Primordial germ cells produced from northern white rhinos

As part of their efforts to save the northern white rhinoceros from extinction, researchers from the BioRescue consortium are racing to create lab-grown egg and sperm cells of the critically endangered subspecies. The team have now reported the creation of primordial germ cell-like cells (PGCLSs) from induced pluripotent stem cells — said to be a world-first in large mammals.

Najin and her daughter Fatu are the last surviving northern white rhinos on the planet, meaning the subspecies is no longer capable of natural reproduction. But a new study published in the journal Science Advances has detailed the cultivation of primordial germ cells (PGCs) — the precursors of rhino eggs and sperm — from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). This represents a major milestone in an ambitious plan.

The BioRescue project, which is coordinated by the Leibniz Institute for Zoo and Wildlife Research (Leibniz-IZW) and has been funded by the German Federal Ministry of Education and Research (BMBF) since 2019, wants to save the northern white rhino from extinction. To this end, the scientists are pursuing two strategies — one of them trying to generate viable sperm and eggs from the skin cells of deceased rhinos. The idea is to implant the resulting embryos into closely related southern white rhino females, who will then carry the surrogate offspring to term.

First success with an endangered species

The study’s co-last author, Professor Katsuhiko Hayashi, leads research labs at Japan’s Osaka and Kyushu Universities, where his teams have already accomplished this feat using mice. But for each new species, the individual steps are uncharted territory. In the case of the northern white rhinoceros, Hayashi is working in close cooperation with Dr Sebastian Diecke’s Pluripotent Stem Cells Technology Platform at the Max Delbrück Center and with reproduction expert Professor Thomas Hildebrandt from Leibniz-IZW.

“This is the first time that primordial germ cells of a large, endangered mammalian species have been successfully generated from stem cells,” said the study’s first author, Masafumi Hayashi of Osaka University. Previously, this had only been achieved in rodents and primates. Unlike in rodents, the researchers have identified the SOX17 gene as a key player in rhinoceros PGC induction. SOX17 also plays an essential role in the development of human germ cells — and thus possibly in those of many mammalian species.

The southern white rhino embryonic stem cells being used in Japan come from the Avantea laboratory in Italy, where they were grown by Professor Cesare Galli’s team. The newly derived northern white rhino PGCs originated from the skin cells of Fatu’s aunt, Nabire, who died in 2015 at Safari Park Dvůr Králové in the Czech Republic. Diecke’s team at the Max Delbrück Center was responsible for converting them into induced pluripotent stem cells.

Next step: cell maturation

Masafumi Hayashi said the researchers are hoping to use the cutting-edge stem cell technology from Katsuhiko Hayashi’s lab to save other endangered rhino species. The international team also used stem cells to grow PGCs of the southern white rhino, which has a global population of around 20,000 individuals. In addition, the researchers were able to identify two specific markers, CD9 and ITGA6, that were expressed on the surface of the progenitor cells of both white rhino subspecies. “Going forward, these markers will help us detect and isolate PGCs that have already emerged in a group of pluripotent stem cells,” Hayashi said.

The BioRescue scientists must now move on to the next difficult task: maturing the PGCs in the laboratory to turn them into functional egg and sperm cells. As explained by Dr Vera Zywitza, from Diecke’s research group, “The primordial cells are relatively small compared to matured germ cells and, most importantly, still have a double set of chromosomes. We therefore have to find suitable conditions under which the cells will grow and divide their chromosome set in half.”

Hildebrandt is also pursuing a complementary strategy: he wants to obtain egg cells from Fatu and fertilise them in Galli’s lab using frozen sperm collected from four now deceased northern white rhino bulls. This sperm is thawed and injected into the egg in a process known as intracytoplasmic sperm injection (ICSI). However, Hildebrandt noted that Fatu is not able to bear her own offspring, as she has problems with her Achilles tendons and cannot carry any additional weight. Najin, meanwhile, is past child-bearing age and also suffers from ovarian tumours.

“And in any case, since we only have one donor of natural eggs left, the genetic variation of any resulting offspring would be too small to create a viable population,” he said. The team’s top priority, therefore, is turning the PGCs they now have at their disposal into egg cells.

“In mice, we found that the presence of ovarian tissue was important in this crucial step,” Zywitza said. “Since we cannot simply extract this tissue from the two female rhinos, we will probably have to grow this from stem cells as well.”

Image caption: The last two surviving females live in the Ol Pejeta Conservancy in Kenya. Image credit: Jan Stejskal, Safari Park Dvůr Králové.