What is the next step for human embryos grown in the laboratory?

This is the 13th day of a series of experiments in Ali Brevanlu’s laboratory, and he is faced with a painful task

These embryos will soon violate the 14-day rule.

"This is one of the hardest decisions I have to make in my life, but it's time to stop this experiment," Brevanlou recalled the research conducted in his laboratory at Rockefeller University in New York in 2015.

Now, Brevanlou and other developmental biologists have a chance to find out

In May of this year, the International Association for Stem Cell Research (ISSCR) issued new guidelines, relaxing the 14-day rule and removing hard obstacles

Opening a window in these later stages allows scientists to better understand nearly one-third of pregnancy losses and many birth defects that are thought to occur during these stages of development

Most researchers expect that Brevanlu’s team and a handful of others will immediately push the technological boundaries of growing human embryos in the laboratory for longer periods of time

Some researchers do not expect a large number of new studies in 14 days-they also do not believe that these experiments are reasonable

As a way to cross borders, researchers have developed a series of human embryo models in the past five years, most of which are formed from a mixture of stem cells

Whether using models or real objects, scientists say they have a lot to learn

The 14-day rule was first proposed in 1979, when in vitro fertilization technology emerged, and human embryos existed outside the body for the first time-even though the longest time they could survive was only a few days

The limitations of human embryos grown in the laboratory are broken by stem cell bodies

This guide has been widely adopted by researchers and funders all over the world

The last update of the ISSCR guidelines was released in 2016, just before the breakthroughs were published by the two research groups

The Magdalena Zernicka-Goetz laboratory at the University of Cambridge in the United Kingdom began to explore human embryos that have been cultured for more than seven days in 2013

Zernicka-Goetz said: "Our ongoing discoveries, such as where humans place our heads in the future, are very important

One of the problems is related to how embryonic cells express genes during growth
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In the largest study of human embryos to date, Zernicka-Goetz’s research team analyzed 4,820 single cells developed in the laboratory from 16 embryos that were normally implanted in the uterus (day 5) to day 11 when they were ready to ovulate.

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Single-cell RNA sequencing revealed which genes were turned off and which genes were turned on when embryonic cells transitioned from pluripotency to pluripotency (a more differentiated state)
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Although these developmental signals have been found in mice, this study is the first to reveal one of the molecules that support human development
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In other experiments that can only be performed with artificially cultured human embryos, Brevanlou and Zernicka-Goetz and their team are tracking the fate of human embryos with aneuploid cells
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These cells have an abnormal number of chromosomes, and this condition is thought to cause up to half of early pregnancy miscarriages
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In vitro fertilization clinics usually only test a small number of embryonic cells to analyze their genetic health
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But experiments show that this may be misleading
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Zernicka-Goetz’s research team found that embryos diagnosed with certain types of aneuploidies continue to develop normally in the laboratory
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Brevanlu’s research team analyzed the gene expression of human embryos from day 3 to day 14, and found that cells with an abnormal number of chromosomes were eliminated-possibly due to development into supporting tissues or due to cell death.

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Both studies have shown that the usual aneuploidy tests for IVF embryos may cause many embryos to be mistakenly considered "unhealthy
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Brevanlu said that the study of human embryos is critical to these insights
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He said: "We can only understand this by observing developments
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" He is planning some experiments-some of which may last until the 14th day-to try to find out how embryos containing certain aneuploid cells are.
Adjusted
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More than two weeks

Researchers of animal embryos have cultured them for more than 14 days of development, which may pave the way for similar advances in human embryo culture
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In March of this year, Jacob Hanna’s team at the Weizmann Institute of Science in Rehovot, Israel, doubled the culture time of mouse embryos in the laboratory, from day 5.
5 to The 11th day (roughly equivalent to the 13th to 30th day in humans)
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Although other researchers call this process too cumbersome, the embryo progresses smoothly during organ development
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Both Brevanlou and Zernicka-Goetz plan to further push the limits of human embryo culture, doing in humans what Hannah's team did in mice
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Brevanlu wanted to unravel the genetic program that transformed stem cells into the first brain cells.
He also wanted to reveal the molecular instructions for the four-chamber beating heart
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Both of these conditions occur on the 14th day, and unraveling them may be the key to understanding neurodevelopmental disorders and common congenital heart defects
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Brevanlu and others say that in the future, researchers will be technically able to observe the implantation of human embryos into uterine tissue in the laboratory
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His team is applying to the management committee of his university to extend their research on human embryos to the 21st day
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In this extra week, the plan for the entire body has been determined, including the structures that will become the spinal cord, brain, bones, heart, blood, muscles, and face
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He said: "I don't know if we can make human embryos more than 14 days old
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But once we get there, we should move forward very carefully
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Model embryo

