BioRescue produces primordial germ cells from northern white rhino stem cells – a world’s first for large mammals

Creation of primordial germ cell-like cells (PGCLSs) from induced pluripotent stem cells of the northern white rhino Nabire nourishes the hope to prevent the extinction of the northern white rhino

LEIBNIZ INSTITUTE FOR ZOO AND WILDLIFE RESEARCH (IZW)

Northern White Rhino Nabire at Safari Park Dvur Kralove — IMAGE: NORTHERN WHITE RHINO NABIRE, FROM WHICH THE PRIMORDIAL GERM CELLS HAVE BEEN PRODUCED, AT SAFARI PARK DVUR KRALOVE view more
CREDIT: PHOTO BY KHALIL BAALBAKI

In its race to advance assisted reproduction and stem cell associated technologies to save the northern white rhinoceros from extinction, the BioRescue consortium announces a major breakthrough: the creation of primordial germ cell-like cells (PGCLSs) from induced pluripotent stem cells of the northern white rhino Nabire. This milestone was led by specialists from Osaka University, Japan, and has never been achieved in large mammals before. Now there is one last step to master for the production of artificial rhino gametes (eggs and sperm) from preserved tissue. If successful, this would boost the availability and genetic diversity of embryos and become a cornerstone for saving the northern white rhinoceros. The scientists describe the culture systems and processes for the induction of the PGCLCs from stem cells in a newly published paper in the journal Science Advances.

Thirty-three-year-old Najin and her daughter Fatu are the last surviving northern white rhinos on the planet. Without a possibility for natural reproduction with only two females left of their kind, the only hope for the northern white rhinoceros lies in advanced assisted reproduction technologies. The scientists of the BioRescue consortium have already produced northern white rhino embryos by in vitro fertilisation of oocytes with sperm. Embryos are destined to be carried to term by southern white rhino surrogate mothers. Crucial to embryo production is the availability of oocytes (egg cells) and sperm. Female Fatu is the only donor of natural oocytes left and frozen sperm is available to the program from only four males – and some of these males are closely related to Fatu. With stem cell associated techniques (SCAT) the BioRescue scientists aim to overcome this bottleneck: By using stored and preserved tissue of rhino skin it should be possible in principle to create induced pluripotent stem cells (iPCSs), primordial germ cells and finally artificial gametes. This could increase the number of individuals in the founder group of a future population to 12 and permit the production of oocytes in much larger quantities. According to their most recent paper published in the journal Science Advances, the team has now successfully cultivated primordial germ cells (PGCs) – the precursors of eggs and sperm – from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

In sexually reproducing organisms such as humans, rhinos or mice, primordial germ In sexually reproducing organisms such as humans, rhinos or mice, primordial germ cells (PGCs) are embryonic precursors of sperm and eggs that pass on genetic and epigenetic information from one generation to the next. In order for them to be developed from stem cells, they need a very specific environment in which signals from hormones or proteins trigger the required morphological and functional transformation. This also includes a migration of the cells in the body (PGC migration). For the first time in large mammals, BioRescue scientists Masafumi Hayashi and Katsuhiko Hayashi and their team from Osaka University succeeded in creating such an environment in a culture system. They established culture systems for the southern white rhino, for which embryonic stem cells are available, and the northern white rhino, for which they used induced pluripotent stem cells derived from tissue samples. In order to succeed they needed to identify the signals which had to be introduced to the system at specific time points and the order in which they should trigger the development into PGCLCs.

The scientists relied on knowledge from the mouse model: In 2016, Katsuhiko Hayashi and his team managed to create primordial germ cell-like cells and finally germ cells from mice that were fertilised in the lab and resulted in healthy offspring being born. In the case of the white rhinoceroses, Hayashi is working in close cooperation within BioRescue with Sebastian Diecke’s Pluripotent Stem Cells Platform at the Max Delbrück Center and with reproduction experts Thomas Hildebrandt from Leibniz-IZW, both of them last authors of the paper, and Cesare Galli from Avantea.

Through repeated refinement, the BioRescue team produced PGCLCs from northern white rhino Nabire under a defined condition. Because PGCs are the founder population for gametes, this accomplishment paves a way to produce functional gametes from induced pluripotent stem cells from northern white rhinos which will contribute to the effort to rewind their extinction. As soon as the creation of artificial gametes is successful, this plan merges with the procedures that BioRescue carries out with natural gametes: Just like with oocytes obtained from Fatu and sperm thawed from frozen samples, the artificially created eggs and sperm would be in vitro fertilised in the lab. The embryos created in this way would be stored safely in liquid nitrogen until a transfer to a surrogate mother is possible.

