| NSF Org: |
CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems |
| Recipient: |
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| Initial Amendment Date: | August 7, 2017 |
| Latest Amendment Date: | July 29, 2018 |
| Award Number: | 1706118 |
| Award Instrument: | Continuing Grant |
| Program Manager: |
Stephanie George
CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems ENG Directorate for Engineering |
| Start Date: | August 15, 2017 |
| End Date: | July 31, 2022 (Estimated) |
| Total Intended Award Amount: | $400,000.00 |
| Total Awarded Amount to Date: | $400,000.00 |
| Funds Obligated to Date: |
FY 2018 = $115,792.00 |
| History of Investigator: |
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| Recipient Sponsored Research Office: |
2601 WOLF VILLAGE WAY RALEIGH NC US 27695-0001 (919)515-2444 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
NC US 27695-7905 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Engineering of Biomed Systems |
| Primary Program Source: |
01001819DB NSF RESEARCH & RELATED ACTIVIT |
| Program Reference Code(s): | |
| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.041 |
ABSTRACT
PI: Rao, Balaji M.
Proposal: 1706118
The 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.
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PROJECT OUTCOMES REPORT
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This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Cells of the trophectoderm, the outer layer of the blastocyst-stage embryo, are precursors of all trophoblast cell types in the placenta. Upon implantation of the embryo, the trophectoderm eventually gives rise to three key trophoblast cell types – the villous cytotrophoblast (vCTB), the syncytiotrophoblast (STB) and the extravillous cytotrophoblast (EVT). The vCTB is a progenitor cell in the villus (putatively with stem cell like properties) that can differentiate into STB or EVT. The multinucleate STB forms the membrane interface between the fetus and the mother and is in direct contact with maternal blood. The EVTs can be further classified as column cytotrophoblasts, which anchor the placenta in the uterus, and invasive cytotrophoblast that originate at the distal end of the column cytotrophoblasts, penetrate the uterine tissue and play a key role in remodeling the uterine arteries. Abnormalities in trophoblast development and invasion are associated with several potentially serious yet relatively common pregnancy disorders. In this project, we have developed experimental systems that will enable quantitative mechanistic studies on early human trophoblast development in vitro. Recently, human trophoblast stem cells (hTSCs) from first trimester placental tissues or blastocyst-stage embryos have gained increasing prominence as a model system for studies on placental development. We have derived hTSCs that are functionally equivalent to those obtained from the placenta or human embryos using human pluripotent stem cells (hPSCs). Our approach provides important advantages. Restricted accessibility of human embryos and placental samples from early gestation, and low genetic diversity of existing lines, limit the use of placenta- or blastocyst-derived hTSCs. In contrast, hPSCs such as human induced pluripotent stem cells (hiPSCs) are a more accessible source for generation of hTSCs from non-fetal sources. Second, unlike hTSCs derived from primary placental samples or embryos where the projected pregnancy outcome is uncertain, hiPSC-derived hTSCs may provide models of validated normal and pathological trophoblast development. Finally, genertically modified hTSCs can be easily generated through use of genetically modified iPSCs; technologies for such modifications in iPSCs are well-established. We have also developed a chemically defined 2D and 3D culture systems for differentiation of hTSCs. These systems will enable investigation of the molecular mechanisms underlying human trophoblast differentiation.
Last Modified: 11/28/2022
Modified by: Balaji M Rao
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