3-D tissue model of placenta as a predictive platform to study microbial infections during pregnancy

During pregnancy, the rapidly developing fetus is enshrouded by a remarkable structure: the placenta. Researchers hope to better understand many peculiarities of placental development, including maternal and fetal pathologies associated with microbial infections during pregnancy. Studying the various cell types making up this unique structure, however, is challenging.

In a new study, Cheryl Nickerson, a researcher at Arizona State University’s Biodesign Institute, along with colleagues from University of Pittsburgh, Johns Hopkins University and the Magee-Womens Research Institute (MWRI), examined a specialized class of placental cells known as trophoblasts — critical players in placental development. The new study looked at the response of trophoblasts to infection with pathogens that can harm the developing fetus.

Given the many dynamic functions performed by the placenta, including protecting the unborn baby against infection, it is not surprising that placental dysfunction can lead to serious complications during pregnancy, including fetal and maternal disease and death. Intrauterine infections, which may result from failures in the placenta’s defensive function, affect 10 percent of all live births.

The new research paves the way for more effective strategies to address placental abnormalities as well as exploring foundational issues in microbiology associated with microbial infection and resistance processes.

The group’s findings appear in the current issue of the journal Science Advances.

“We are excited to participate in this multidisciplinary collaboration with our colleagues, which leverages my lab’s previous work in the development of 3-D cell-based modelsof human tissues, including placenta, that structurally and functionally mimic in vivo tissues and their application as predictive platforms to study host-pathogen interactions and infectious disease,” Nickerson said.  “I believe that the advanced 3-D placental cell culture developed in this study offers an improved model system for the study of human placentation development and microbial infections during pregnancy that can threaten the mother and her unborn child.”

According to Carolyn Coyne, who led the new study, the human placenta is unique and unlike that of other placental mammals. Coyne is an associate professor of microbiology and molecular genetics at the Pittsburgh School of Medicine and member of the MWRI. “Until now, we have not been able to use many cell-line based models to learn more about the structure and function of the human placenta, and that has kept us from answering several fundamental questions about how the placenta can prevent most, but not all, maternal infections from causing problems for the baby,” Coyne said.

During pregnancy, a protective layer of placental cells is continually replenished by underlying cells known as cytotrophoblasts. The biological mechanisms causing cytotrophoblasts to fuse into multi-nucleated syncytiotrophoblasts are unknown and until now have been tricky to study in the laboratory. Animation by Jason Drees.

The new study explores trophoblastic cells, following a trajectory during which some of them fuse to form a continuous cell layer known as the placental syncytia. The fused cells, with their multiple cell nuclei, are known as syncytiotrophoblasts. Using a 3-D cell culture method to grow trophoblasts in combination with another cell type that makes up blood vessels, this study developed a placental model that closely mirrors the structure and behavior of trophoblasts in the living placenta, including their transition into syncytiotrophoblasts.

A crucial property of these specialized placental cells is their ability to protect the developing embryo from microbial infection. The 3-D cell cultures used in the new study to model trophoblast behavior likewise exhibited infection-thwarting properties when challenged with pathogens that can cross the placenta, and displayed functional similarities to primary human trophoblasts found in the body. The study further identifies an important suite of syncytiotrophoblast-specific genes that were expressed in the 3-D placental models in a manner that was similar to that of cells derived from full-term human placentas. 

Life-giving membrane

The placenta exists in all so-called eutherian mammals, including humans. It is sometimes referred to as a chimerical organ — one composed of both maternal and fetal cells. As such, the placenta acts as a vital nexus between mother and developing offspring, performing duel roles by providing oxygen, glucose and other nutrients to the fetus while filtering waste products from the developing baby’s blood.

Once the fetus uses up oxygenated blood, it is returned to the placenta, where carbon dioxide is removed and new oxygen is picked up. Waste products from the baby are released into the mother’s circulation, ultimately to be expelled in urine. Every minute during pregnancy, around a pint of blood is pumped into the uterus, where it exchanges nutrients with the placenta. 

The placental barrier also envelops the developing embryo, shielding it from viruses, bacteria and parasites. This biophysically protective layer is formed when individual trophoblast cells fuse together to form a continuous cell aggregate known as the placental syncytia, which is bathed in maternal blood.

