A slight note of frustration creeps into Hiromitsu Nakauchi’s voice when he discusses his research. But it’s not because the experiments aren’t working, or that he’s out of ideas, or that his ultimate goal is just too audacious.
On the contrary, it just might work.
Nakauchi dreams of growing transplantable human organs in large animals like sheep or pigs. Recent advances in stem cell technology would ensure that each organ would be a genetic match for its recipient and would take only months to generate — alleviating the current desperate need for organ donors and subsequent lifelong immunosuppression.
“If we are able to generate human organs in animals we could help many, many people,” says Nakauchi, MD, PhD, a professor of genetics at Stanford. “Furthermore, we could also use animal-grown human cells or tissue for toxicology studies or drug screening. Surgeons could practice surgery on intact human organs before operating on patients, and we could study aspects of early human development that have never before been accessible to researchers.”
So what’s the problem?
Many people cite ongoing ethical concerns about creating sheep, pigs or primates sporting human cells. Could any of these “humanized” animals take on human attributes, intellect or consciousness? Could human cells find their way into an animal’s reproductive system to create human eggs and sperm? What are our ethical obligations, if any, to these newly created human-animal chimeras?
Concerns like these prompted the Japanese government to ban the in-animal experiments necessary to determine whether Nakauchi’s plan would work. As a result, Nakauchi, who at the time was the director of the Center for Stem Cell Biology and Regenerative Medicine at the University of Tokyo, uprooted his laboratory in early 2014 to come to Stanford, where federal laws about chimeric research and its funding were less restrictive.
But in September 2015 the U.S. National Institutes of Health abruptly announced a moratorium on funding studies in which developmentally flexible, or pluripotent, human stem cells are injected into early animal embryos — again impeding the very work to which Nakauchi has devoted his scientific career, and for which he had already moved across an ocean. Continuing the research without NIH support is legal, but it requires other sources of reliable, substantial funding.
“We can repeat experiments quite quickly in rats and mice,” says Nakauchi. “But, although they mature fairly quickly, big animals are not easy to handle and they have specific breeding seasons around which we have to work. The research is very slow and expensive.”
To the NIH, the timing of the funding moratorium made sense. “We were seeing stem cell researchers beginning to pursue lines of inquiry that would eventually lead to the need to introduce human pluripotent stem cells into the early embryos of large vertebrates,” says Carrie Wolinetz, PhD, the director of the NIH’s Office of Science Policy. “Coupled with the rise of new gene-editing technologies, we felt it was the right time to take a deep breath and carefully consider the potential impact of such work from scientific, ethical and animal welfare angles.”
In August 2016, the NIH asked for public comment on proposed changes to the funding guidelines that would allow some of this work to move forward, after a case-by-case review by a committee of experts. They received over 22,000 comments. Nearly all were against allowing the work to proceed. Many cited a reluctance to permit scientists to “play God” by pulling the developmental strings necessary to create human-animal chimeras.
“Although chimeras in general are quite common in biomedical research, I don’t know that this was common knowledge to the general public,” says Wolinetz. “Also, it seems that many commenters mistakenly believed that the proposed research involved human, rather than animal, embryos.”
Regardless of whether you’re a philosopher, a biologist or a parent-to-be, the potential of a fertilized egg is staggering. Formerly dormant, the egg has now achieved pluripotency and can give rise to all the tissues of a growing embryo as well as the placenta. The resulting animal or human goes on to romp across the savannah, root in the dirt or govern a nation.
The early developmental dance is strictly choreographed. Fertilization is followed by cell division and, like clockwork, the egg becomes one cell, two cells, four cells, eight cells. Within about three or four days (in humans), the dividing cells have formed a solid spherical clump; after about five days a sphere of about 200 to 300 cells encloses a hollow, fluid-filled cavity called a blastocyst. After 17 days, the embryo enters a stage called gastrulation. During this mind-blowing, three-dimensional contortionist trick, a subset of cells on the surface of the blastocyst first dimple into and then burrow through the sphere to emerge on the other side, forming a tube that will become the digestive tract. (Say hello to your mouth and anus!)
