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 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.

Publication
Hayashi M, Zywitza V, Naitou Y, Hamazaki N, Goeritz F, Hermes R, Holtze S Lazzari G,
Galli C, Stejskal J, Diecke S, Hildebrandt TB, Hayashi K (2022): Robust induction of
primordial germ cells of white rhinoceros on the brink of extinction. Science Advances.
DOI: 10.1126/sciadv.abp9683

Statements
Masafumi Hayashi, Osaka University:
“This is the first time that primordial germ cells of a large, endangered mammalian species have been successfully generated from stem cells. Previously, it has only been achieved in rodents and primates. Unlike in rodents, we 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. Additionally, we found that two specific markers, CD9 and ITGA6, 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.”

Katsuhiko Hayashi, Osaka University:
“Developing a culture system that delivers robust results has been extremely challenging since the precise orchestration of the specific signals required to induce the desired cellular differentiation is unique to every species. It was also necessary to confirm that the primordial germ cell-like cells were genetically identical to the cells from which they originated – this can be a daunting task.”

Jan Stejskal, Safari Park Dvůr Králové:
“We are thrilled that this milestone was achieved by the BioRescue scientists and that Nabire, that deceased in Dvůr Králové in 2015, is still in the race for saving her kind. Unfortunately, she didn’t give birth to an offspring during her life, but with the recent successes in stem cell associated techniques it seems perfectly possible that a direct descendant of Nabire will be born at some point in the future and can play an important role in repopulating central Africa with northern white rhinos.”

Thomas Hildebrandt, Leibniz Institute for Zoo and Wildlife Research:
“We knew from the beginning that relying on natural gametes for the assisted reproduction technologies would not be sufficient to save the northern white rhino from the brink of extinction on the long run. So, it is crucial that we pursue a complementary strategy for making gametes available with significantly higher genetic diversity and in much larger numbers – even making it possible to create embryos from Najin through artificial gametes, which is something that was not possible with her natural gametes. It is encouraging to see that the stem cell specialists in our consortium, from the Osaka University and the Max Delbrück Center for Molecular Medicine, achieved this important step already now. It is also important to note that the plans for natural gametes and artificial gametes are not alternative paths to go, but interconnect and merge at the point where in vitro fertilization yields embryos.”

Cesare Galli, Avantea
“The successful derivation of embryonic stem cells (ESCs) in our laboratory in 2018 by our colleague Dr. Giovanna Lazzari from the first SWR embryos that we were able to obtain, proved to be instrumental for the success of the work of Prof. Hayashi team because ESCs have been studied and differentiated for a long time and provided a template for the iPSCs.”

Vera Zywitza, Max Delbrück Center
“After we succeeded in creating the PGCs, we 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. We therefore have to find suitable conditions under which the cells will grow and divide their chromosome set in half. In mice, we found that the presence of ovarian tissue was important in this crucial step. Since we cannot simply extract this tissue from the two female rhinos, we will probably have to grow this from stem cells as well.”

Contacts
Osaka University, Germline Genetics in the Department of Genome Biology, Graduate School of Medicine
Masafumi Hayashi
Email: mhayashi@gcb.med.osaka-u.ac.jp
Katsuhiko Hayashi
Email: hayashik@gcb.med.osaka-u.ac.jp

Leibniz Institute for Zoo and Wildlife Research (Leibniz-IZW)
Thomas Hildebrandt
BioRescue project head and head of Department of Reproduction Management
Phone: +49305168440
Email: hildebrandt@izw-berlin.de
Steven Seet
Head of Science Communication
Phone: +491778572673
Email: seet@izw-berlin.de

Jan Zwilling
Science Communication
Phone: +49305168121
Email: zwilling@izw-berlin.de
Safari Park Dvůr Králové
Jan Stejskal
Director of Communication and International Projects
Phone: +420608009072
Email: jan.stejskal@zoodk.cz
Max Delbrück Center for Molecular Medicine in the Helmholtz Association

