The mice scurrying around their cage in Katsuhiko Hayashi’s laboratory do not look remarkable. They run, eat and sleep like others of their kind. But these eight rodents have an unusual origin story, one that Hayashi, a reproductive biologist at Kyushu University in Japan, revealed three years ago in the pages of Nature. The tawny-colored mice, he and his colleagues announced, did not spring from the mating of sperm and egg. On their mother’s side, their roots trace to a reprogrammed skin cell.

The advance, called “amazing” by other researchers, delivers on a promise hinted at in 1997, when scientists managed to clone Dolly the sheep. That accomplishment built on earlier cloning work in frogs from the 1970s and taught scientists that every animal cell has the same basic set of instructions. By transforming a sheep’s mammary cell into a living animal, Dolly’s creators showed that every mammalian cell has the same genes—and that the difference between a breast cell and any other cell is simply which genes are turned on or off.

For Hayashi and other scientists, that work created the prospect that they might be able to reprogram mammalian cells to become anything from a neuron to an egg if only they could devise the right instruction manual. A small number of researchers around the world, including Hayashi, are using this legacy to tackle in vitro gametogenesis—generating eggs and sperm from adult cells.

Reproductive scientists and some couples struggling with infertility are closely tracking Hayashi’s progress, as well as similar efforts that have successfully converted rodent stem cells (progenitor cells that can develop into any type of specialized cell) into rudimentary sperm. If these egg and sperm techniques can be made to work in humans, we may one day be able to replace our faulty gametes with blood or skin cells. In that future, men would not have to worry about a lack of healthy sperm. And instead of watching their chances of motherhood fade with their 30s, women of virtually any age could give a little blood and end up with a batch of eggs. Gay couples, too, might one day be able to have children to whom they are both biologically related.

The hope remains tantalizing but distant. Years of animal experiments aimed at finding a reliable substitute for the egg and sperm cells essential to creating most mammalian life have not yet succeeded. But even this very preliminary work in mice and human cells is prompting a wave of ethical questions from the scientific community about eventual human applications.

Planning parenthood

To make this reproductive process work in mice, Hayashi’s team needed to tie together several earlier discoveries. In 2010 it practiced hitting the “reset” button on cells, sending them back to a stage before they had found their identity. The team began by retracing a process developed by Shinya Yamanaka of Kyoto University in Japan, for which he won a Nobel Prize in 2012.

First, the researchers scraped skin cells off an adult mouse’s tail. They then injected them with a chemical cocktail containing four specific genes to transform adult cells back into stem cells capable of becoming many different kinds of cells. Next, they employed genetic insights established in the early 2000s by Azim Surani of what is now the Gurdon Institute in England and Mitinori Saitou, who was then working in Surani’s lab. (Both men would later mentor Hayashi.) This work, and related experiments with embryonic cells derived from regular mouse embryos, eventually helped Hayashi’s team understand which genes would be needed to coax stem cells into becoming egg progenitor cells called primordial germ cells.

Credit: Tami Tolpa

There was a catch: primordial germ cells, which can develop into either sperm or eggs, still have two sets of chromosomes like any typical animal cell. To form sex cells, which have just one set of genes from each parent, germ cells must twice undergo cell division in a process called meiosis. In females, the first cell division happens in the embryo as the primordial germ cell enters the reproductive system. The second division happens during ovulation when the egg is finally matured after exposure to a number of hormones. After creating the primordial germ cell, Hayashi and his co-workers were able to place them back into a live mouse to complete their development—reaching what was then the boundary of science. To create viable eggs in a dish, however, researchers would need to understand and re-create each step along the pathway to maturation.

The key, the scientists discovered, was to more carefully mimic nature. They spent several years tweaking the solution in which the converted egg cells were grown. One breakthrough came when the team added cells from ovaries of other mouse fetuses into the medium while they matured the cells in a dish. The ovaries released a mixture of hormones—basically creating an ovarylike environment to fool the cells into thinking they were in the body. Furthermore, the scientists altered the viscosity of the fluid medium to mimic what would be found in the mouse.

Once they got that medium right, and the eggs were matured in the lab, the next steps were akin to any other in vitro fertilization (IVF) procedure. The researchers first married the mature eggs and normal mouse sperm together in the lab. After a few days, they selected a promising embryo using a tiny pipette and injected it into a female mouse that would incubate the mouse fetus for 20 days. Finally, after many failed attempts in which the mouse would miscarry, or the embryo would not implant, or it would become stillborn, the process at last led to one healthy pup. Eventually more followed.

The process is still far from perfect, however. Only 16 of the hundreds of stem cells Hayashi’s group created survived the five-week mouse-egg-maturation process. And when the scientists coupled the successful lab-made eggs with sperm, only an extremely small percentage of those skin-cells-turned-eggs went on to become healthy mouse pups (compared with a 62 percent success rate for eggs taken from adult mice and fertilized in vitro). Yet the scientists proved that their methods could work. Those eight pups grew up to be normal and healthy. They even went on to have mates and to birth lively pups of their own.

