Stem cell model offers first glimpse of early human embryonic development

It's one of life's most defining moments -- that crucial step in embryonic development, when an indistinct ball of cells rearranges itself into the orderly three-layered structure that sets the stage for all to come. Known as gastrulation, this crucial process unfolds in the third week of human development. "Gastrulation is the origin of our own individualization, the emergence of our axis," says Rockefeller's Ali Brivanlou. "It is the first moment that separates our heads from our behinds."

Observing the molecular underpinnings of this pivotal event would go a long way toward helping scientists prevent miscarriages and developmental disorders. But studying human gastrulation has proven both technologically difficult and ethically complicated, and thus current approaches have had limited success in expanding our understanding of early human development. Now Brivanlou and colleagues have demonstrated how a stem cell model system known as a blastoid can allow the study of the nuances of human gastrulation in the presence of pre-implantation extra-embryonic cell types. Their study, published in Stem Cell Reports, describes the scientific and clinical potential of this new platform.

"Gastrulation was a tremendous black box. We had never seen ourselves at that stage," Brivanlou says. "This moves us closer to understanding how we begin."

A better blastocyst

Prior to implantation, an embryo is a ball of about 250 cells organized as a blastocyst. This elusive ball of cells was difficult to study directly, so scientists developed blastoids -- stem-cell-based blastocyst models. Blastoids can be cloned, experimentally manipulated, and programmed, allowing scientists to study identical blastoids over and over again.

The question was whether blastoids could gastrulate in vitro. Unlike a blastocyst in vivo, which rolls around in the uterus until it attaches to maternal tissue, blastoids were good at modeling the ball of cells from which life emerges, but it remained unclear whether this in vitro model could model later stages of human development. That is, until Brivanlou developed a platform to allow blastoids to attach in vitro, and thereby progress toward gastrulation.

"We were then able to see epiblast symmetry breaking, marked by BRA expression, for the first time with the high molecular resolution," says Riccardo De Santis, a research associate in the Brivanlou lab and lead author on the study. "This allowed us to start asking more detailed questions about the earliest moments of life."

With this unprecedented clarity, the team directly observed two key moments in gastrulation: the first epiblast symmetry-breaking event and the emergence of the molecular markers of the primitive streak and mesoderm upon in vitro attachment.

The primitive streak is a structure that marks the beginning of gastrulation and lays the foundation for the three primary layers of the embryo. One of those layers, the mesoderm, forms during gastrulation and gives rise to muscles, bones, and the circulatory system. The team discovered that, as early as seven days after attachment, they were already able to use molecular markers to detect the earliest signature of a nascent primitive streak and mesodermal cells.

To confirm their findings, the team also compared the blastoid results with data from in vitro attached human embryos and demonstrated that blastoids express the same genes in vitro that a regular embryo would at that stage in vivo, a strong demonstration of the power of blastoids as models for human embryonic development. Further highlighting the power of the lab's in vitro attached blastoid system, the team then used it to demonstrate that pathways that regulate the rise of the primitive streak and mesoderm in vivo also regulate blastoids symmetry breaking in vitro -- all with nothing but stem-cell-derived blastoid models.

Along the way, the team also demonstrated that gastrulation in vitro can begin at day 12, earlier than once thought. "This will change textbooks," Brivanlou says. "We've contributed to redefining the molecular signature and timing of the onset of gastrulation upon in vitro attachment."

Therapeutic possibilities

The results demonstrate that blastoids, when combined with the Brivanlou lab's unique attachment platform, are now capable of conveying insights into early human development that have long been inaccessible. De Santis envisions a future in which blastoid-based research leads to advancements in diagnosing and treating developmental disorders, or offers insights into potential causes of early miscarriages during gastrulation.

"Many couples can't have babies because the embryo doesn't attach properly, and many miscarriages occur in the first few weeks of pregnancy," De Santis explains. "We now have a model system that can help us understand the molecular mechanism that defines whether a pregnancy will be successful or not." In the near future, De Santis hopes to combine this method with machine learning to help predict pregnancy outcomes and the trajectories of developmental disorders by observing how model blastoids built with particular genetic makeups fare in vitro.

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Computer model IDs roles of individual genes in early embryonic development

Computer software developed at Washington University School of Medicine in St. Louis can predict what happens to complex gene networks when individual genes are missing or dialed up more than usual. Such genetic networks play key roles in early embryonic development, guiding stem cells to form specific cell types that then build tissues and organs. Mapping the roles of single genes in these networks is key to understanding healthy development and finding ways to regrow damaged cells and tissues. Likewise, understanding genetic errors could provide insight into birth defects, miscarriage or even cancer.

Such genetic experiments -- typically conducted in the laboratory in animal models such as mice and zebrafish -- have been a mainstay of developmental biology research for decades. Much can be learned about a gene's function in animal studies in which a gene is missing or overexpressed, but these experiments are also expensive and time-consuming.

In contrast, the newly developed software called CellOracle -- described Feb. 8 in the journal Nature -- can model hundreds of genetic experiments in a matter of minutes, helping scientists identify key genes that play important roles in development but that may have been missed by older, slower techniques. CellOracle is open source, with the code and information about the software available at this link.

"The scientific community has collected enough data from animal experiments that we now can do more than observe biology happening -- we can build computer models of how genes interact with each other and predict what will happen when one gene is missing," said senior author Samantha A. Morris, PhD, an associate professor of developmental biology and of genetics. "And we can do this without any experimental intervention. Once we identify an important gene, we still need to do the lab experiments to verify the finding. But this computational method helps scientists narrow down which genes are most important."

CellOracle, which was included in a recent technology feature in the journal Nature, is one of a number of relatively new software systems designed to model insights into cellular gene regulation. Rather than simply identify the networks, CellOracle is unique in its ability to let researchers test out what happens when a network is disrupted in a specific way.

Morris and her team harnessed the well-known developmental processes of blood cell formation in mice and humans and embryonic development in zebrafish to validate that CellOracle works properly. Their studies, in collaboration with the lab of co-author and zebrafish development expert Lilianna Solnica-Krezel, PhD, the Alan A. and Edith L. Wolff Distinguished Professor and head of the Department of Developmental Biology, also uncovered new roles for certain genes in zebrafish development that had not previously been identified.

And in a related paper online in the journal Stem Cell Reports, Morris and her colleagues used CellOracle to predict what happens when certain genes are dialed up beyond their usual expression levels.

"We found that if we dialed up two specific genes, we can transform skin cells into a type of cell that can repair damaged intestine and liver," Morris said. "In terms of regenerative medicine, these predictive tools are valuable in modeling how we can reprogram cells into becoming the types of cells that can promote healing after injury or disease."

According to Morris, most laboratory methods for converting stem cells into different cell types, such as blood cells or liver cells, are inefficient. Maybe 2% of the cells arrive at the desired destination. Tools like CellOracle can help scientist identify what factors should be added to the cocktail to guide more cells into the desired cell type, such as those capable repairing the gut and liver.

At present, CellOracle can model cell identity in more than 10 different species, including humans, mice, zebrafish, yeast, chickens, Guinea pigs, rats, fruit flies, roundworms, the Arabidopsis plant and two species of frog.

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