DAVIS, Calif. — Like many of the scientists who helped usher in the groundbreaking creation of a part-human, part-animal chimera earlier this year, biologist Dr. Pablo Juan Ross is no stranger to cutting-edge tools such as CRISPR and stem cells. But he also knows his way around the inside of a pig uterus.
While growing human cells inside fetal pigs involved some of science’s fanciest new tricks, it also required something decidedly more mundane: a farm, stocked with livestock and staffed with people like Ross who know how to handle them.
Trained as a veterinarian and animal scientist, Ross works at the University of California, Davis, where hay is piled two-stories high on Dairy Road, signs read “Meat sales today,” cows graze next to soccer fields, and the swine facility, filled with squealing black and white Hampshire piglets, has its own Facebook (FB) page.
Without Ross and his deep understanding of the peculiarities of various livestock species and their embryos, the daunting chimera experiments might not have been possible.
“He was indispensable,” said Jun Wu, a human stem cell expert at the Salk Institute who found himself in the unlikely position of helping hoist a 200-pound sow onto an operating table during one of his visits here to work with Ross.
The experiments, led by Wu and his Salk colleague Juan Carlos Izpisua Belmonte and published in January, were hailed as a tremendous accomplishment. The first-ever creation of organisms that were part human and part large animal represents a huge and hopeful first step in the quest to grow human organs for transplant. But that goal remains far out of reach: The chimeras now contain just a tiny fraction of human cells — maybe 1 in 100,000 — leaving the creators grappling to find ways to boost the proportion of human cells in their chimeras to useful levels. Ross is doing what he can, with limited funding, to puzzle out the animal side of the equation.
His expertise is so in demand, Ross has also been recruited by the Salk’s main rival, Stanford University’s Dr. Hiromitsu Nakauchi, who broke open the chimera field with a 2010 experiment showing he could grow a rat pancreas in a mouse and followed up with subsequent experiments showing mice could be cured of diabetes using chimeric mouse pancreases grown in rats. Nakauchi is now working with Ross on human-sheep chimeras, hoping that sheep, which have proven more receptive than pigs for growing human blood cells, might be a better template for growing human organs.
“It’s much harder than I originally thought,” Nakauchi said. “But if our ultimate goal is to make functional human organs for transplant, mice are much too small. We need to size up.”
Ross is something of a chimera himself: He’s part Irish from a family that made plum pudding every Christmas, but he speaks with a lilting accent from his native Argentina and occasionally trips over English idioms, saying things like “it sparkled something in my mind.”
Described by colleagues as modest, ferociously efficient, but most of all pragmatic, Ross is the first to admit the path to growing human organs — or even cells — in animals remains difficult. “The field made its first move,” he said. “But it’s like going to Mars. It’s not going to work the first time.”
Ross, 40, was born to be a large animal vet. His father was one, and he was raised in a cow town in Argentina so small and rural that as a small child, he never saw a TV. He hoped to be an engineer at first. He was good at math, and desperately wanted to leave his hometown and see the wider world. Vet school eventually won out, but his passion for problem-solving would prove invaluable.
Ross decided to pursue a Ph.D. in animal science, and after some wrangling, got a position at Michigan State University with Jose Cibelli, a fellow Argentinian who had graduated from the same veterinary school as Ross and became famous for cloning the first human embryo in 2001.
He plunged into the lab’s research — working on cloning, stem cells, transgenic cells, and figuring out how to trick cloned cow embryos into developing without the input of sperm. He was perfecting a skill that the chimera experiments would demand — manipulating finicky animal embryos for experiments, injecting them with the tiniest of needles, sometimes hundreds of times a day.
Ross had thought himself lucky to land in the lab of a star as bright as Cibelli. But Cibelli says he was the lucky one. “His learning curve was amazing. He published eight or nine papers,” Cibelli said. “He just keeps his head down and pushes forward.”