During pregnancy, this protective layer of cells is continually replenished by underlying cells known as cytotrophoblasts. The biological mechanisms causing cytotrophoblasts to fuse into syncytiotrophoblasts are unknown and until now have been tricky to study in the laboratory.

Conventional cell cultures are usually grown on flat dishes, where they adhere and spread out laterally in two dimensions. While enormous strides in understanding how tissues develop normally or transition to disease have resulted from 2-D cell culture study, the contrived nature of these cultures produce cell behaviors that can differ radically from their physiologically relevant counterparts in the human body.  This is not surprising, given that tissues and organs function in a 3-D environment.

Nickerson is a pioneer in the culture of cells in 3-D in an effort to more closely approximate the architecture and behavior of tissues in the body, and thus the environment that pathogens experience in the infected host. The technique more faithfully replicates the complex behavior and unique cell interactions taking place in living systems, making 3-D cell cultures vastly more relevant to the study of human health and disease, compared with conventional approaches. Such cultures may someday obviate the need for animal studies, which do not always provide a reliable surrogate for the behavior of human cells. Nickerson’s establishment and characterization of a variety of 3-D tissue models as human surrogates for infectious disease have helped to advance the understanding of infectious processes.

Probing the third dimension

In the current study, primary human trophoblasts were cultured in three dimensions using a dynamic cell culture device known as a rotating wall vessel (RWV) bioreactor. The device was originally designed by NASA to mimic the low fluidic shear force conditions experienced by cells in the quiescent environment of microgravity. However, Nickerson and colleagues previously showed that the RWV also provides a low-fluid-shear growth environment similar to that encountered by pathogens in certain regions of the body that are commonly infected by pathogens (including in utero); and it is known that fluid shear is important for cell and tissue differentiation and development in humans.

The RWV bioreactor is a rotating cylindrical apparatus that contains cells attached to porous bead scaffolds and bathed in a medium of nutrients. As the device rotates, the cells are cultured under conditions of physiological fluid shear that closely mimic that of their native tissue, thereby allowing the cells to differentiate into well-organized 3-D tissue-like structures which replicate many of the in vivo tissue characteristics.

The researchers used RWV 3-D cell culture to grow a type of trophoblast cell known as JEG-3 in conjunction with microvascular endothelial cells. They demonstrated for the first time that culturing with 3-D methods caused populations of JEG-3 cells to successfully transition into terminally differentiated syncytiotrophoblasts, capable of resisting microbial infection like their natural counterparts. 

Results showed high rates of spontaneous trophoblast fusion in 3-D cells, with 25 to 50 percent of the observed nuclei occurring in multinucleated cells. When the latter are exposed to pathogens, including the bacterium Toxoplasma gondii — a major source of congenital infections worldwide — they display a similar level of resistance found in their naturally-occurring counterparts in the human body.  The authors believe their 3-D culturing system provides a useful platform for investigating the resistance of syncytiotrophoblasts to viral infection as well.

Compared with 2-D-cultured cells, the JEG-3 cells grown as 3-D cultures showed a similar profile of transcribed genes to syncytiotrophoblasts in the living placenta, including a number of genes exclusively expressed in syncytiotrophoblasts, when subjected to RNA sequencing analysis.  Specifically, 55 genes with unique transcriptional profiles associated with primary human trophoblastic cells were found in the 3-D JEG-3 cells, several of which are exclusive to syncytiotrophoblasts, reaffirming the utility of 3-D cultures for effectively modeling in vivo cell types.

Nickerson stresses the broad applicability of the research:

“The results of this and future studies using 3-D tissue models will offer new insight into the transition between normal and pathological pregnancies, with implications for the development of safe, efficacious therapeutics to protect against the significant morbidity and mortality associated with congenital infections, including those caused by cytomegalovirus, HIV and Zika virus.”  

Cheryl Nickerson is co-director of the Biodesign Institute’s Center for Infectious Diseases and Vaccinology and is a professor at ASU’s School of Life Sciences. She is editor-in-chief of Nature Partner Journals (npj) Microgravity.