Extracellular matrix surface regulates self-assembly of three-dimensional placental trophoblast spheroids

Wong, Michael K.; Shawky, Sarah; Aryasomayajula, Aditya; Green, Madeline A.; Ewart, Tom; Selvaganapathy, P. Ravi; Raha, Sandeep

doi:10.1371/journal.pone.0199632

Cited by 35 publications

(33 citation statements)

References 55 publications

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“…Accumulating evidence in other organ systems and cancer models suggest that culturing cells in three-dimensions (3D) can provide more anatomically- and physiologically-relevant results compared to traditional 2D monolayer cultures 7 . While some researchers have begun to culture placental cells as 3D spheroids or organoids 8–12 , our overall understanding of the transcriptomic changes and functional outcomes associated with its formation remains preliminary. Characterization studies contrasting novel 3D spheroids against 2D monolayers are especially lacking – yet, these are necessary to justify its use and advancement, as the theorized advantages ( e .…”

Section: Introductionmentioning

confidence: 99%

Transcriptomic and functional analyses of 3D placental extravillous trophoblast spheroids

Wong

Wahed

Shawky

et al. 2019

Sci Rep

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Placental extravillous trophoblast (EVT) invasion is essential in establishing proper blood supply to the fetus during pregnancy. However, traditional 2D in vitro systems do not model the in vivo invasion process in an anatomically-relevant manner. Our objectives were to develop a 3D spheroid model that would allow better emulation of placental invasion in vitro and to characterize the transcriptomic and functional outcomes. HTR8/SVneo EVT cells were self-assembled into 3D spheroids using ultra-low attachment plates. Transcriptomic profiling followed by gene set enrichment and gene ontology analyses revealed major global transcriptomic differences, with significant up-regulations in EVTs cultured as 3D spheroids in canonical pathways and biological processes such as immune response, angiogenesis, response to stimulus, wound healing, and others. These findings were further validated by RT-qPCR, showing significant up-regulations in genes and/or proteins related to epithelial-mesenchymal transition, cell-cell contact, angiogenesis, and invasion/migration. A high-throughput, spheroid invasion assay was applied to reveal the dynamic invasion of EVTs away from the spheroid core into extracellular matrix. Lastly, lipopolysaccharide, dexamethasone, or Δ 9 -tetrahydrocannabinol exposure was found to impact the invasion of EVT spheroids. Altogether, we present a well-characterized, 3D spheroid model of EVT invasion and demonstrate its potential use in drug and toxin screening during pregnancy.

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Section: Introductionmentioning

confidence: 99%

Transcriptomic and functional analyses of 3D placental extravillous trophoblast spheroids

Wong

Wahed

Shawky

et al. 2019

Sci Rep

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“…Tissue stiffness is now well-established as a critically important regulator of a wide variety of cellular processes, including differentiation 23 and disease progression 24 ; but the role of extracellular tissue mechanics on trophoblast fusion has not previously been defined. Recent studies have demonstrated that ECM thickness affects fusion-related markers of mRNA and secreted proteins in trophoblasts 25 suggesting that mechanics may play a role, but whether trophoblast fusion is mechanically sensitive to disease-specific physiological cues remains undefined. Interestingly, recent evidence from in vivo imaging studies demonstrates that tissue stiffness varies significantly across patients diagnosed with pregnancy complications, including preeclampsia [26][27][28] , intra-uterine growth restriction 29 , and gestational diabetes mellitus 30 compared to normal placental tissue 26 (literature values presented in Fig.…”

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confidence: 99%

Mechanobiological regulation of placental trophoblast fusion and function through extracellular matrix rigidity

