The Ephemeral Life of the Placenta

ABOVE: © shutterstock.com, SciePro

We have all had one, and we owe our lives to it. It’s the first organ to develop and it simultaneously serves as the lungs, kidneys, immune system, and digestive tract, to name a few, in a fetus while it develops these systems. Despite being one of the most important organs, the placenta is one of the least understood.

“It’s such a fascinating organ,” said Norah Fogarty, a developmental biologist at King’s College London. “We know so little about it, but there’s also this kind of intrigue about the placenta.” This mysterious organ has inspired lore and customs for centuries.

Throughout gestation, the fetus depends entirely on the placenta. The discoid-shaped organ serves as a barrier between the parent and child. Although some researchers describe the placenta as an evolutionary battleground due to the mix of maternal and paternal DNA both vying for resources, it is also a space where compromise prevails to ensure the health of both parties. Research has linked abnormal placental development to a number of pregnancy complications, including preeclampsia, fetal growth restriction, placental abruption, and preterm labor, collectively referred to as the great obstetrical syndromes.¹ Preeclampsia, characterized by high blood pressure and increased protein in the urine after 20 weeks of pregnancy, occurs in 3-5% of pregnancies.² Preeclampsia ranges from mild to severe and can be life-threatening for the parent and child. Currently, the only cure is delivery of the baby and placenta.

We also don't know what's happening in normal placenta development.

 —Norah Fogarty, King’s College London

The dearth of treatments for preeclampsia stems from a greater gap in our knowledge. “We also don’t know what’s happening in normal placenta development,” said Fogarty. The lack of physiologically relevant models of placental development stymies efforts to close these knowledge gaps. Although animal models have provided valuable insight into the organ’s development, the placenta is one of the most evolutionarily divergent organs, and considerable differences in the developmental trajectory, morphology, and degree of placental invasion into the uterine wall demand caution when extrapolating data from other species to humans.³ Additionally, several ethical and logistical obstacles hinder the study of early placenta development in humans. 

These obstacles led researchers to develop in vitro models, but until recently it wasn’t clear how robust these models could be. “The placenta has been difficult to capture in the dish,” said Fogarty. Now, a few key advancements in cell culture techniques over the last decade have breathed new life into the field, and many hope that these models hold the key to unlocking the secrets of the space between. 

A black box

Following fertilization, one cell becomes two, and those become four and so on, until the zygote transforms into a blastocyst around six days post fertilization (dpf).⁴ The blastocyst, or the preimplantation embryo, comprises of an inner cell mass (ICM) swaddled by an outer layer of cells that make up the trophectoderm. The trophectoderm, which is home to nearly 90 percent of the blastocyst cells, develops into the placenta while the ICM gives rise to the fetus. 

Fogarty wants to understand the molecular processes that orchestrate these early developmental stages. As an undergraduate student at Trinity College Dublin, Fogarty’s interest in fetal health and development began when she enrolled in a course on molecular medicine that focused on treating diseases of adulthood. “It led me to think ‘you know, we’re focusing all this time and research into treating diseases in the adult, but if we can help babies be born as healthy as possible and grow up to be healthy adults, then we would likely eradicate a lot of these diseases,’” said Fogarty.

Near the end of her studies, she came across an email that piqued her interest: It was an advertisement about a PhD project on placenta development at the University of Cambridge. She applied and got the position where she studied transcriptional dynamics in the human placenta under the joint supervision of Graham Burton and Anne Ferguson-Smith. Following her doctoral studies, Fogarty joined the lab of stem cell and developmental biologist Kathy Niakan at the Francis Crick Institute to continue her investigations into the molecular drivers of early cell fate. 

