Osaka University and others working toward the conservation of the northern white rhinoceros, an endangered species, using germ-cell precursors

A research group consisting of Specially Appointed Researcher Masafumi Hayashi and Professor Katsuhiko Hayashi of the Graduate School of Medicine, Osaka University and their colleagues has announced that they have succeeded in the in-vitro induction of primordial germ cell-like cells (PGCLCs), the 'origin' of eggs and sperm, from embryonic stem cells of the southern white rhinoceros and the iPS cells of the northern white rhinoceros, which are endangered species. This was realized by creating cell material and considering culture conditions. The research was carried out as part of the BioRescue Project, in which five countries are involved, particularly Germany. It is hoped that this will lead to the breeding of the northern white rhinoceros, which is in danger of extinction. The group's outcomes were published in the December 10 edition of the international science journal Science Advances.

It is said that Earth is now in the sixth mass extinction event, in which human activity is causing living species to decrease at a faster rate than the extinction of the dinosaurs. Rhinoceros are one of these. There are five species of rhinoceros, including the black rhinoceros and the white rhinoceros, and each of these has subspecies, but almost all of these are designated endangered species. Rhinoceros' gestation period is long - 16 months - and their reproductive age is high, so once the number of individuals is reduced it is difficult for them to recover naturally.

Due to poaching and environmental destruction, only two female northern white rhinoceros remain, so assisted reproduction technology and cellular engineering technology is required for their numbers to increase.

The BioRescue Project was started in 2019 with the aim of recovering the number of northern white rhinoceros, and numerous research institutions and zoos are involved. In addition to the goals of rhinoceros conservation and the establishment of assisted reproductive medicine, the project aims to establish technology for the creation of rhinoceros iPS cells and embryo cultures, as well as technology for the induction of gametes in vitro. To date, they have succeeded in creating ES cells from the southern white rhinoceros, a related species, and northern white rhinoceros iPS cells, among others.

In these developments, the research group explored methods of inducing PGCLCs, the origin of eggs, from southern white rhinoceros ES cells in vitro. They engaged in research observing the expression of a pluripotency gene (OCT3/4) and the PGC gene (BLIMP1) by using gene editing on ES cells.

First, when investigating culture conditions, they succeeded in inducing PGCLC differentiation at a level of around 7% after preculture through cultivation in the conditions for the induction of human and mouse PGCLCs. As a result of analyzing and comparing the features of these cells with those of humans and mice, the group found that changes in the expression of the representative pluripotency gene and PGC gene were similar. In particular, during the differentiation process leading to human PGCs, the characteristic SOX2 expression was downregulated and SOX17 expression was upregulated. This was different from the mouse cells, but the human and rhinoceros cells were extremely similar. Moreover, the upregulation of SOX17, the key to differentiation into human PGCs, was also upregulated in rhinoceroses via the same pathway as in humans.

It was thought that more PGCLCs were needed to obtain eggs and sperm, so the group next explored ways of improving the production rate of these. Investigating WNT inhibition and culture conditions led to them gaining a production rate that was around 20 times the initial rate. They were able to differentiate nearly half of the cells of the original cell clump into PGCLCs.

The group investigated whether these southern white rhinoceros PGCLCs have the properties of PGCs, whether they have migratory capabilities, and whether they will propagate. Using these as indicators, they confirmed that all of these conditions were met. They then applied these cultivation conditions, etc. to northern white rhinoceros iPS cells supplied from a German research group.

By doing this, the group confirmed that, similar to the southern white rhinoceros cells, these cells were able to differentiate into PGCLCs with around 40% efficacy, and they followed the same differentiation pathway.

Previous experiments had been carried out with gene-edited cells, but it is not possible to carry out similar investigations with actual wild animals. Thus, the group investigated the possibility of isolating PGCLCs by using endogenous proteins as indicators. When they did so, they found that isolation is possible using CD9 and ITGA6 as indicators. They also confirmed that it is possible to create PGCLCs with high efficiency from both northern white rhinoceros iPS cells and southern white rhinoceros ES cells that haven't undergone gene editing using this method.

As fertilized eggs are needed to create ES cells, the project aims to create gametes using skin-derived iPS cells from sources other than the existing freeze-preserved northern white rhinoceros. If the project can create eggs, it will be possible to obtain northern white rhinoceros offspring by fertilizing the eggs with freeze-preserved sperm and transplanting them into southern white rhinoceros.

Hayashi explained, "Our challenge for the future is to establish a method for the differentiation and induction of sperm and eggs from PGCLCs. Ultimately, if we are able to induce gametes, we hope to hand them over to the BioRescue Project group that handles the living rhinoceros so they can transplant embryos."

Journal Information
Publication: Science Advances
Title: Robust induction of primordial germ cells of white rhinoceros on the brink of extinction
DOI: 10.1126/sciadv.abp9683