Sagrillo-Fagundes

Mok

et al. 2020

Sci Rep

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the syncytiotrophoblast is a multinucleated layer that plays a critical role in regulating functions of the human placenta during pregnancy. Maintaining the syncytiotrophoblast layer relies on ongoing fusion of mononuclear cytotrophoblasts throughout pregnancy, and errors in this fusion process are associated with complications such as preeclampsia. While biochemical factors are known to drive fusion, the role of disease-specific extracellular biophysical cues remains undefined. Since substrate mechanics play a crucial role in several diseases, and preeclampsia is associated with placental stiffening, we hypothesize that trophoblast fusion is mechanically regulated by substrate stiffness. We developed stiffness-tunable polyacrylamide substrate formulations that match the linear elasticity of placental tissue in normal and disease conditions, and evaluated trophoblast morphology, fusion, and function on these surfaces. our results demonstrate that morphology, fusion, and hormone release is mechanically-regulated via myosin-ii; optimal on substrates that match healthy placental tissue stiffness; and dysregulated on disease-like and supraphysiologically-stiff substrates. We further demonstrate that stiff regions in heterogeneous substrates provide dominant physical cues that inhibit fusion, suggesting that even focal tissue stiffening limits widespread trophoblast fusion and tissue function. These results confirm that mechanical microenvironmental cues influence fusion in the placenta, provide critical information needed to engineer better in vitro models for placental disease, and may ultimately be used to develop novel mechanically-mediated therapeutic strategies to resolve fusion-related disorders during pregnancy. The human placental barrier is responsible for several critical functions during pregnancy including nutrient transport, gas exchange, waste elimination and hormone secretion 1. The placenta hence directly impacts fetal development 2 , immune tolerance 3 , and gestational length 4 , each of which can profoundly affect long-term quality of life and healthcare economics for both mother and baby 5-8. Transport across this fetal-maternal interface is regulated by the syncytiotrophoblast, a multinucleated layer that forms the outer surface of the placental villi 9. The syncytiotrophoblast arises and is maintained by continuous fusion of mononuclear villous cytotrophoblasts (vCTBs) 10 , through a tightly regulated process that can only be partially recreated in vitro 11. Disruption of fusion results in placental malformation and aberrant villous trophoblast turnover 10 , which is associated with life-altering pregnancy complications such as preeclampsia 12 and intrauterine growth restriction 13. Several biochemical factors are known to regulate placental trophoblast fusion in vitro and in vivo, including growth factors 14-16 , hormones 17 , proteases 18-20 , transcription factors 21 and membrane proteins 22. Despite this wealth of information, fusion remains a stochastic and poorly controlled process in cult...

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“…Pioneer work on placental spheroids from, for example, immortalized EVTs, trophoblast Jar cell line, and the choriocarcinoma cell line BeWo have been used as 3D models to study aspects of human placental development in vitro [135][136][137] and, when co-cultured with human immortalized endometrial stromal cell lines, have served as a model for TB invasiveness [138,139]. Moreover, attempts have been performed to develop 3D models for human endometrial cells that could also be used to investigate endometrial cancer [140][141][142].…”

Section: Stem Cells For Human Placental Development and Organoidsmentioning

confidence: 99%

The TGFβ Family in Human Placental Development at the Fetal-Maternal Interface

2020

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Emerging data suggest that a trophoblast stem cell (TSC) population exists in the early human placenta. However, in vitro stem cell culture models are still in development and it remains under debate how well they reflect primary trophoblast (TB) cells. The absence of robust protocols to generate TSCs from humans has resulted in limited knowledge of the molecular mechanisms that regulate human placental development and TB lineage specification when compared to other human embryonic stem cells (hESCs). As placentation in mouse and human differ considerably, it is only with the development of human-based disease models using TSCs that we will be able to understand the various diseases caused by abnormal placentation in humans, such as preeclampsia. In this review, we summarize the knowledge on normal human placental development, the placental disease preeclampsia, and current stem cell model systems used to mimic TB differentiation. A special focus is given to the transforming growth factor-beta (TGFβ) family as it has been shown that the TGFβ family has an important role in human placental development and disease.

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Extracellular matrix surface regulates self-assembly of three-dimensional placental trophoblast spheroids

Cited by 35 publications

References 55 publications

Transcriptomic and functional analyses of 3D placental extravillous trophoblast spheroids

Transcriptomic and functional analyses of 3D placental extravillous trophoblast spheroids

Mechanobiological regulation of placental trophoblast fusion and function through extracellular matrix rigidity

The TGFβ Family in Human Placental Development at the Fetal-Maternal Interface

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