Researchers already have some methods to observe the process of gastrulation in mammals, not by using real embryos, but by building a model with a 3D mixture of stem cells
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In the past five years, researchers have made a variety of embryo models in the laboratory that can be used to observe the stage after the 14th day
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In most cases, these embryo models are not subject to the 14-day rule or any special review
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In 2017, Jianping Fu, a biomedical engineer at the University of Michigan In Ann Arbor, and his team simply put embryonic stem cells into a 3D culture to create the first human embryo model
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In 3D culture, embryonic cells self-organized to form the first signs of amniotic sacs and primitive stripes
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Fu said that this discovery has aroused great excitement in the field
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"This possibility exists in human embryonic stem cells-it's amazing
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Models like Fu Jianping continue to proliferate and can now simulate parts of the earliest embryonic stages of mice and humans-implantation, gastrulation, and the initial stages of brain, spinal cord, and heart development
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For example, Fu and others have created human neurolike bodies that mimic the formation of spinal cord and brain precursors.
Fu said this will help researchers cultivate functional neurons that can one day be implanted into patients.
In vivo
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The mixture of these cells is usually similar in appearance and behavior to embryos at the same stage, but whether they reproduce the molecular and cellular activities during normal development is still a huge knowledge gap
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Fu and others admit that as human embryo models form more complex structures and advance on the developmental schedule, they raise new ethical issues
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For example, their neurons will start to excite, and heart cells will start beating
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Or they can gain development potential beyond the limited stage currently imitated
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Most models lack the complete life support tissue required to form a complete embryo, but in the past few years, some laboratories have built mouse and human models of the blastocyst stage, called "blastocysts
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" These precursors containing supporting tissues can theoretically form entire organisms
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The structure grown in the laboratory mimics the early stages of a human embryo

ISSCR focuses on this area, and its guidelines state that the models containing these supporting organizations must be specially supervised and grow in the shortest time required to meet scientific goals
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Robin Lovell-Badge, a stem cell biologist at the Francis Crick Institute and chairman of the ISSCR steering committee, said that as they develop, these models will require ethical reconsideration
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"Obviously, this area must receive attention
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With the deepening of research on real embryos and model embryos, scientists are eager to know how similar these two embryos are
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Researchers hope to study in real embryos for more than 14 days.
The main reason is to find out the differences in the molecular details of the models and how their cells behave
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"We can learn a lot from a model," said Jesse Wienflitt, a developmental biologist at the Max Planck Institute for Molecular Cell Biology and Genetics in Dresden, Germany
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"But it's important to know what went wrong
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In 2020, Morris and his colleagues compared mouse embryos and mouse gastric body side-by-side gene expression, and found that the signal timing waves used to establish the human body were surprisingly similar
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She said that this precise benchmark should also be used for human stomach-like bodies, which will require culturing human embryos until around the 21st day
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Rivron envisions a molecular map for every stage of human development
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Once sufficient benchmarks are established, researchers can reduce the number of human embryos used, and only turn to them when there are good reasons
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Stop the clock

But how long should researchers observe human development in a petri dish? Critics say that ISSCR is irresponsible for relaxing the 14-day rule without giving researchers a new stopping point-it gives embryos The research gave the green light
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Fu Jianping is a member of the ISSCR committee, which developed new guidelines in 18 months and participated in more than 100 Zoom meetings
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He said: "Science is progressing so fast, as a scientific community, it is difficult to draw a stop sign
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" On the contrary, the guidelines open the door for research, as long as a proper review process is carried out
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Brevanlou agreed with this decision
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"From my conscience, I know that the past 14 days have been very beneficial," he said, referring to the study of abnormal chromosomes
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"It may really save the lives of the next generation
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" He and Lovell-Badge believe that it is unethical not to allow some research after 14 days, because it can reveal how organ cell types are produced, as well as miscarriages and birth defects.
How it happened
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As for which experiments and models should be given the same status as embryo research after 14 days, it has become increasingly blurred
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The ISSCR guidelines set clear ethical boundaries, and only include models that include supporting tissues-theoretically with the potential for full development-to be classified into the same category of embryos after 14 days
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(The guidelines also prohibit the transfer of human research embryos, human-animal chimeric embryos, or human embryo models into the uterus of animals or humans
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Some researchers are conservative about canceling the 14-day baseline
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For example, Morris believes that the public has no real opportunity to measure the consequences of the experiment 14 days later
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Bioethicist Josephine Johnston went a step further: "I think it is wrong to give up the 14-day rule without proposing another rule
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" Johnston is a bioethicist at the Garrison Hastings Center in New York.
Home, he said, the restrictions show that the scientific community understands that society values ​​human embryos and respects it
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Remove this restriction "There could really shake the public trust"
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In addition, she said, researchers need to better explain "how studying embryos older than 14 days can really help humans
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" Johnston said they also need to be candid about the details of embryo research
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"A lot of the content of this research feels remote, but if there are no restrictions, it would be too careless
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