LA JOLLA, Calif. — It was the salamanders.
Juan Carlos Izpisua Belmonte had spent years probing the inner workings of embryos, ferreting out the genes that give a body its shape or allow wings to form instead of legs. He’d tracked wafting chemical messengers that, like traffic police, guide streams of dividing cells either left or right. He’d even found a way to tweak animals to grow extra limbs. But one thing he never stopped thinking about was how salamanders could lose parts of their bodies and then regrow perfect replacements. Was it possible, he wondered, that humans might do the same?
The dogged pursuit of that question has pushed Izpisua Belmonte to the forefront of biology as he’s made one stunning discovery after another — many before the world is ready to deal with their far-reaching ethical implications.
Early this year, seeking a way to grow human organs for transplant, his group announced it had created pig-human chimeras — fetal pigs with human cells mixed in. His Salk Institute lab has discovered two new kinds of stem cells, including one considered the pinnacle of stem cells because, in addition to being able to create every type of cell in the body, it can also form tissues like the placenta and amniotic sac that embryos need to survive. Last December, they used a technique in mice that may help reverse aging by reprogramming adult cells back to their youth.
His lab also played a supporting role in two extremely controversial studies conducted at Oregon Health and Science University: the work reported last week on the editing of a heart-disease gene in human embryos, as well as the creation last year of “three parent” embryos in an effort to eliminate the mutations that lead to mitochondrial disease.
“It’s hard to know where to start, he’s doing so many different things,” said Paul Knoepfler, a stem cell researcher at University of California, Davis. “You get the sense he’s sort of fearless.”
There are those who remain skeptical about these early findings. The pig chimera, for example, contained so few human cells that Stanford stem cell researcher Hiromitsu Nakauchi said it seemed “like more a negative result.” Others say the many barriers to creating human organs in pigs seem insurmountable. And the work is revolting to people who oppose any dabbling with human embryos or mixing of human and animal tissue.
But among scientists, Izpisua Belmonte has a growing legion of fans. They applaud him for his audacious work, his pushing of boundaries, his keen intellect, and his willingness to conduct difficult experiments everyone tells him will never succeed.
“Scientists have this way of predicting what can’t be done, then being proven wrong by other scientists who are not bound by conventional wisdom,” said Dr. George Q. Daley, a stem cell researcher and dean of Harvard Medical School. “Juan Carlos is both rigorous and intrepid enough to take bold risks and then produce science that surprises us all.”
Izpisua Belmonte, now 57, is an unlikely star of this futuristic arena. He was born in rural Spain to a farm family so poor that he had to drop out of elementary school at age 8 and work in the fields. His mother could neither read nor write. When he finally re-enrolled in school at 16, he quickly caught up — and caught the notice of teachers who encouraged him to attend the University of Valencia.
He had no interest in science. He wanted to study philosophy. But on his way to enroll in the philosophy department, a beautiful building caught his eye and, on a whim, he enrolled there instead. It was the pharmacy department.
Izpisua Belmonte went on to earn a Ph.D., studying adipose tissue in Bologna, Italy — but fat research frankly bored him. Only when he moved to a lab in Heidelberg, Germany, and helped with pioneering work on the genes that rigorously control the body patterns of embryos, did he find a spark.
“That’s when it clicked,” he told STAT. “This is what I want to do.”
It was there, fiddling with genes and embryonic tissues, that Izpisua Belmonte finally found some questions he could sink his teeth into. They were biological questions, but they were also deeply philosophical: How could a single cell unfold into a unique individual? Where did the instructions come from? And how much, really, separated humans from other animals?
After more training at UCLA — to learn about frogs — Izpisua Belmonte was heading to Europe to start his own lab. But first, he attended a conference where three of his mentors happened to be speaking. All mentioned the young scientist’s work.
Salk Institute scientist Ron Evans was at the conference and decided he had to meet the person everyone was buzzing about. “He thought differently,” said Evans, who recruited Izpisua Belmonte on the spot, ”and the combination of his thinking and meticulous execution was exciting.”
The famed institute is now being roiled by a very public gender discrimination lawsuit, but it has long been known for the quality of the scientists it hires; the 53-member faculty includes three Nobelists, four Lasker Award winners, and more than a dozen members of the National Academy of Sciences. Izpisua Belmonte joined the faculty in 1993, and, according to Evans, has more than lived up to his promise.
“It’s hard to know where to start, he’s doing so many different things. You get the sense he’s sort of fearless.”
Paul Knoepfler, stem cell researcher at University of California, Davis
There are many reasons for that success: a strong work ethic (“science 25 hours a day” is how Izpisua Belmonte describes his schedule), large labs in both the U.S. and Spain, the embrace of new tools like CRISPR gene-editing as soon as they emerge, and a focus on some of life’s most fundamental questions. Perhaps the most important reason is his depth of understanding of embryos and how they work — and his ability to coax them to not only give up their secrets but do things even millions of years of evolution never had.
