The scientists of the MDC Technology Platform “Pluripotent Stem Cells” and the Helmholtz Zentrum München were able to generate and describe in detail so-called induced pluripotent stem cells (iPSCs) from preserved skin cells of the northern white rhinoceros Nabire. Nabire lived in the Safari Park Dvůr Králové (Czech Republic), where she died in 2015 at the age of 31. On the very day of her death, scientists secured skin and tissue samples and cryopreserved them for later use. Stem cell scientist Dr Micha Drukker and his team at Helmholtz Zentrum München and at Leiden Academic Centre for Drug Research at Leiden University succeeded in producing iPS cells from this tissue using the method of episomal reprogramming. To do this, Drukker and his team introduced foreign DNA molecules into the genome of the skin cells, so-called plasmids. These contain genes that reprogram the skin cells into iPS cells. It is the first time that the creation of iPSCs has been successful with samples obtained from a rhinoceros at this age. Since northern white rhino tissue donors are or have been rather of old age, this significantly improves the prospects of a stem-cell approach to create artificial oocytes for advanced assisted reproduction.

A second and no less important advance to the process and protocols of stem-cell creation in rhinoceroses are the insights gained by the team into the differentiation of stem cells into different states: iPS cells have different states: they can be naïve – the “ground state” of pluripotency – or primed. Cells in the latter state are thought to have reached a slightly more advanced stage of embryonic development. Experiments with stem cells generated from mice show that they are particularly good at producing germline cells when converting from the primed to the naïve-like state. However, when the scientists first attempted to convert the rhino cells to a naïve-like state, the cells died. The team therefore introduced a gene into the rhino cells that prevents cell death – and with this, they successfully obtained naïve iPS cells. “We have characterized the cells in detail by, among other things, analyzing transcriptome data,” explains first author Dr Vera Zywitza from the Technology Platform “Pluripotent Stem Cells” led by Dr Sebastian Diecke at the MDC. “The successful conversion to a naïve-like state of pluripotency is a promising starting point for generating germline cells.”

Nevertheless, Zywitza and her colleagues cannot yet move onto the next stage. “The iPS cells we have cultivated contain persistent foreign genetic material – namely, the reprogramming factors and the 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, Diecke’s team has generated further iPS cells. They 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. “This work contributes significantly to the understanding of pluripotency – the ability of stem cells to differentiate into any body cell,” says Zywitza. “It marks a promising start for germline cell cultivation and thus a significant milestone on the way to the artificially generated rhino egg.”

All of BioRescue’s procedures undergo a thorough ethical assessment in order to systematically evaluate the balance of animal welfare and conservation value of the procedures. Since this is particularly important when boundary-breaking new technologies for conservation are being developed, a team of wildlife ethics specialists lead by Prof Barbara de Mori at Padua University is part of the BioRescue team. This team assessed the ethical dimension of stem-cell-related procedures within BioRescue and will continue to closely accompany any further step of the mission.

Natural reproduction is no longer possible for the northern white rhinoceros given a remaining population of only two animals and both of them being females. However, the BioRescue consortium led by the Leibniz Institute for Zoo and Wildlife Research (Leibniz-IZW) is developing methods that could make offspring possible despite these adverse circumstances. They retrieve so-called oocytes (immature egg cell cells) from the females, fertilise 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 is 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 the oocytes and the low genetic diversity of the represented population are particularly challenging,” says BioRescue project leader Prof Thomas Hildebrandt, head of the Department of Reproduction Management at Leibniz-IZW. Oocytes could only be successfully retrieved and fertilised from one individual, which is why strategies are being sought for obtaining a higher number of oocytes from several unrelated animals. As part of the BioRescue consortium, the MDC and Kyushu University, together with cooperation partners such as Helmholtz Zentrum München, are developing methods to produce gametes (eggs and sperm) from skin cells. In 2016, Prof Katsuhiko Hayashi (Kyushu University) succeeded in generating oocytes from the skin of mice, artificially fertilising them and implanting them in female mice. The mice produced with this method were healthy and 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

In the following months and years, the BioRescue scientists are faced with the challenge of reprogramming the iPS cells so that they actually develop into eggs or sperm. If this succeeds, the further procedure would be the same as in 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 of the northern white rhinoceros. These embryos would also need to be cryopreserved in liquid nitrogen before being thawed again for transfer into a surrogate. “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,” Hildebrandt concludes.

The BioRescue project can be financially supported at www.biorescue.org.