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Saving the Northern White Rhino from Extinction
The international BioRescue Consortium will save the northern white rhinoceros from extinction by using stem cell associated techniques. Scientists are on their way to producing gametes out of skin cells. Additionally, oocytes are collected from the last northern white rhinos to produce embryos in the lab. After transferring these embryos into surrogate mothers, a new viable population of rhinos can be created and introduced into the wild.
by Dr Sebastian Diecke and Steven Seet


Nabire at Safari Park Dvur Kralove, photo by Khalil Baalbaki.

The BioRescue consortium develops advanced methods of assisted reproduction to save the northern white rhino from extinction. Oocytes from the last remaining females play a key role in this mission, as embryos are created from them by in vitro fertilisation with sperm from deceased bulls. Within BioRescue, the Max Delbrück Center for Molecular Medicine (MDC) in Berlin is working with partners in Munich and Kyushu (Japan) on a second strategy for obtaining oocytes – developing them from stem cells. The team created induced pluripotent stem cells (iPSCs) from the northern white rhino Nabire, characterised them thoroughly, and transferred them into a naïve-like state of pluripotency, which may be beneficial for subsequent germ cell differentiation.

“This work contributes significantly to the understanding of pluripotency in rhinos,” says Vera Zywitza, a scientist in the lab of Sebastian Diecke, group leader of Technology Platform Pluripotent Stem Cells at the MDC. “The identified culturing conditions are a promising starting point and thus a significant milestone on the way to the artificially generated rhino egg.”

Nevertheless, Zywitza and her colleagues cannot yet move on to the next stage. “The iPS cells we generated contain persistent foreign genetic material – namely, the reprogramming factors and a gene that prevents cell death,” Zywitza explains. “This means we can’t use them to make germ cells, as there is a risk these would be pathologically altered.” In the meantime, the Diecke lab has generated further iPS cells. The scientists used RNA viruses instead of plasmids to introduce the reprogramming factors. These new iPS cells do not contain anything that does not belong there. Now, the scientists are trying to produce primordial germ cells from them.

Natural reproduction is no longer possible for the northern white rhinoceros since the remaining population only consists of two females. This is why the international BioRescue consortium led by the German Leibniz Institute for Zoo and Wildlife Research (Leibniz-IZW) develops methods that could make offspring possible despite these adverse circumstances. They retrieve immature egg cell cells (oocytes) from the two females, fertilised them in the laboratory with thawed sperm from already deceased bulls and create embryos in this way – 14 of them are already stored in liquid nitrogen. Using a technology and method that is entirely new for rhinos and currently in development by the BioRescue team, the embryos can be implanted in southern white rhino females, which then act as surrogate mothers to bear the longed-for offspring for the world’s rarest rhinos.

“Every step of this mission is uncharted scientific territory, and the limited availability of oocytes and the low genetic diversity of the represented population are a particular challenge,” says BioRescue project leader Professor Thomas Hildebrandt, head of the Department of Reproduction Management at the Leibniz-IZW.

Oocytes could only be successfully retrieved and fertilised from one individual, which is why strategies are being sought to obtain a larger number of oocytes from several unrelated animals. As part of the BioRescue consortium, the MDC and Osaka University, together with cooperation partners such as the Helmholtz Zentrum München, are developing methods to produce gametes (eggs and sperm) from skin cells.

In 2016, Professor Katsuhiko Hayashi (at this time Kyushu University) succeeded in generating oocytes from the skin of mice, artificially fertilising them and implanting them in female mice. The baby mice produced with this method were healthy and after completion of their development, fertile.

“If we succeeded in doing the same for the northern white rhinoceros, we could stop the cumbersome egg collection from living animals and still produce embryos in larger numbers,” says Hildebrandt. “This strategy would also significantly expand the number of animals that we could use to produce the embryos.” So far, this group is limited to two living females of which only one is an egg donor and four bulls from which sperm have been cryopreserved. Cell cultures are available not only from these six individuals, but from six further northern white rhinos – such as from Nabire.

All of BioRescue’s procedures undergo a thorough ethical assessment in order to systematically evaluate the perspectives of animal welfare and the conservation value of the procedures. Since this is particularly important when breakthrough technologies for conservation are being developed, a team of wildlife ethics specialists led by Professor Barbara de Mori from Padua University in Italy is part of the BioRescue team. These specialists assess the ethical dimension of stem cell-related procedures within BioRescue and will continue to closely accompany every further step of the mission.

In the following months and years, the BioRescue scientists are faced with the challenge of differentiating the generated iPS cells so that they actually produce eggs or sperm. If this succeeds, the follow-up procedure would be the same as the approach carried out by BioRescue so far. The artificial oocytes would be matured and fertilised in the laboratory via intracytoplasmic sperm injection (ICSI) and would develop into embryos. These embryos would also need to be cryopreserved in liquid nitrogen before being thawed again for transfer into a surrogate mother from the southern white rhino.

“The stem cell approach is a hugely important piece of the puzzle for our mission, but it does not exempt us from having to master challenging steps such as embryo transfer and thus creating a pregnancy in a surrogate mother,” Hildebrandt concluded. The BioRescue project can be financially supported at www.biorescue.org.

References

  1. Zywitza V, Rusha E, Shaposhnikov D, Ruiz-Orera J, Telugu N, Rishko V, Hayashi M, Michel G, Wittler L, Stejskal J, Holtze S, Göritz F, Hermes R, Wang J, Izsvak Z, Colleoni S, Lazzari G, Galli C, Hildebrandt TB, Hayashi K, Diecke S & Drukker M (2022): Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction. Sci Rep 12, 3100. https://doi.org/10.1038/s41598-022-07059-w>.
  2. Biasetti P, Hildebrandt TB, Göritz F, Hermes R, Holtze S, Galli C, Lazzari G, Colleoni S, Pollastri I, Spiriti MM, Stejskal J, Seet S, Zwilling J, Ngulu S, Mutisya S, Kariuki L, Lokolool I, Omondo P, Ndeereh D, de Mori B (2022): Ethical Analysis of the Application of Assisted Reproduction Technologies in Biodiversity Conservation and the Case of White Rhinoceros (Ceratotherium simum) Ovum Pick-Up Procedures. Front Vet Sci, 831675. https://doi.org/10.3389/fvets.2022.831675.

About the Authors

Dr Sebastian Diecke, Group Leader Technology Platform Pluripotent Stem Cells, Max Delbrück Center for Molecular Medicine in the Helmholtz Association.

Steven Seet, Head of PR and Science Communication, Leibniz Institute for Zoo and Wildlife Research (Leibniz-IZW) – Steven is a senior science communicator for conservation science.

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