Transcription factors orchestrate trophectoderm development and differentiation. Using comparative analyses, researchers previously demonstrated that two such factors, octamer-binding transcription factor 4 (OCT4) and caudal-type homeobox-2 (CDX2), exhibit temporally and spatially distinct expression patterns in the embryos of mice and humans.⁵ Considering the divergent expression patterns between the two species, Fogarty was curious about the function of OCT4 during human embryo development. To study this, she turned to CRISPR-Cas9-mediated genome editing. Deletion of the gene encoding OCT4 from early zygotes donated from patients of IVF clinics led to a downregulation of trophectoderm genes, including CDX2, and compromised the development of the blastocyst.⁶ In contrast, when the researchers manipulated mouse embryos in a similar manner, the blastocyst formed but its maintenance was compromised. Fogarty’s research detailed functional consequences of species-specific gene expression patterns, further illustrating why mouse models may fail to capture key developmental events in humans.

Around six to seven dpf, the blastocyst implants into the surface of the uterine wall and begins its expansion.⁴ This area of the endometrium is transformed early on in pregnancy and acts as a fluffy bed in which the embryo grows. As the blastocyst burrows, the trophectoderm begins to differentiate into subtypes of trophoblast cells, starting with cytotrophoblasts, which are progenitor stem cells in the placenta that give rise to other trophoblasts. 

     

This point in development, approximately 14 dpf, corresponds to around the time of the first missed period, and it is typically the earliest point that most people realize that they are pregnant. Early zygote and blastocyst donations from patients of IVF clinics have helped shed light on this black box period of development, but human embryos are a limited resource and ethical concerns restrict their long-term use in the lab. These earliest days of development, although temporally distant from the clinical manifestation of preeclampsia, may lay the foundations for future problems. “In the last few years, there have been more hypotheses being developed that it’s a defect in the cytotrophoblast cell that sets the track for whether preeclampsia will develop or not,” said Fogarty.

There are a number of available in vitro models for the study of human placental development with varying degrees of physiological relevance.^2,4,7 Choriocarcinoma cells, derived from malignant tumors of trophoblasts, are genetically abnormal and mouse trophoblast stem cells, while a valuable tool, do not fully recapitulate the genetic and molecular milieu orchestrating human placental development. 

However, 2018 saw a major breakthrough: For the first time, researchers successfully generated bona fide human trophoblast stem cells (hTSC).⁸ The researchers demonstrated that either trophectoderm from the blastocyst or first-trimester placentas could be used to generate bipotent trophoblast stem cells. Now, researchers finally have access to pure trophoblast cells with the capacity for self-renewal. By tweaking what they feed the hTSC, researchers can transform the cells into different trophoblast subtypes. 

The hTSC are useful models for studying trophoblasts, while the embryo provides unrivaled access to studying trophectoderm development. Fogarty uses the hTSC alongside human embryos to study the signaling pathways that regulate trophectoderm and early trophoblast development and differentiation. Furthermore, hTSC are a useful platform for optimizing tools and developing hypotheses before testing them in valuable human embryos. “These experiments will further our understanding of hTSC and how they differ from the trophectoderm, but will also give us insights into trophoblast biology,” said Fogarty. She hopes that these tools can one day help reveal how defects emerging early in development set the stage for placental diseases like preeclampsia. 

Miniplacentas in a dish

As the invasion into the uterine wall wages on, cytotrophoblasts differentiate into syncytiotrophoblasts (SCT), which carve out villi, or frond-like structures that soon house the fetal capillary system.⁷ As SCT build larger and larger villi, cytotrophoblasts march forward to conquer a new frontier in search of nutrients to fuel the continued expansion. These rogue cytotrophoblasts go deeper into the uterine wall and differentiate into incredibly invasive extravillous trophoblasts (EVT). Once there, EVT hunt down uterine arteries, enlarge them, and hook them up to the placenta. Finally, around 10 weeks into the pregnancy, the parental circulation reaches the intervillous space.⁹ By 12 weeks, the placental blueprint is in place.