The Ph.D. work in animal science showed Ross firsthand that the eggs, embryos, and reproductive quirks of different livestock can vary wildly and be far more challenging than those of more commonly studied laboratory animals or humans.
Take in vitro fertilization procedures, for example. Compared with cows and pigs, “humans are a piece of cake actually,” said George Seidel Jr., an animal reproductive physiologist at Colorado State University and working cattle rancher who has nearly 20 former animal science students now running human IVF labs. “People who work with humans or mice and try to work on a cow, they run into a brick wall real quickly,” he said. “That’s why you need people like Pablo.”
Cibelli believes veterinary training, which involves studying the anatomy, physiology, and reproduction of many different species, was critical in helping Ross succeed. “It’s not the mouse only,” said Cibelli, noting that James Thomson of the University of Wisconsin, Madison, who is famous for deriving the first line of embryonic human stem cells in the late ’90s, also has veterinary credentials. “We’re not afraid of other species.”
Ross worked with human stem cells as well. Ironically, for someone now helping create chimeras, he spent a lot of time trying to figure out how to keep human cells in his experiments from getting contaminated with animal material. Since research with human eggs was a no-no, he and Cibelli wondered whether there might be a way to nurture human stem cells inside of animals instead. (In an early chimera experiment, Cibelli put his own DNA into a cow egg; the experiment generated a lot of controversy but not much in the way of results.)
In the mid 2000s, such chimeric possibilities seemed the stuff of fantasy. “But that stood in my head all this time,” Ross said.
Jump forward to 2014, when Izpisua Belmonte and Wu were preparing to test their dream of growing human cells and organs in pigs. Realizing they needed a university with livestock, they were scrolling through the UC Davis website when they stumbled upon Ross and his background with embryos and cloning.
“We were so excited to find him,” Wu said. “His expertise was perfect for the project.”
The Salk team talked with Ross over Skype and hit it off immediately. The Salk scientists explained that they had methods to make various kinds of human stem cells that might survive better inside of animal embryos. And they were using the CRISPR genome-editing technology to remove the genes for organs from animal embryos, hoping that the human cells would fill the gap.
“Juan Carlos said, ‘Do you think it will work?’ I just said, ‘Yes, it has to work. It makes so much sense,’” Ross recalled. He was ready to start the experiments the next day.
But first, review boards at Davis had to sign off on the thorny experiments, which raise concerns among some ethicists because they potentially blur the line between humans and other animals. A number of safeguards were put in place, including ensuring that the sows implanted with chimeric embryos would be kept separate from other pigs and destroyed at the end of the experiment. Even though the host pigs would not contain human cells, they were not allowed to be used for meat after they were killed or even rendered for fat as a precaution.
Once the approval came, Ross jumped in, setting aside tasks such as writing papers and grant applications. “For two years, I completely neglected my office,” he said.
Because none of his students had enough experience, Ross conducted the difficult experiments himself. The procedure required sitting at a microscope and injecting human stem cells into a pig embryo through a protective layer of cells called the trophoblast. That’s easy to do in a mouse embryo, where the trophoblast is thin. But the layer is much thicker in larger animals like pigs and cows; pushing a needle in requires so much force that the whole ball of cells can just collapse.
“Manipulating embryos, micro-injecting embryos. He’s one of the rare people who can do this kind of work,” Nakauchi said.
Ross set up a system to blast embryos with a laser pulse, which created a small hole through which he could inject human stem cells or molecules to CRISPR the genes inside, depending on what the experiment called for.
He had other specialized knowledge the chimera research required. He knew which slaughterhouses could provide cow and pig ovaries needed for harvesting eggs. He knew the recipes for cell cultures — mixtures of sodium, glucose, and albumin — that could keep pig embryos alive in a Petri dish.
And Davis has spectacular facilities and personnel to raise and nurture livestock, including the 9,000-square-foot Swine Teaching and Research Center filled with hundreds of pigs of all ages that manager Aaron Prinz keeps tidier than any pig barn has a right to be.