Izpisua Belmonte was not content to work just with chicks and mice, the standard lab animals for embryology. His new lab was filled with salamanders, including Mexican axolotls, amphibians that can regenerate not only limbs, but jaws, spines, and brains. His was one of the first labs in San Diego to study zebrafish, valuable because they have see-through embryos that develop outside their bodies. “The lab,” he said, “was like a zoo.”
His quest for regeneration started with limbs. They could regrow, even though they were made of so many different cell types, including muscle, bone, skin, and nerve. They had specific and complex patterns. To Izpisua Belmonte, they were like “small embryos outside the body.”
They were also a lot like organs — even using the same genes and pathways to develop. He thought being able to create organs for the tens of thousands of desperate people on transplant waiting lists might be one of the most important things he could do.
When embryonic stem cells burst onto the scene in the late 1990s, Izpisua Belmonte immediately saw their potential. Like many scientists, though, he didn’t start working with them right away because of ethical and funding issues.
But his native Spain jumped on board in 2003, passing a law endorsing work on the cells. The next year, officials asked Izpisua Belmonte to create and run a new center for regenerative medicine where he could work on them. (He was becoming something of a legend in Spain. That same year, his hometown, Hellin, named the local high school after him.)
In 2006, Japanese scientist Shinya Yamanaka discovered that adult cells could be reprogrammed into stem cells, abating most ethical issues and igniting the field. (He was later awarded a Nobel Prize for this work.) At the outset, stem cells promised to cure everything and produce anything, including human organs to order. But many labs, including Izpisua Belmonte’s, found the trial and error attempts to create different cell types or grow specific organ shapes on scaffolds hugely frustrating.
“We could be 100 years doing that,” Izpisua Belmonte said. “We were trying to imitate nature with very little knowledge.” He couldn’t, as he puts it, “educate” the cells in the Petri dish.” And they just wouldn’t form the complex 3-D structures he needed to build working organs.
Then the idea hit. In one of his first published papers, back in Germany, Izpisua Belmonte had grafted tissue from an embryonic mouse limb bud onto the wing bud of a chick embryo and found development proceeded normally. The experiment showed that the signals for development appeared similar in widely differing species. It also suggested that mixing tissues from different species might work.
“This idea of chimera was already in my mind,” Izpisua Belmonte said. “It was in my first paper and has stayed there for many years.” And animals grow perfect organs inside of them all the time, he noted.
Geneticists had long used chimeras made from genetically distinct mice as tools. The mid-’80s saw the debut of the shaggy “Geep” or goat-sheep chimera. And a handful of labs, including Izpisua Belmonte’s, had succeeded in getting a small number of various types of human stem cells to grow in mice. But there were problems with using mice for his organ project. Even if you could grow a human liver in a mouse, he thought, it would be far too tiny to be of any use.
Izpisua Belmonte decided it was far better to grow human organs in pigs than mice. The group had early success with cows, too, but found pigs much cheaper and easier to use. Pigs also have organs about the size of humans, large litters, and a long history of human health applications.
Still, getting human cells to grow inside of pig hosts seemed so impossible the team didn’t think their first experiments would work. “The pig embryo sees the human cells as an invader. The natural response is to find a way to kill them,” said Jun Wu, a senior researcher in Izpisua Belmonte’s lab who was lead author on the chimera paper. “We didn’t expect we’d find any human cells.”
But they did. Only a tiny percentage of the human cells, about 1 in 100,000 cells, survived in the pig embryos — so few that some in the field wonder whether those cells were just flukes that survived but would never really function. Some, like Harvard’s Daley, say many barriers remain before complex organs that require multiple types of tissue, not to mention nerve inputs and blood vessels, could ever be created for humans in animals and not be rejected by the immune system.
“It remains a highly speculative and risky proposition,” Daley said. But he and others also see the study as a critical first step.
How did Izpisua Belmonte get the chimera experiment to work against all odds? One reason is its scale. He pulled together a team of 40, including pig farmers, to run the chimera experiments on a massive farm of some 9,000 pigs in Spain. The effort took four years and involved injecting stem cells into some 2,000 embryos.
There was also his deep understanding of development. Pigs are so different from humans. They gestate in just three months while humans take nine. Izpisua Belmonte likens this to cars entering freeways at vastly different speeds: Accidents are bound to happen.
To overcome this problem, Izpisua Belmonte tried using stem cells of different ages in the experiments. While theory might suggest that the earliest, most embryo-like stem cells would work best because they have the most potential to become any type of cell, the results showed that the pig embryos were more open to accepting intermediate, or slightly aged, human stem cells.