Sebastian Diecke
Head of the Technology Platform “Pluripotent Stem Cells”
email: sebastian.diecke@mdc-berlin.de

Jana Schlütter
Editor, Communications Department
phone :+49-(0)30-9406.2121
email: jana.schluetter@mdc-berlin.de or presse@mdc-berlin.de

Avantea
Cesare Galli
Director
phone: +390 / 372437242
email: cesaregalli@avantea.it

University of Padua
Barbara de Mori
Director of the Ethics Laboratory for Veterinary Medicine, Conservation and Animal Welfare, Department of Comparative Biomedicine and Food Science
Phone: +39-3403747666
Email: barbara.demori@unipd.it
Boilerplates

Osaka University, Germline Genetics in the Department of Genome Biology, Graduate School of Medicine
Our goal is to better understand germ cells, the only cells that pass genetic and epigenetic information across generations. This includes understanding how the quality of inheritable information in the germ cell lineage is maintained; elucidating the different mechanisms underlying anisogametogenesis; elucidating the mechanism for sustainable oogenesis in the ovaries; and developing new technology that models the differentiation of germ cell lineages.
www.med.osaka-u.ac.jp/eng/introduction/research/genome/germline

Leibniz Institute for Zoo and Wildlife Research (Leibniz-IZW)
The Leibniz-IZW is an internationally renowned German research institute of the Forschungsverbund Berlin e.V. and a member of the Leibniz Association. Our mission is to examine evolutionary adaptations of wildlife to global change and develop new concepts and measures for the conservation of biodiversity. To achieve this, our scientists use their broad interdisciplinary expertise from biology and veterinary medicine to conduct fundamental and applied research – from molecular to landscape level – in close dialogue with the public and stakeholders. Additionally, we
are committed to unique and high-quality services for the scientific community.
www.izw-berlin.de

Safari Park Dvůr Králové
Safari Park Dvůr Králové is a safari park in the Czech Republic. It’s one of the best rhino breeders outside of Africa and the only place where the northern white rhino bred in human care - both remaining females, Najin and Fatu, were born here. Dvůr Králové Zoo coordinates efforts to save the northern white rhinos.
https://safaripark.cz/en/

Max Delbrück Center
The Max Delbrück Center for Molecular Medicine in the Helmholtz Association (Max Delbrück Center) is one of the world’s leading biomedical research institutions. Max Delbrück, a Berlin native, was a Nobel laureate and one of the founders of molecular biology. At the Center’s locations in Berlin-Buch and Mitte, researchers from some 70 countries analyze the human system – investigating the biological foundations of life from its most elementary building blocks to systems-wide mechanisms. By understanding what regulates or disrupts the dynamic equilibrium in a cell, an organ, or the entire body, we can prevent diseases, diagnose them earlier, and stop their progression with tailored therapies. Patients should benefit as soon as possible from basic research discoveries. The Max Delbrück Center therefore supports spin-off creation and participates in collaborative networks. It works in close partnership with Charité – Universitätsmedizin Berlin in the jointly run Experimental and Clinical Research Center (ECRC), as well as with the Berlin Institute of Health (BIH) at Charité and the German Center for Cardiovascular Research (DZHK). Founded in 1992, the Max Delbrück Center today employs 1,800 people and is funded 90 percent by the German federal government and 10 percent by the State of Berlin.
www.mdc-berlin.de

Avantea
Avantea is a laboratory of advanced technologies for biotechnology research and animal reproduction based in Cremona, Italy. Avantea has over twenty years of experience and the know-how in assisted reproduction of livestock developed through years of research conducted in the biomedical and animal reproduction fields.
www.avantea.it/en/

University of Padua
University of Padua in Italy is one of the oldest in the world, celebrating 800 years. Its Department of Comparative Biomedicine and Food Science is developing leading research and education in the field of wildlife conservation and welfare with a special focus on ethical assessment and evaluation of research projects and educational programs developed by the Ethics Laboratory for Veterinary Medicine, Conservation and Animal Welfare.
https://www.unipd.it/en/