When Berna Sozen, PhD, assistant professor of Developmental Stem Cell Biology at Yale, was in her first weeks of pregnancy, she couldn’t help but wonder about what exactly was going on in the “black box” of human development occurring inside her, especially since her lab studies embryogenesis with human pluripotent stem cells (hPSCs). Months later (and just a few weeks before the due date), Sozen is providing a new lens into this process with a model for human post-implantation development, a period in human development that is very difficult to study.

According to the study published today in Nature, the technique gives rise to human extra-embryoidsstructures developed from human pluripotent stem cells (hPSCs) that resemble human embryos at days 9–14 after fertilization and include embryonic and a few extra-embryonic tissues of the post-implantation embryo. These human embryo-like structures provide spatially and temporally organized model generation, enabling researchers to record a variety of intricate and interrelated cell state transitions as they shape the early human embryo in a context that is unique to humans.

“Our model platform captures a specific snapshot of human development about which we know perhaps the least,” senior author Berna Sozen, assistant professor of Developmental Stem Cell Biology at Yale University, told GEN. “This is the stage where the progenitor cells of the future human body form and the body axes are set to establish, and the stage where most pregnancies fail for unknown reasons. So far, the basic molecular mechanisms controlling embryonic development at this stage carry enormous importance for both basic science and biomedical research.”

Expedited delivery

The publication of the article from Sozen’s lab—which was submitted on July 7, 2022, before Sozen’s pregnancy began, and was accepted in principle on May 30, 2023, and officially accepted on June 21, 2023—was accelerated after a wave of research demonstrating lab models of early human embryos was announced nearly two weeks ago. The rush was triggered when the leader of one group, developmental biologist Magdalena Zernicka-Goetz, briefly described her team’s results at the International Society for Stem Cell Research (ISSCR) meeting in Boston on the morning of June 14, 2023.

Over the next two days, several research teams posted preprints of work using various kinds of human stem cells, some genetically modified, to create models of post-implantation embryos in the lab. One of the preprints that went online on June 15, 2023, came from Zernicka-Goetz’s group, which used transgenes to direct the induction of a stem cell-derived human embryo model. That same day, researchers from the University of Pittsburgh showed that they used an “engineered synthetic gene circuit” to produce early human embryos. Then on June 16, 2023, two preprints, one from Kunming University of Science and Technology in China and another from Jacob Hanna’s group at the Weizmann Institute of Science in Israel, reported creating artificial human embryos without transgenes.

“All stem cell platforms vary in strategy and approach,” said Monique Pedroza, a graduate student in Sozen’s lab and first author of the Nature study. “Our system is unique as it is a reductionist, robust platform starting from a single stem cell type. Our system, as well as existing and emerging human stem cell-based systems, has the unifying goal of progressing the field forward towards a greater understanding of early human development.”

“In the future, it will be important to define the specific developmental process that is to be explored and to use a stem cell-derived model appropriate for the question,” Janet Rossant, Chief of Research Emeritus and Senior Scientist at The Hospital for Sick Children in Toronto told GEN.

Test tube “extra-embryoids”

Studying early human embryonic development is extremely challenging due to ethical and technical restrictions, which have fueled sustained efforts to create embryo models. In the past, most of the work into early human development has used embryoid bodies—three-dimensional aggregates of pluripotent stem cells—that have allowed for differentiation into key lineages from primed hPSCs. However, according to Sozen, aggregate assembly and subsequent differentiation are typically disordered and disorganized, sharing little in common with the in vivo human embryo. 

Sozen’s lab demonstrated that primed hPSCs can be triggered to self-organize into 3D extra-embryoid structures that mimic events in early human development that occur following implantation into the uterus. The researchers report that extra-embryoids feature spontaneous differentiation and co-development of embryonic epiblast and extra-embryonic hypoblast-like lineages, as well as the setting up of key signaling hubs with secreted modulators. These artificial early embryos can go through events where symmetry along a certain axis is lost to set up polarity, which are also called symmetry-breaking-like events.

“In our new platform, we use a single stem cell type and show spontaneous formation (without using genetic modification) of the two key embryonic cell populations: epiblast, the precursor of the future embryo, and hypoblast, the precursor of the future yolk sac (supportive tissue),” said Sozen. “This reductionist approach allows us to study the critical crosstalk between the key two lineages as they shape the future embryo, as well as their differentiation process. It is critical to understand these processes because they tell us how human development occurs at the stages that are normally hidden within the body of the mother.”

The Yale team used single-cell transcriptomics to confirm that hPSCs can differentiate into the cell states of the peri-gastrulating human embryo without forming placental cell types. The extra-embryoids showed striking transcriptional overlap with signatures recovered from human and non-human primate embryos. They found the transcriptomic signatures of the post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, and initial yolk sac induction.

But the epigenetic landscape of the extra-embryoids did not differ significantly from that of the primed hPSCs. Sozen said that the fact that the extra-embryoid cells do not seem to be able to capture the dynamic epigenetic events that happen at the same time during implantation may be another thing that needs to be improved in the future.

Uncharted territory

This new platform demonstrates fresh possibilities for addressing as-yet unstudied phases of human development in a way that takes into account a variety of crucial factors, from gene expression to spatial patterning. The Yale team reveals a previously unknown interaction between the extra-embryonic and embryonic lineages in the coordination of early human development. And this takes place in the absence of the trophectodermal cell types required for implantation in the uterus.

This system provides a repeatable, tractable, and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new ways to study congenital pathologies with high throughput.

Pedroza said, “Our system is significant as it provides a platform to dissect the mechanisms underpinning early fate decisions occurring at inaccessible stages of our species’ development, allowing for opportunities to understand the origins of developmental disorders.”

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