Izpisua Belmonte is also the first, said University of Cambridge developmental biologist Magdalena Zernicka-Goetz, to think about stem cells not just in terms of time, but also in space. Last year, he also published the discovery of “region specific” stem cells, finding some are more powerful depending on where they originate from in an embryo.
“It’s a very novel concept,” she said. “I like the way he uses developmental biology to tackle important human health problems.”
Izpisua Belmonte readily admits that he’s a long, long way from growing any human organs in pigs. First, the team will need to create chimeras with a much higher percentage of human cells. Izpisua Belmonte thinks that’s coming. He already has a few powerful tricks up his sleeve.
One is the use of CRISPR to edit out the genes involved in the development of an organ, leaving the host with a vacancy that can then be preferentially filled with donor cells. Izpisua Belmonte calls this “emptying the niche.”
The technique has worked well in mice-rat chimeras to create mice with pancreases, hearts, and eyes that are rich in rat cells. Mice even developed gallbladders with rat cells — a finding that stunned Izpisua Belmonte because normal rats don’t have gallbladders and haven’t for millions of years.
“That’s something fantastic,” he said in an interview here in an office decorated with sculptures of eggs and huge colorful photos of human and zebrafish embryos. “It’s evolution taking place not in millions of years but in 19 days” — the length of a time a mouse gestates.
The team is now working on using the CRISPR technique in pig embryos to increase the human cell count in organs. They’re also using their newest stem cells in tests; experiments now underway show they survive far better in chimeras as well.
In 2015, Izpisua Belmonte applied for a prestigious NIH Director’s Pioneer Award that comes with $500,000 a year for five years. He was thrilled when he got a call telling him his application had received the highest ratings. It had been hard for him to get governmental funding for research involving human embryos and embryonic stem cells. Much of his work had been funded by private foundations, with some contributions from California’s stem cell agency, CIRM.
But a few weeks later, he got another call. The grant couldn’t be funded, he was told, because the National Institutes of Health would not support work on human chimeras. The issue had the agency scrambling to sort out its policy; it convened a workshop for the following month on the issue, inviting Belmonte and other chimera researchers to make a case for their work.
It’s one of many examples of regulatory agencies, and society for that matter, scrambling to keep up with Izpisua Belmonte. “There are no policies in place for what he’s working on,” said UC Davis’s Knoepfler, who has called for more guidance in the area. “It’s all uncharted territory, and he’s one of the folks who is pushing us into that territory.”
The creation of human-animal chimeras is deeply upsetting to many people. Animal rights groups vehemently oppose the work. Some bioethicists say the work is an affront to human dignity. The U.S. Council of Catholic Bishops calls it the creation of “beings who do not fully belong to either the human race or the host animal species.”
Hank Greely, director of the Center for Law and the Biosciences at Stanford University, said many people are fine with the idea of growing organs in animals, but problems arise when the research starts involving what he’s termed “brains, balls, and beauty.” Most people would object, Greely said, to a pig with a human brain, a pig with human cells that can reproduce, or a pig with a human nose.
“The big issue is this question of humanness and if we are conferring humanness on nonhumans,” he said. “That is going to make people upset.”
Greely has served with Izpisua Belmonte on panels to address ethical issues and co-authored articles with him on the ethics of chimeras. “Juan Carlos has always seemed aware of these issues. He gets that people can get freaked out,” Greely said. “I’m quite convinced he’s sensible and responsible.”
Izpisua Belmonte said he wants more than anyone to see clear guidelines. He understands the importance of what the new technologies, many of them from his lab, portend.
“We’re in a critical moment in human evolution. Everything that has happened in the past billion years follows two rules: random mutation and natural selection,” he said. “We’re now at a moment in history where we don’t have to follow Darwin’s rules. We need to be conscious of that.”
But he also thinks fears of the potential humanness of chimeras are greatly overblown, in part because of biology: So few human cells have survived in embryos so far, he said, and chimeras are highly likely to be sterile. He said technology could be used to prevent human cells from migrating into an animal’s brain, and any reproduction of chimeras could be avoided by not bringing the animals to term. (His lab stops the pig experiments after just four weeks even though Spain would allow them to go further.)
“Scientists have the most to lose by not being careful,” he said.
He worries that restrictions on the research mean that U.S. science will fall behind in this area. An internationalist who fondly recalls the babble of foreign languages spoken in the European labs where he trained, Izpisua Belmonte said he has no problem turning to colleagues in foreign countries for their expertise or to conduct work that might not be funded or allowed in the U.S.
“If that means we knock on the door of another lab in China, we need to,” he said. “If it’s in Saudi Arabia, let’s go there.”
Izpisua Belmonte did end up getting that Pioneer Award from the NIH late last year. But he had to agree to keep away from human tissue and use the funds to work only on creating organs for primates.
He’s agreed, but hopes future NIH policy will fund research on animal-human chimeras. “In the end,” he said, “we want to cure humans, not monkeys.”