Now the embryo has the structure necessary for the next, and possibly greatest, phase of development: the beginnings of organ formation. This is the stage of most interest for many regenerative medicine specialists seeking to grow human livers, pancreases or other organs in animals.
The steps taken by the human embryo are shared among most mammals, with variations in timing. A cow, a sheep, a mouse or a rat start out the same, and, at the outset, the earliest embryos of all species are difficult to distinguish from one another.
That similarity is key to what Nakauchi and other regenerative medicine specialists are trying to achieve. If they can successfully integrate human pluripotent stem cells into the very early developmental stages of animal embryos, they could potentially generate biological chimeras composed of a mosaic of human and animal cells. But merging two species is tricky. The evolutionary distances between species can garble the transmission of developmental signaling pathways.
The eventual location and potential functional contributions of the human cells are also wild cards dependent in part on the timing of their introduction into the developing embryo. Those joining the developmental dance early in embryonic development can participate in more steps, and become more different types of tissues, than those cutting in later. In 2010, Nakauchi successfully generated adult mouse-rat chimeras by injecting rat pluripotent stem cells into mouse blastocysts. Further experiments showed the viability of his dream of one species’ organs developing in the bodies of another species: When mouse embryos unable to make their own pancreases were supplemented with rat pluripotent stem cells, the adult animals had a functioning pancreas comprised of rat cells.
Similarly, in March 2013, Nakauchi showed that pig pluripotent stem cells were able to grow a pancreas in a pig that had been genetically engineered to be unable to generate one of its own. The stage was set for similar experiments using human cells.
At the time, those experiments could not be conducted in Japan, despite Nakauchi’s outspoken advocacy. Although there were signs of change (a national scientific advisory board recommended in June 2014 that the restrictions be lifted), Nakauchi was concerned that revising the guidelines could take a year or more — time he thought he couldn’t afford to lose. His instincts, at least on this matter, were correct. As of January 2018, the restrictions in Japan have not changed. The United States, he believed, would support his research.
The idea of chimeras has captured human imagination since Greek mythology introduced the Chimera —sometimes conceived as afire-breathing dragon-goat-lion. Often the stuff of monsters or fables, they invoke a knee-jerk negative response in many people. Biologically, chimeras are organisms made up of a mosaic of cells that contain two or more distinct genomes. This can occur naturally when fetal or maternal cells cross the placenta during pregnancy to take up residence in the baby or the mother, or after medical procedures such as organ transplantation or blood transfusion. The term interspecies chimera is used when the genomes come from different species.
Many people are taken aback when first introduced to the idea of deliberately creating interspecies chimeric animals with human cells. But interspecies chimeras, whether naturally occurring or scientifically generated, have been around for a long time. Thousands of people are walking around with heart valves from pigs after their own have failed. Conversely, “humanized” mice genetically engineered to lack their own blood and immune system can develop a human immune system when researchers introduce human hematopoietic stem cells. Mice are also commonly used to test the responses of implanted human tumor cells to various drugs or growth conditions.
But introducing pluripotent human stem cells into an early animal embryo, as Nakauchi proposes, is somewhat different than these examples. These cells have the capacity to become nearly any tissue in the body, and, when injected into a developing embryo, they have the opportunity and the means to do so.
NIH funding blocked
In 2014, Nakauchi arrived at Stanford eager to continue his experiments with large animals with the expectation of receiving NIH funding to advance his research. In the interim his move was supported by a $6 million research leadership grant from the California Institute for Regenerative Medicine. The award was designed to help bring stem cell researchers from outside California to the state, and to allow them to pursue high-risk, high-reward research. The recruitment marked a return to Stanford for Nakauchi, who studied immune-cell genes as a postdoctoral scholar in the laboratory of the late Stanford geneticist Leonard Herzenberg, PhD.
But the 2015 NIH funding ban was implemented before Nakauchi was able to receive a grant from the agency. Shortly after the funding moratorium, seven Stanford faculty members, including Nakauchi and cardiologist and stem cell researcher Sean Wu, MD, PhD, authored a letter in Science magazine describing the detrimental effects of the ban.
