International team creates first chimeric human-monkey embryos

A team of scientists from the U.S., China, and Spain reported Thursday they have created the first embryos that were part human and part monkey and kept them alive for up to 20 days in laboratory dishes.

The ethically controversial creation of chimeras — containing cells from multiple species — is part of a drive to make experimental models to help scientists better understand early development, devise new treatments for human disease, and possibly find methods to grow organs for transplant inside other animals, such as pigs or sheep. Such chimeras can be used in experiments that can’t be done with human embryos, for example, but combining human cells with those of primates ushers in new and urgent ethical concerns.

To be clear, the work, published in the journal Cell, is in very early stages. The team did not create living, breathing part-human animals. They created very early embryos and never implanted them into the uterus of any animal. Concerns about human-animal chimeras center on what Stanford bioethicist and legal scholar Hank Greely calls “brains, balls, and beauty” — worries that an animal could look at all human, be born with a human brain, or be able to reproduce.

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This experiment carried none of those risks, but makes it clear that the time to start discussing the ethics of human-animal chimeras is now. “It’s a first-in-kind experiment,” Nita Farahany, a professor of law and philosophy at the Duke University School of Law who co-authored a perspective piece on the ethics of the research, told STAT. “Whenever you cross a major technological or biological hurdle like this one, it’s a good moment to stop and reflect.”

The report provides the scientific details of work that began several years ago in Spain and China and made waves after it was leaked to the El País newspaper in 2019. (The Covid-19 pandemic delayed the completion and publication of the work, the researchers said.)

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In the experiment, researchers injected 25 of a specific kind of human pluripotent stem cell (called “pinnacle stem cells” because they can turn into any kind of cell, including extraembryonic cells such as placenta) into each of 132 6-day-old macaque monkey embryos. The researchers stained the human cells with an exceptionally bright red fluorescent protein called tdTomato so they would be detectable within the monkey embryo as it developed.

The scientists took advantage of a new technique that allowed them to grow monkey embryos outside the womb for up to 20 days, a stage where the embryos were still largely undeveloped, but had formed layers and cavities. Survival of the embryos declined during the experiment; after 10 days, 103 embryos were still developing, and by day 19, just three of the chimeric embryos remained alive. (After 20 days, monkey embryos grown outside the womb, even those that are not chimeric, simply collapse.)

Still, the researchers found a high number of human cells in the chimeric embryos that survived. On average, 3% to 4% of the cells in the embryos were human, and in one embryo, up to 7% were. “When we observed human cells were alive in monkey embryos, that was amazing,” Tao Tan, a principal investigator at the State Key Laboratory of Primate Biomedical Research and Kunming University of Science and Technology in Yunnan, China, and one of the paper’s lead authors, told STAT.

Work published earlier this year by a research group in France showed that human cells did not survive well in monkey embryos. The key to the success in the new paper may be the particular stem cells that were used, as well as the way they were cultured.

The work, other scientists said, is the most successful to date in keeping a relatively large number of human cells alive and healthy in a nonhuman embryo. “This paper is a dramatic demonstration of the ability of human pluripotent stem cells to be incorporated into the embryos of the cynomolgus monkey,” said Magdalena Zernicka-Goetz, a developmental biologist at Caltech whose lab first developed, in mice, the technique of growing embryos in lab dishes. Such work gained increasing attention with the announcement last month that Jacob Hanna, a developmental biologist at the Weizmann Institute of Science in Israel, had kept mouse embryos alive and seemingly healthy for an extended period in an artificial womb.

The results published Thursday showed a far higher rate of survival for human cells in a nonhuman embryo than in previous experiments with sheep-human or pig-human chimeras, where sometimes just one in 100,000 cells were human.

It was those disappointing results that pushed the researchers to turn to monkeys, which are much more closely related to humans and have a similar developmental timeline, said the project’s leader, Salk Institute developmental biologist Juan Carlos Izpisua Belmonte. “Human cells do not robustly contribute to chimera formation in species evolutionarily distant to humans,” he said.

In monkeys, it was a different story. “We found the human and monkey cells cooperate with each other in building the embryo,” said Jun Wu, a research team member who is now an assistant professor at UT Southwestern Medical Center in Dallas, but was a staff scientist at the Salk when he worked on the project. Wu’s research has shown that when human cells are placed in culture with mouse cells, “we see the mouse cell actively kill the human cell, but when we place human and monkey cells together, there is no competition.”