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

All BioRescue procedures are accompanied by ethical risk assessments, conducted under the guidance of the Ethics Laboratory for Veterinary Medicine, Conservation and Animal Welfare at the University of Padua, Italy

Northern White Rhinos Najin and Fatu in their enclosure at Ol Pejeta Conservancy (Kenya). They are are the only two remaining individuals of their kind on the planet.

CREDIT

Photo by Jan Zwilling

The graphic outlines the researchers’ plan (A), shows day 4 of the induction of primordial germ cells from rhinoceros pluripotent stem cells (B), and a comparison of the gene expression profiles of these cells in humans, mice, and southern white rhinoceroses. Cell differentiation proceeded similarly in humans and rhinos. Here, embryonic stem cells of the southern white rhinoceros were used (C).
CREDIT
Graphic: Masafumi Hayashi, Osaka University

CAPTION
Prof. Katsuhiko Hayashi at Stem Cell Lab at Osaka University
CREDIT
Photo by Antony Mwangi

JOURNAL

Science Advances

DOI

10.1126/sciadv.abp9683

METHOD OF RESEARCH

Experimental study

SUBJECT OF RESEARCH

Cells

ARTICLE TITLE

Robust induction of primordial germ cells of white rhinoceros on the brink of extinction

ARTICLE PUBLICATION DATE

9-Dec-2022

A big step toward producing rhino gametes

Peer-Reviewed Publication

MAX DELBRÜCK CENTER FOR MOLECULAR MEDICINE IN THE HELMHOLTZ ASSOCIATION

Fata and Najin — IMAGE: THE LAST TWO SURVIVING FEMALES LIVE IN THE OL PEJETA CONSERVANCY IN KENYA. view more
CREDIT: JAN STEJSKAL, SAFARI PARK DVŮR KRÁLOVÉ

To save the northern white rhinoceros from extinction, the BioRescue team is racing to create lab-grown egg and sperm cells of the critically endangered subspecies. The team has now reported a milestone in Science Advances: they have generated primordial germ cells from stem cells – a world’s first.

Thirty-three-year-old Najin and her daughter Fatu are the last surviving northern white rhinos on the planet. They live together in a wildlife conservancy in Kenya. With just two females left, this white rhino subspecies is no longer capable of reproduction – at least not on its own. But all hope is not lost: according to a paper published in the journal Science Advances, an international team of researchers has successfully cultivated 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. And so the northern white rhino subspecies, which humans have already effectively wiped out through poaching, may yet be saved thanks to state-of-the-art stem cell and reproductive technologies.

First success with an endangered species

To get from a piece of skin to a living rhinoceros may be a true feat of cellular engineering, but the process itself is not unprecedented: the study’s co-last author Professor Katsuhiko Hayashi leads research labs at the Japanese universities of Osaka and Kyushu in Fukuoka, 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. The two Berlin-based scientists are also co-last authors of the current study.

“This is the first time that primordial germ cells of a large, endangered mammalian species have been successfully generated from stem cells,” explains the study’s first author, Masafumi Hayashi of Osaka University. Previously, it has 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 Cremona, Italy, where they were grown by Professor Cesare Galli’s team. The newly derived northern white rhino PGCs, meanwhile, 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 says that they are hoping to use the cutting-edge stem cell technology from Katsuhiko Hayashi’s lab to save other endangered rhino species: “There are five species of rhino, and almost all of them are classified as threatened on the IUCN Red List.” 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 explains.

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. “The primordial cells are relatively small compared to matured germ cells and, most importantly, still have a double set of chromosomes,” explains Dr. Vera Zywitza from Diecke’s research group, who was also involved in the study. “We therefore have to find suitable conditions under which the cells will grow and divide their chromosome set in half.”

Genetic variation is key for conservation

Leibniz-IZW researcher Hildebrandt is also pursuing a complementary strategy. He wants to obtain egg cells from 22-year-old Fatu and fertilize them in Galli’s lab in Italy 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 explains that Fatu is not able to bear her own offspring, as she has problems with her Achilles tendons and cannot carry any additional weight. Her mother Najin, meanwhile, is past child-bearing age and also suffers from ovarian tumors. “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 adds.

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 explains. “Since we cannot simply extract this tissue from the two female rhinos, we will probably have to grow this from stem cells as well.” The scientist is hopeful, however, that ovarian tissue from horses could come in useful, as horses are among the rhinos’ closest living relatives from an evolutionary standpoint. If only humans had taken as good care of the wild rhino as they had of the domesticated horse, the immense challenge now facing the BioRescue scientists could perhaps have been avoided altogether.

The SOX17 gene played a key role in inducing primordial germ cells from pluripotent stem cells of the white rhinoceros.