When sperm meets egg

Plenty of people require reproductive help. More than 10 percent of American men and a similar percentage of women are considered infertile. Options for overcoming infertility are arduous and often unsuccessful. IVF, for example, requires a woman to undergo a week or two of hormone shots designed to release multiple eggs. A handful of those eggs will then be fertilized with sperm in a lab, and one or two will be implanted. The cost, largely paid out of pocket, can easily top $20,000, and yet approximately 65 percent of in vitro fertilization cycles still fail, often because of poor egg quality. Moreover, IVF cannot help if someone has no healthy eggs or sperm.

It is obvious why mining human blood or skin cells to make a baby is an alluring alternative. Instead of extracting human eggs, a health care worker could draw a small vial of a potential mother’s blood. (Blood, which is routinely drawn in medical facilities, might be easier to access in human patients than skin cells, Hayashi says, although either could be used.)

Scientists in a lab could transform those blood cells into stem cells and then, after a few more steps, into eggs or sperm. Next, the manufactured egg could be fertilized with normal sperm, or vice versa, and implanted into the woman using the same method as IVF—leaving the child with the same genetic inheritance he or she would normally get from each parent.

Hayashi says that right now the procedure is too risky to apply to humans and would become acceptable only if the eggs that scientists create can lead to healthy embryos as often as natural ones do. To begin with, researchers will have to show that they can keep the eggs alive in the lab long enough to closely emulate what would be necessary for human development. (In mice, egg cells mature in five days; in women, they require roughly 30.) Yet before reproductive scientists can even think about creating babies this way, they will have to confirm that the process will work in larger animals that more closely resemble people.

Growing together

To overcome that hump, Hayashi’s team is already working with primates: marmoset monkeys. But several major challenges have hindered its progress. Mice are good research subjects because they ovulate every five days and are pregnant for 20. Marmoset pregnancies last more than 140 days, so making a baby would take longer, even if all the science worked perfectly. It takes much longer for primordial germ cells in a marmoset to mature into eggs than it does for mouse eggs to mature, and Hayashi and his team have yet to find a lab environment that keeps the cells alive long enough for that to happen.

In their rodent work, the researchers learned how to mature primordial germ cells outside of living mice, but they still needed ovarian cells from mice fetuses to aid the process. To make sure the primordial germ cells survive and mature in monkeys—and to be able to scale up this work to ultimately create many more lab-grown eggs—Hayashi thinks he will have to do more than simply transfer ovarian cells to a lab dish. He will have to identify the specific ovarian cells that send key signals for maturation and figure out how to derive them from stem cells. That way, in future stages of the work, he would be able to grow all the necessary ingredients—rather than remain dependent on mining other fetuses for their ovarian cells.

Surani, director of germ-line and epigenomics research at Gurdon and a pioneer in this field, has been experimenting with different combinations of these key “helper” cells to support the germ cells’ maturation and communication. “The [germ] cells actually go through to a certain point, and then they need something very specific to break through the next point—they need a change of signal or environment—or something,” Surani explains. He and his team have been making educated guesses about which cells may be particularly significant in that process, but it is slow, painstaking work. To help guide their next steps, they are now studying aborted human fetuses for clues about each step of egg cell maturation. The lab has also started using pigs, instead of mice, because porcine development more closely resembles that of humans and because pigs are cheaper to work with than monkeys. Saitou and his lab recently published a paper in Science showing that they had created human egg precursor cells from adult cells. The process took a long time and yielded few egg-precursor-like structures, suggesting that making eggs in humans might be far more complicated than it was in mice.

Rather than tweaking lab dish protocols, there might be another way to further the process. Some researchers think they will get better results by moving their manufactured cells in vivo as soon as possible to piggyback off the body’s natural quality-control systems that eliminate flawed gametes and leave more resources for the remaining cellular contenders. Renee Reijo Pera, a stem cell scientist at Montana State University, is taking that tack in her research with sperm. In nature, only the fittest sperm survive to fertilize the egg, but making and maturing sperm in a lab dish does away with that competition, increasing the risk that defective sperm will fertilize, she says. Because the human body is exquisitely tuned to weed out bad sperm, Reijo Pera focuses her work on making primordial sperm that can be matured in the testes. “We think the body should do the selecting,” she says. “In a dish, I’m worried we’ll force things to go forward that wouldn’t in the natural environment.”

No matter what precautions scientists take, some critics say artificial eggs or sperm should never be used to create human life. Marcy Darnovsky, for example, does not think that lab-generated germ cells could ever be safe enough to justify their risks. Darnovsky is executive director of the Center for Genetics and Society, a public-interest organization advocating for the responsible use of human genetic technologies. She says she fully supports research that leads to a better understanding of human and animal development. But she draws a line at using engineered eggs and sperm to generate a new life—especially a human one. “I think it’s likely to be extremely biologically risky for any resulting children,” she says, citing the example of mammalian cloning: many of the cloned embryos failed to develop, and some animals were born with terrible health problems. Darnovsky believes that public policy is needed to make sure that the scientific progress Hayashi, Surani, Reijo Pera and others are pursuing does not go too far.