Experiments such as this one cannot be funded with federal research dollars, though they break no U.S. laws. The work was conducted in China, not because it was illegal in the United States, the researchers said, but because the monkey embryos, which are difficult to procure and expensive, were available there. The experiment used a total of 150 embryos, which were obtained without harming the monkeys, “just like in the IVF procedure,” Tan said.

But such experiments, which combine human cells with those of animals, are nevertheless controversial. This work, and other work by Izpisua Belmonte, has moved so rapidly, bioethicists have had trouble keeping up.

“The complicated thing is that we need better models of human disease, but the better those models are, the closer they bring us to the ethical issues we were trying to avoid by not doing experiments in humans,” Farahany said. “Remarkable steps forward require urgent public engagement.”

In addition to considerations about research that may blur the line between humans and animals, the work on monkeys raises issues about both animal welfare and human consent. While many “de-identified” human cells can be used in research without consent for specific experiments, that may not be the best approach for highly controversial experiments such as this one, she said.

Izpisua Belmonte and the other researchers agreed that ethical debate and guidelines for such work is critical. They said they discussed the work with ethicists before proceeding and received extensive review and approval from all scientific institutions involved. The paper’s discussion section starts not with scientific findings as is usual in a research paper, but with a statement about ethical considerations.

The researchers say animal-human chimeras could prove a powerful tool in biomedicine and that the new research is important for helping understand what roadblocks stand in the way of merging human cells into animal embryos. Their hope is to understand the principles in monkey-human chimeras, which seem easier to create, and then try to apply that knowledge to the more technically challenging but ethically more acceptable pig-human chimeras.

The researchers, and other scientists STAT contacted for comment, said one of the most exciting things about the new paper was the discovery of “crosstalk” between human and monkey cells in the developing embryo, something that did not occur so readily in human-pig chimeras. “Human cells in pig tissue is akin to communications between two distant languages, such as Chinese and French, whereas human cells in macaques is like communications between two closely related languages, like Spanish and French,” said Izpisua Belmonte. “We do think understanding interspecific cross-talk could be the key for generating more successful chimeras.”

One major goal, they say, is to find a way to grow human organs in pigs or other animals to ease the shortage for transplants. Currently, more than 100,000 Americans are awaiting organs for transplant. But some scientists question whether that goal is achievable given how hard the research has proven so far.

“How realistic is it to think that someday this line of research could actually be able to produce organs for transplants into human patients?” asked Paul Knoepfler, a stem cell scientist and professor at the UC Davis School of Medicine. “If that is extremely unlikely given technical challenges, even setting aside ethical considerations, how much further should this research be pushed just to advance our knowledge of human development?”

Knoepfler said the research posed a host of other questions as well. “Is there a point at which a chimera is “too human”? For instance, what if a monkey-human chimera ended up being more human than monkey, should special rules apply?” he asked. “What if there are many human cells in the developing brain of a chimera?”

The issue of the brain merits special consideration, in part because many consider the brain the organ that makes humans unique. Because of these considerations, the National Academy of Sciences convened a group to weigh in on the ethics and governance of the emerging field of neural organoids, transplants, and chimeras. The report, released earlier this month, stated that many ethical issues in the field could be addressed by existing mechanisms, but that it was critical to continually reassess concerns as the science developed.

The Cell paper is interesting, said the report committee’s co-chair, Joshua Sanes, a neuroscientist and professor of molecular and cell biology at Harvard, but it is not pertinent to a discussion about neural chimeras because the less-than-3-week-old embryos had not reached the point of having a nervous system. Still, he said the new work reinforced the committee’s urging that ethical conversations “get ahead of the curve.”

“It’s pretty clear the entire field is moving ahead with great rapidity,” Sanes said. “It’s not clear how fast it will go, or in which direction, but we do need to revisit these issues regularly.”

Work is already afoot to loosen internationally recognized ethical limits that prohibit scientists from growing any human embryo outside the body for longer than 14 days. The rule had been in place for decades, and was developed in a time when it was technologically impossible to keep embryos alive outside the body for more than a few days. But that’s changed dramatically in recent years, and with increased interest in powerful gene editing tools such as CRISPR to genetically modify embryos to cure disease, motivation is strong to push biological limits further — and to have clear guidelines about what may be going too far.

Said Knoepfler: “Leaving it up to every researcher and institutional ethics review board to entirely come up with their own rules on human embryo research seems to me like a recipe for big trouble.”

Source: STAT