The uterine wall is home to glands, vessels, stromal cells, and immune cells that interact with the invading fetal cells to create a boundary between the parent and fetus.⁴ The relationship between the parent and the growing fetus is often portrayed as parasitic or antagonistic, a 9-month war waged from within. This is due in part to the highly invasive nature of EVT leaching nutrients, but also the presence of the fetus’ foreign DNA. But Ashley Moffett, a reproductive immunologist at the University of Cambridge, said that the relationship between the parent and the placenta isn’t simply friend or foe. “It’s a compromise, actually.”

Moffett, a doctor and pathologist by training, didn’t set out to study the placenta. In the 1980s, she was looking for a job at the hospital in Cambridge. The only available job at the time was in the maternity ward. “I was sort of banished to the maternity hospital without any interest at all in this, but I then, of course, realized that there were these major disorders and that they were completely understudied,” recalled Moffett. “Nobody knew anything about them really.”

While working in the maternity ward, Moffett recalled influential papers published in the early 1980s that suggested that preeclampsia results from a failure of EVT to properly invade the uterine wall.¹ She also remembered some peculiar looking cells that she came across in pathology training.  “I looked at every single organ in the body under the microscope,” said Moffett. “I realized that there were some cells in the uterus that I’ve never seen anywhere else.” 

She thought that they might be a kind of natural killer (NK) cell, so Moffett contacted Charlie Loke, one of her undergraduate professors from the University of Cambridge and an expert in reproductive immunology, to study these cells in the lab. After only a month in the lab they figured out that these cells were, in fact, a type of NK cell. “And [Loke] said, ‘you know, you’ll never go back to clinical medicine,’ and I didn’t ever go back to clinical medicine,” said Moffett. “That was the end of my medical career.” 

Uterine NK cells, which differ substantially from blood NK cells, dominate the immune cell landscape of the uterine wall bordering first trimester placentas.¹⁰ Moffett and others went on to characterize this unique immune cell and demonstrate its importance as a mediator between the needs of the mother to retain resources and the needs of the baby to grow. 

I realized that there were some cells in the uterus that I’ve never seen anywhere else.

 —Ashley Moffett, University of Cambridge

To explore the boundary between the parent and fetus, Moffett and her team used single-cell RNA sequencing on placental and endometrial samples donated by patients who underwent elective pregnancy termination in the first trimester.¹¹ They identified transcription factors that orchestrate cytotrophoblast differentiation into SCT or EVT but also uncovered three subtypes of NK cells with distinct immune regulation and cell-cell communication profiles. Their findings further highlighted the compromise between parent and fetus, suggesting that NK cells keep a check on EVT expansion while these cells protect the fetus from parental immune responses. 

Around the same time in 2018, Moffett and her team and Martin Knofler’s research team at the Medical University of Vienna separately published the first organoid models for trophoblasts.^12,13 These three-dimensional organoid models offered another step towards a physiologically relevant model that recapitulates certain aspects of the in vivo environment. To build a mini-placenta, the researchers isolated proliferative cells from first trimester placenta tissue and cultured them in a special cocktail chock full of growth factors that coax trophoblast development and assembly into a three dimensional blob of cells. Not only did the trophoblast organoids retain transcriptomic and methylation patterns characteristic of in vivo first trimester trophoblasts, but they also developed hormone-secreting SCT with intricate structures akin to villi as well as migratory EVT. These self-replicating mini-placentas even produced enough of the hormone chorionic gonadotropin to test positive on an at-home pregnancy test.¹² 

In the 1980s, Moffett gained access to rare first trimester pregnancy hysterectomies, which included the entire uterus. She safely tucked these samples away with the hope that one day new tools would emerge to explore their cellular intricacies. Earlier this year, Moffett returned to her historical hysterectomy samples and published a spatially resolved multiomics single-cell atlas that captures the trajectory of trophoblast differentiation as the cells invade and transform the arteries in the uterine wall.¹⁴ This rich resource identified transcription factors and key cell-cell interactions, including uterine NK cells in close proximity with EVT. Furthermore, they found many of the same factors expressed on EVT derived from hTSC and primary trophoblast organoids. “We now have a trajectory of the whole invading trophoblast in humans for the first time, and I think the organoids do recapitulate that quite well,” said Moffett. 