 

Other concerns persist about what this methodology might mean for our understanding of parenting. If anyone’s cells could be manipulated into becoming sperm or egg, for example, could that portend a future where individuals’ cells could become both sperm and egg—creating a uniparent? Or might someone be able to snatch up a stray skin cell from a person’s napkin or bed to create a child without his or her consent or knowledge? Moreover, as George Daley, dean of Harvard Medical School, and his colleagues wrote in 2017 in Science Translational Medicine, such a technology could enable the creation of embryos on a previously unimagined scale—raising the specter of the devaluation of human life, as well as vexing policy challenges.

Ethical concerns have thus far constrained any human-related work on in vitro gametogenesis and have kept funding to a minimum, researchers say. Science involving embryos has long been restricted in the U.S. Whereas the Obama administration was friendlier to stem cell research than its predecessor, the pendulum has swung back somewhat under Donald Trump. In other countries as well, the lack of funding for such research and difficulty in accessing tissue samples of natural embryos for comparison add an extra layer of challenge to the research, according to Surani and Helen Picton, who does related work at the University of Leeds in England. Hayashi, for example, says it would be very difficult for him to do human germ-cell studies in his native Japan. (Japanese law forbids fertilizing human germ cells, even for research purposes.) But Jacob Hanna, a stem cell scientist at the Weizmann Institute of Science in Israel, says he has an easier time because of cultural interest in advancing reproductive technologies.

An ethical conundrum

Even if they never produce a human baby, though, scientists say simply pursuing the goal of making eggs and sperm will have payoffs: in treating infertility, understanding early development and unraveling the effect that toxins can have on human inheritance. “It’s a voyage of discovery,” says Picton, who specializes in ovarian physiology and reproduction. Figuring out how to identify high-quality eggs and sperm may help improve the selection process for IVF, for example. And the process of refining the recipe for gamete creation will provide the first real insights into where cells go wrong to cause disease, birth defects or cellular death.

Learning how to make eggs and sperm from skin or blood cells might also help scientists better unravel genetic inheritance known as epigenetics—changes not to the genes but to gene expression. Understanding how sperm and eggs are formed in their earliest days might allow us to scour those cells for any methyl groups or other changes that have accumulated in the genes. Right now questions abound regarding how some traits seem to get passed down without altering the underlying genetics. In a 2016 study, for instance, epigenetic changes to areas of genes associated with regulating stress hormones were found in the children of Holocaust survivors born years after their parents’ trauma. The genes were unchanged, but how the genes acted seemed to get passed down. Being able to generate eggs and sperm from stem cells could allow scientists to dig into this epigenetic process, Surani says, and could offer insights into diseases of aging, which are often caused by the accumulation of epigenetic markers. Treatments for aging-related diseases might even come out of a new understanding of how these marks are erased in the developing germ cell.

Surani is now researching how mitochondria—the cells’ energy source—perform during the egg-making process. Mitochondria go through a selection process during reproduction, with the child receiving only his or her mother’s genetic material. The process of correcting defects in mitochondria is not well understood, but Surani hopes that by studying how the germ cell corrects such errors, he and his colleagues can learn a lot about cellular energy and related diseases. “Along the way, we can gather knowledge that could have a huge impact on human health,” he says.

Hayashi hopes that such efforts will also be useful for rescuing and restoring nearly extinct species, such as the white rhinoceros. By improving their understanding of the process of forming gametes, researchers will be better poised to work with species that are likely to die out, he says. He is currently trying to reproduce his mouse research in northern white rhinos. Although progress is coming slowly, Hayashi thinks artificial insemination will eventually allow scientists to rescue the nearly extinct species. But the wait time is much longer. A mouse is pregnant for 20 days; a white rhino is pregnant for 16 months, he notes.

When Hayashi talks to audiences about his white rhino work, everyone looks happy, he says. But when he mentions doing similar research in humans, “some people are very skeptical, and some are very afraid.” Hayashi understands their concerns. A lot of human germ cells and embryos would be wasted before human stem cells could successfully be transformed into viable eggs and sperm. Even the viable gametes might still carry the risk of birth defects, he cautions.

Reijo Pera believes that ethics do support studying this work for human applications—and, if it can be done safely enough, even using it to create humans. A cancer survivor who is infertile herself, Reijo Pera believes that helping couples have children justifies the quest.

Yet thorny questions remain about exactly what should be considered safe and who should decide that. When scientists developed other contentious technologies, such as IVF and the gene-editing CRISPR system, formal meetings among researchers, ethicists and members of the public helped to develop recommendations and guidelines for their potential applications. The same will likely be required for in vitro gametogenesis, researchers and ethicists note. Moreover, those conversations should take place long before the science is at a stage where humans can use it. “Before the inevitable, society will be well advised to strike and maintain a vigorous public conversation on the ethical challenges of [in vitro gametogenesis],” Daley and his colleagues wrote in their January 2017 paper. “With science and medicine hurtling forward at breakneck speed, the rapid transformation of reproductive and regenerative medicine may surprise us.”