Nakauchi is using the funding from CIRM as a stopgap measure to continue his preliminary studies of human pluripotent stem cells in pigs and sheep. He also continued his groundbreaking research in mice and rats. In early 2017 he showed that islet cells from mouse pancreases grown in rats could reverse diabetes in mice, normalizing their blood glucose levels for over a year without the need for ongoing immunosuppression.
In August 2016, the NIH published proposed changes to the funding guidelines after extensive consultations with experts, including Nakauchi and Hank Greely, JD, a Stanford law professor who works on bioethics. Prior to the 2015 funding moratorium, the guidelines prohibited the funding of any research in which human pluripotent stem cells would be introduced into primate blastocysts. They also prohibited funding the breeding of animals whose reproductive systems might have incorporated pluripotent human cells.
The suggested changes broaden the funding restrictions to include injecting human pluripotent stem cells into primate embryos at any early developmental stage up to and including the blastocyst stage, and expand the breeding restriction to include any instance in which any type of human cells (not only those that are pluripotent) may contribute to the production of egg or sperm cells. They also proposed the creation of a new NIH steering committee of federally employed, scientific experts to review research proposals in which human pluripotent cells are introduced into any vertebrate embryo prior to the end of gastrulation. Some scientists were encouraged by the agency’s move.
“Their statement made it sound that NIH is opening to the possibility that people can do this kind of work,” says Wu. But the wheels of change move slowly, and more than a year later no new guidelines have been issued.
“What I find so frustrating,” says Greely, “is that NIH did have that conversation with scientists, held workshops, gathered expert opinion on both the science and ethics sides, and reached a conclusion, but then just stopped.”
For now, any work that involves the injection of human pluripotent stem cells into an early animal embryo is ineligible for NIH funding.
When asked about the delay, Wolinetz says, “I do understand scientists’ frustration. We do see value in this research, it’s just a matter of making sure the appropriate guidance is in place,” she says. “We are still in the policy development process, and I don’t really know when we will finalize new guidelines or what the final policy will look like. It is still in limbo.”
As he waits, Nakauchi continues to dream. He’s experimenting with ways to introduce what are known as committed progenitor cells — cells that are already a few steps along a pathway of specialization toward specific organs or tissues. These cells have closed many of the developmental doors that are accessible to pluripotent stem cells and should be unable to distribute themselves willy-nilly throughout a developing embryo. Not only would these committed progenitor cells circumvent the NIH ban, they also might be more efficient at generating specific organs and lead to more healthy, more viable embryos.
“We are trying to ensure that the human cells contribute only to the generation of certain organs,” says Nakauchi. “With our new, targeted organ generation, we don’t need to worry about human cells integrating where we don’t want them, so there should be many fewer ethical concerns.”
He also envisions the possibility of tinkering with molecules on the surface of the human cells that our immune systems use to recognize self from nonself in ways that would render immunologically invisible, or “universal” organs that could be accepted by any ailing patient. Or how about a cow with human blood for transfusions? The lack of funds, however, has slowed the research. “This is a really expensive project,” he says of the large-animal studies. “Eventually we need to get more funding.”
Public education about research is key
For better or for worse, more funding may require better education of the public.
“Chimeras are very important research tools and disease models, but the creation of animal-human chimeras often comes across with a big ick factor. In some ways the onus is on us to communicate to the public what this research entails,” says the NIH’s Wolinetz.
“Advances in technology are making the ethical issues both easier and harder,” says Greely, who points out that some scientists are eager to study human brain cells in animals as a way to learn more about diseases such as Alzheimer’s or Parkinson’s, or other conditions such as autism or depression. Although these experiments will likely reignite the national discussion of what it means to be human and what our moral obligations to such research animals may be, human consciousness doesn’t reside in our pancreases or livers or hearts.
“Thoughtful discussion is always important,” says Greely. “But evidence is mounting that it may well be possible to transplant human stem cells into nonhuman animals to create organs in ways about which few people would have ethical concerns.”
Nakauchi is more blunt.
“More than 116,000 patients are on the waiting list and 20 people die each day in the United States alone due to a lack of donor organs,” says Nakauchi. “Animal-grown organs could transform the lives of thousands of people facing organ failure. I don’t understand why there continues to be resistance. We could help so many people.”