embryonic stem cells (hESCs) as an in vitro surrogate for TB development in vivo. Second, quantitative analysis of TB differentiation and migration in 3D cultures is experimentally challenging. The project will develop a microfluidic platform that enables live cell imaging in 3D cell culture and allows recovery of specific cells for transcriptome analysis. The approach for live cell imaging draws from experimental strategies used in studies on C. elegans. The project has three objectives: 1) Investigate the effect of an environmental contaminant bisphenol A (BPA), a chemical shown to have an inhibitory effect on TB invasion, on TB invasion in the microfluidic platform, thus serving as a testbed to validate the microfluidic platform, 2) Investigate the effect of oxygen concentration and oxygen gradients on TB differentiation and invasion and 3) Investigate the effect of intercellular communication between macrophages and TB on TB differentiation and invasion. The project is potentially transformative due to two expected outcomes. First, the proposed in vitro system will enable the investigation of molecular mechanisms underlying pregnancy disorders and evaluation of potential therapies. In particular, the experiments will help elucidate the role of oxygen gradients and TB-macrophage interactions in TB development. Second, the proposed microfluidic platform and strategies for tracking live cells will be broadly applicable to cells in 3D culture, including co-culture of different cell types.

PI：Rao，Balaji M.提案：1706118胎盘是母胎界面的复杂器官，在妊娠期间维持胎儿发育。在人类胎盘发育早期，胎盘滋养层（TB）细胞的一个子集，称为侵袭性TB，穿透子宫组织并改变子宫动脉的结构。这种动脉重塑对于使母体血液有效灌注胎盘和确保胎儿营养至关重要。结核病侵袭不足和过度均与潜在的严重妊娠障碍相关;因此结核病侵袭受到高度管制。精确的调节是由胎盘界面上TB细胞和其他细胞类型之间复杂的相互作用协调的。侵袭性TB的形成及其侵袭行为也受到外部刺激的影响，例如氧气浓度或环境污染物的变化。 该项目的目标是开发一种微流体支持的人类结核病体外模型，用于定量分析结核病在3D培养中的分化和侵袭。所提出的方法的一个关键特征是使用来自人类胚胎干细胞（hESC）的结核病作为人类结核病的真正替代品。该模型系统将用于评估外部刺激和其他细胞类型与TB之间的细胞间通讯对TB分化和迁移的影响。所获得的知识有可能指导胎盘疾病的治疗，从而影响孕妇及其婴儿的健康。将研究与教育活动相结合，包括：针对高中生的关于“怀孕工程学”的互动式讲座讨论模块;旨在提高对多能干细胞研究的科学、技术、道德和监管的普遍认识的高中生讲习班;与北卡罗来纳州的Juntos方案合作，以增加拉丁美洲青年对STEM学科的参与;该提案的重点是创建一种微流体装置，该装置将能够在体外分析早期人类胎盘发育，在此期间，胎盘滋养层（TB）细胞的一个子集，（侵入性结核病）穿透子宫组织，改变子宫动脉的结构并建立重塑，使胎盘能够有效地灌注母体血液并确保胎儿营养。 TB分化为侵袭性表型和随后的侵袭的精确调节是由TB细胞与胎盘界面处的其他细胞类型（特别是子宫蜕膜细胞和某些类型的免疫细胞（蜕膜自然杀伤（dNK）细胞和蜕膜巨噬细胞））之间的动态相互作用协调的。此外，TB分化和侵袭受外部刺激如氧浓度变化或环境污染物的影响。 系统地和定量地探测外部刺激和/或细胞间通讯对TB分化和侵袭的影响的实验平台在很大程度上缺乏。 该项目解决了两个主要的限制，阻碍了现实的体外模型的发展。 首先，从妊娠早期获得结核病的机会非常有限。该项目将使用来自人类胚胎干细胞（hESC）的结核病作为体内结核病发展的体外替代品。 其次，在3D培养中TB分化和迁移的定量分析在实验上具有挑战性。该项目将开发一个微流体平台，使活细胞成像在3D细胞培养，并允许特定细胞的转录组分析回收。活细胞成像的方法借鉴了C.优美的 该项目有三个目标：1）研究环境污染物双酚A（BPA）（一种显示对TB侵袭具有抑制作用的化学物质）对微流体平台中TB侵袭的影响，从而用作验证微流体平台的测试平台，2）研究氧浓度和氧梯度对TB分化和侵袭的影响，以及3）研究巨噬细胞与结核菌之间的细胞间通讯对结核菌分化和侵袭的影响。 由于两个预期结果，该项目具有潜在的变革性。首先，所提出的体外系统将能够调查潜在的妊娠疾病的分子机制和评估潜在的治疗方法。特别是，这些实验将有助于阐明氧梯度和TB-巨噬细胞相互作用在TB发展中的作用。其次，所提出的用于跟踪活细胞的微流体平台和策略将广泛适用于3D培养中的细胞，包括不同细胞类型的共培养。