After a long career, Moffett recently handed over the keys to her lab, but she hopes these organoids will go on to provide a relevant platform for studying important placental biology questions that have relevance to placental disorders like preeclampsia. 

Early Placenta Development Sets The Stage During early pregnancy, the placenta remodels the uterine environment to support fetal growth.

DAYS 5-6 Approximately five days post fertilization (dpf), the blastocyst develops. The inner cell mass gives rise to the fetus, while the surrounding trophectoderm transforms into the placenta. DAYS 6-7 Six to seven dpf, the blastocyst attaches to the uterine wall and begins its invasion. DAYS 7-9 After implantation, the trophectoderm starts reshaping the endometrium. A layer of cytotrophoblasts—trophoblast progenitor cells—emerges around the same time as the invading primitive syncytium. DAYS 10-12 By 12 dpf, cytotrophoblast cells begin to penetrate the primitive syncytium to form primary villi, which later form the villous placenta.

From weeks three to 10, cytotrophoblast cells escape into the decidua, a specialized layer of endometrium, and differentiate into extravillous trophoblasts. These invading cells remodel spiral arteries to reroute parental blood to the intervillous space.

By the beginning of the second trimester, the cytotrophoblast plug breaks down and parental blood begins to enter the intervillous space.

Shallow roots 

As the pregnancy progresses, cytotrophoblast cells keep dividing, and the placenta keeps getting bigger and bigger to keep up with the needs of the growing fetus. Shallow implantation of EVT early in development might be one cause of preeclampsia.¹ This results in an insufficient or poor transformation of the arteries and paves the way for a sparsely branched villous tree and weak perfusion network for blood and waste products to travel between the parent and fetus.⁷ This can cause serious problems later in pregnancy. 

Mariko Horii trained as an obstetrician in Japan before coming to the University of California, San Diego in 2013 to work with Mana Parast, a placental pathologist. Horii arrived searching for answers to why the placenta grows so poorly and has devastating consequences for some of her patients. At the time, Parast was gearing up to develop in vitro models for studying preeclampsia. 

A lot of what researchers in the field know about human placental development comes from morphological, immunohistochemical, and transcriptomic analyses of primary first-trimester placental tissue.⁴ While an incredibly rich source of information, access to these tissues is limited, and isolated cells did not survive for long in a dish, making it difficult to run experiments in the lab. This left scientists with a choice between genetically abnormal cancer cell lines or mouse trophoblast stem cells for their research.

     

A major advance came in 1998 when James Thomson, a stem cell biologist at the University of Wisconsin-Madison, successfully isolated stem cells from human blastocysts.¹⁵ Shortly after, his research team demonstrated that they could differentiate human embryonic stem cells (hESC) into hormone-secreting cells akin to SCT by feeding the cells a special media spiked with bone morphogenetic protein-4 (BMP4).¹⁶ The subsequent development of induced pluripotent stem cells (iPSC) via the reprogramming of somatic cells provided scientists with a less controversial, more accessible source of human pluripotent stem cells.¹⁷ Since then, Horii and others have demonstrated that both hESC and iPSC can transform into trophoblasts and subtypes of trophoblasts by altering the environmental conditions and feeding the cells different molecular cocktails.^7,18

While the advent of hTSC and trophoblast organoids in 2018 are major stepping stones, they come with limitations. “Since we have primary cells, then why not use the primary cells for a model system instead?” said Horii. Scientists are still struggling to produce hTSC and trophoblast organoids from full  term placentas and currently derive them from either blastocysts or first trimester placentas. Both sources are limited and, in some countries, laws restrict their use. Horii raised another limitation of using cells sourced from early pregnancy. “We don’t have the scientific knowledge to predict from the early first trimester pregnancy materials whether the patient would have developed pregnancy complications or not.”