Implanting a sow with chimeric embryos isn’t easy. It take a full surgical team, strong folks to correctly position large animals on operating tables, and experts like UC Davis veterinarian Joan Dean Rowe, who has the deft touch to implant embryos — chimeric or otherwise — into precisely the right place within a pig’s huge and complex double-horned uterus.
A lot can go wrong in these experiments, and for Ross, it often did. It was a delicate dance — synchronizing animals to ovulate, having the human stem cells ready, carefully injecting embryos without damaging them, and having a surgical team on hand to precisely implant 50 embryos in a sow. Sometimes an animal would be ready for surgery but the embryos would die before they could be implanted. Sometimes the stem cells would be ready to inject at a time an animal couldn’t be prepped for surgery.
“It’s very frustrating,” Ross said. “It took so much work, so many people. And there were many times it didn’t work.”
Even when surgeries went well, Ross’s frustrations weren’t over: Pigs often lose implanted embryos, especially when those embryos are carrying human cells or have been kept for days outside of a uterus. But Ross was eventually able to get a number of chimera embryos to survive inside their pig hosts for 28 days — the length of time the ethics panels allowed him to gestate the chimeras.
In June 2016, he harvested a half-inch-long embryo and placed it under his microscope, looking for the tell-tale fluorescence that the human stem cells had been labeled with. It was studded with fluorescent dots.
“I was like, this is the real thing,” Ross recalled. “I still have that image in my mind.”
Tests by his Salk collaborators confirmed that human cells had survived in a pig embryo. The experiments were then scaled up at a vast pig farm in Spain, where Ross and Wu trained teams to work with stem cells and inject embryos. The final experiment required some 2,000 pig embryos.
Publication of the findings in January created enormous excitement, but Ross knows better than anyone how much work remains. He knew the pig embryos wouldn’t have a huge number of surviving human cells because pigs and humans are such distant relatives. Even chimeras between closely related rats and mice contain only between 20 percent to 30 percent of introduced cells. He was hoping for 3 percent. But the team got less than .001 percent — far too few for generating human organs.
What he’d like to do most is find out what happened to the human cells and why they died. But he’s stymied by an National Institutes of Health moratorium on funding of animal-human chimera research. The ban means he’s unable to apply for a large, multi-year grant that might let him get traction on the problem.
“All this work has really been done with no real money, no real support,” Ross said. “It’s all been done when we have some extra embryos and we say, ‘Here, let’s do this.’ Nothing is done the way it should be.”
Strapped for funding, Ross does a lot of work himself that better funded labs might have a technician do, from pulling his own pipettes to making the micromanipulators he needs to inject embryos to aspirating eggs from discarded ovaries. Five or six times now, he’s rebuilt the two-decade-old, inherited microforge he uses to make tools. “He’s done things at a fraction of a cost it would take other labs,” Seidel said.
In the meantime, Ross is forging ahead with Stanford’s Nakauchi, trying to up the production of human cells useful for organs in sheep. They’re using human stem cells already committed to becoming organs, and looking for ways to protect the human-derived cells from natural cell death mechanisms.
Ultimately, he believes transplants can be done with in-between organs. He thinks a pig-looking organ loaded with human cells is a more realistic end result and might work just fine because pig organs are so similar to humans in size and shape. He’s also open to the idea that certain treatments based on chimeras might not require organs at all. For diabetes, for example, researchers might be able to use a chimera to grow pancreatic islets, clumps of cells that produce insulin and could then be implanted in lieu of an entirely new organ.
But for any of this to become a clinical reality, Ross and his colleagues need to figure out how to get more human cells to survive inside of their sheep, pig, or cow hosts. “What’s missing?” asked Ross. “It could be one thing. It could be a hundred things.”
He’s optimistic, he said, because the work is really just beginning. Think of Dolly the cloned sheep, he said, one of animal science’s biggest success stories. Creating her took 277 attempts.