More than a decade ago, scientists succeeded for the first time in generating primordial germ cells—the precursors of sperm and eggs—from embryonic stem cells, a process that led to functional sperm capable of producing offspring. The milestone, achieved in mice, had not been repeated in any other species since.
Now, rats join the club. A study published today (April 7) in Science reports that researchers have produced healthy, fertile rat offspring with sperm made from stem cells. The process began with the induction of primordial germ cells from rat stem cells which, when transplanted into rat testes, developed into sperm, which in turn resulted in healthy and fertile offspring when injected into rat oocytes. The achievement may be helpful for biomedical research, where rats are widely used. The study, as well as the length of time that passed between success in mice and rats, also highlight the challenges involved in translating the protocol from one species to another, suggesting that it might still be a long time before a similar reproductive technology could be developed for humans.
“I think it was a really important breakthrough to be able to show this technology in a different species,” says Amander Clark, a stem cell biologist at the University of California, Los Angeles, who did not participate in the study.
The team applied part of the protocol designed for mice, but given the physiological and developmental differences between the rodent species, many steps had to be tweaked—something that only became possible after many years of research.
A few years ago, “we didn’t have enough information about the rat’s development,” says University of Tokyo’s Toshihiro Kobayashi, one of the leaders of the new study. This was partially due to the limited efforts to construct genetically modified rats compared with what has been done in mice, he explains.
In recent years, though, Kobayashi and colleagues have been constructing rat mutants that have allowed them to visualize the development of primordial germ cells in vivo. Using fluorescent markers to trace the expression of genes that are key to the transition from stem cells to primordial germ cells, they have learned more about how gene expression changes over time, all of which was helpful to finally recapitulate the process in vitro, he says.
The first step of the recipe was to induce epiblast-like cells—those giving rise to all the cells in the embryo—from rat embryonic stem cells. Once this transition was accomplished, the epiblast-like cells were placed on a medium to induce what they call primordial germ cell–like cells. Among other ingredients, the medium contained BMP4, a signaling molecule critical for this step in both mice and rats. Kobayashi explains that performing these transitions in vitro was challenging: he and his colleagues had to optimize the culture conditions to induce the desired cell fates because the recipes used for mice were not the most appropriate for rats.
Then, in order to mature the primordial germ cell–like cells derived from this process into a later stage of germ cell development, Kobayashi and colleagues cultured them alongside gonadal somatic cells, simulating the environment they would normally be in during maturation. After a few days, the cells had a pattern of gene expression associated with this later stage of development. Both early and late primordial germ cell–like cells were then transplanted into rat testes lacking endogenous germ cells, where the team confirmed that they developed into mature sperm. Finally, to assess if the sperm derived from this protocol was actually functional, the team injected it into rat oocytes, yielding normal offspring capable of reproducing.
However, the rats were unable to successfully produce offspring with the lab-grown sperm via normal mating. Primordial germ cells require further maturation to achieve this, explains Kobayashi. “I assume that we may need another type of gonadal cells as well as [an] optimized culture condition,” he writes in a follow-up email—a problem he is interested in exploring further.
University of Southern California stem cell researcher Qi-Long Ying, who did not participate in the study, says that the new report demonstrates that this approach to in vitro gametogenesis is reproducible and solid. It might be eventually possible to apply this to other species, such as those at risk of extinction, he adds—it will just take time to improve and optimize the conditions to do so.
But more work will need to be done before these methods might be applied to humans, says Ying. Clark explains that in addition to the challenges of translating the technique posed by developmental differences among species, attempts to generate human germ cells have to date involved cells from two different species: the stem cells come from humans while the supporting somatic cells needed to promote maturation are from mice. In these scenarios, so far, germline development stops before the sperm is mature, Clark explains, and she hypothesizes this might be partly due to not having interactions with the right somatic cells.
What has now been achieved in rats is very similar to what was observed in mice back in 2011, adds Clark. Both successes show the importance of maturing cells in the right environment, including doing so alongside somatic cells from the same species at the appropriate stage.
Even if not immediately applicable to humans, in vitro gametogenesis could be a tool for making rat models of human disease for biomedical research. “Some human diseases can only be . . . modeled in the rat but not in the mice”—for instance, a number of neurological diseases, says Ying. Before the advent of transgenic and gene targeting technologies—where mice have played a predominant role—many more papers were published “using rats as a model to study human diseases” than those using mice, he adds, because rats are more physiologically similar to humans.
Being able to recapitulate the germline development in a dish may also help in gaining a better understanding of human reproductive biology and diseases associated with it. Infertility, for example, affects millions of people, says Clark, and many of its causes “are not well understood because the field lacks a variety of models to be able to study the formation of the germline.” New in vitro models like this one may become “important tools . . . to understand the basis of disease,” she says.