To build this knowledge, Horii and her team turned to full term placentas for iPSC. Either mesenchymal stem cells derived from the umbilical cord or cytotrophoblasts can transform into iPSC when fed a special cocktail.¹⁷ Using iPSC derived from placental cells, Horii and others have worked doggedly to refine culture protocols over the years to generate trophoblasts.^7,18 Eventually, researchers working with these cells demonstrated that their putative trophoblasts secreted key hormones and expressed the EVT marker HLA-G alongside other key genes expressed by trophoblasts.^19,20 

Recently, Horii and her team modified their protocol to include a WNT-inhibitor alongside BMP4 to ensure the exclusion of mesoderm cells and differentiation in trophoblast cells resembling cytotrophoblasts.²¹ However, they struggled to maintain primed stem cells, or iPSC-derived trophoblasts, in a state of self-renewal. To fix this, Horii and her team fed their iPSC the usual fare of BMP4 plus a WNT-inhibitor but swapped the main culture media for one they whipped up for the newfangled hTSC.²² “We were able to finally derive the self-renewing trophoblast stem cells,” said Horii. They further differentiated their new and improved cytotrophoblasts into EVT or SCT using cell type specific differentiation protocols. 

Horii thinks that the iPSC-derived trophoblast models will be particularly useful for disease modeling because scientists can use iPSC to produce cell types beyond trophoblasts, like blood vessels or stromal cells. Currently, in her own lab, she uses this revamped protocol on cells isolated from term placentas of patients with preeclampsia. Her prior work using an earlier version of the culture protocol suggested that this will be a fruitful avenue for modeling preeclampsia in a dish. iPSC derived from placentas of pregnancies with preeclampsia recapitulate several defects observed in primary placenta tissues, including failure to respond to changes in surrounding oxygen levels and abnormalities in EVT differentiation.²³

Fleeting but indelible 

Following the birth of the baby comes the birth of the placenta as it sheds away from the lining of the uterus. By the end of gestation, the SCT region is incredibly invaginated and convoluted to provide a large surface area for diffusion to the baby. “If you were to spread it out it would be 13 square meters in size,” said Fogarty. That’s about the size of a parking space.

Just like that, this transient organ that helped the fetus survive in the womb for the last nine months is gone. Scientists are increasingly appreciating the link between the in utero environment, including placental health, and susceptibility to chronic diseases later in life.²⁴ For example, babies that are born too big or too small relative to their growth potential are at a higher risk for developing cardiovascular disease, diabetes, and obesity in adulthood. These long-lasting effects further emphasize the need for improved screening, prevention, treatment options, and of course, physiologically robust and relevant models. 

“We now have the tools,” said Fogarty. Trophoblast stem cells, trophoblast organoids, iPSC-derived trophoblasts, extended embryo culture, CRISPR Cas9-mediated genome editing, advanced imaging technology, scRNAseq, and spatial transcriptomics all have a role to play in the study of the placenta. 

While no model perfectly captures the complexities of this mysterious organ, recent advances, including refined culture protocols and new in vitro systems, will facilitate the continued study of human placentation. Some researchers are even developing placenta-on-a-chip models using human iPSC-derived trophoblasts to study placental perfusion dynamics.²⁵

“There’s a renewed interest in the field, an energy in the field, that will allow us in the next 10-20 years to make these breakthroughs and bring our understanding of the placenta up to speed with a lot of the other organs that we know so much about,” said Fogarty. 

“Just over half of all pregnancies are uncomplicated, normal pregnancies,” said Fogarty. The other half are affected by miscarriage, intrauterine growth restriction, fetal growth restriction, and preeclampsia. “If we can make these insights, there’s going to be massive numbers of patients who can potentially be helped in the future. There’s potential to make a big impact.”  

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