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

胎盘是位于母胎交界处的一个复杂器官，在怀孕期间支持胎儿的发育。在人类胎盘发育早期，胎盘滋养细胞(TB)的一个亚群，称为侵袭性TB，穿透子宫组织，改变子宫动脉的结构。动脉的这种重塑对于用母体血液有效地灌流胎盘和确保胎儿营养至关重要。结核病侵袭不足和过多都与潜在的严重妊娠障碍有关；因此，结核病侵袭受到高度管制。这种精确的调控是通过结核病细胞和胎盘界面上其他类型细胞之间的复杂相互作用来协调的。侵袭性结核病的形成及其侵袭行为也受到外部刺激的影响，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启用微流体的人类结核病发展的体外模型，用于在3D培养中定量分析结核病的分化和侵袭。拟议方法的一个关键特征是使用来自人类胚胎干细胞(HESCs)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界刺激和其他细胞类型与结核病之间的细胞间通讯对结核病分化和迁移的影响。所获得的知识有可能指导胎盘紊乱的治疗，从而影响孕妇及其婴儿的健康。研究与教育活动的结合包括：针对高中生的关于“孕期工程”的互动讲座-讨论模块；旨在培养对多能性干细胞研究的科学、技术、伦理和监管的普遍认识的高中生讲习班；与北卡罗来纳州立大学的Juntos方案合作，增加拉丁裔青年参与STEM学科；该提案侧重于创建一种微流体设备，该设备将能够在体外分析早期人类胎盘发育过程中，胎盘滋养层(TB)细胞的一部分(侵袭性TBS)穿透子宫组织，改变子宫动脉的结构，并建立重塑，使胎盘能够有效地与母体血液灌流，并确保胎儿营养。结核分化为侵袭性表型和随后侵袭的精确调控是通过结核细胞与胎盘界面其他类型细胞之间的动态相互作用来协调的，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)。此外，结核分枝杆菌的分化和侵袭受到外界刺激的影响，例如氧气浓度的变化或环境污染物。缺乏系统和定量研究外界刺激和/或细胞间通讯对结核病分化和侵袭的影响的实验平台。该项目解决了阻碍现实体外模型发展的两个主要限制。首先，从怀孕早期就可以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(HESCs)的结核病作为体内结核病发展的体外替代品。其次，在3D培养中对结核病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控平台，该平台能够在3D细胞培养中进行活细胞成像，并允许回收特定细胞用于转录组分析。活细胞成像的方法借鉴了线虫研究中使用的实验策略。该项目有三个目标：1)研究环境污染物双酚A(BPA)对结核杆菌侵袭的抑制作用，从而作为验证微流体平台的试验台；2)研究氧气浓度和氧气梯度对结核杆菌分化和侵袭的影响；3)研究巨噬细胞和结核杆菌之间的细胞间通讯对结核杆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，拟议的体外系统将使研究妊娠障碍的分子机制和评估潜在的治疗方法成为可能。特别是，这些实验将有助于阐明氧气梯度和结核-巨噬细胞相互作用在结核病发展中的作用。其次，所提出的跟踪活细胞的微流控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。

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.