There are only two northern white rhinos alive in the entire world, the result of decades of senseless poaching in Africa. Normally, conservationists would spare no effort to ensure this remaining pair breeds and save the species from total obliteration had they not faced an insurmountable roadblock: both rhinos are female. But all hope is not lost. Scientists have now announced they’ve made good progress on their mission to grow artificial rhino eggs from stem cells. The idea is to inseminate these eggs and breed new northern white rhino calves and restore the population to sustainable numbers.

Back to the roots

This latest study is the result of painstaking efforts undertaken by the BioRescue consortium, a collaboration that includes researchers from the Max Delbrück Center for Molecular Medicine (MDC), Imperial College London, the AVANTEA Laboratory of Reproductive Technologies, the Universita degli studi di Padua, and Kyushu University. The consortium is developing new methods and technologies for conservation — and the critically endangered northern white rhinos are at the top of its priority list.

Writing in the journal Scientific Reports, researchers led by Dr. Vera Zywitza of the Pluripotent Stem Cells Platform at the MDC claim they’ve successfully managed to make pluripotent rhino stem cells from normal skin cells.

Stem cells are the body’s “master cells.” They are the building blocks from which all other cells with specialized functions are generated, including those in organs, tissues, blood, and the immune system. In many tissues they serve as an internal repair system, regenerating to replace lost or damaged cells for the life of a person. For this reason, stem cells have immense therapeutic potential.

Until not too long ago, scientists could only harvest stem cells from embryos, which comes with a host of logistical, not to mention ethical, challenges. But that all changed in 2006 when Japanese researcher Shinya Yamanaka found a way to reverse the course of development of adult cells, thereby turning them back into stem cells — so-called induced pluripotent stem cells (iPS). By using four genetic factors, Yamanaka demonstrated how virtually any type of cell, be they skin cells or pancreas cells, could be coaxed into transforming into embryonic-like stem cells, which can then be transformed into any type of cell. For this monumental discovery, Yamanaka was awarded the 2012 Nobel Prize for Medicine.

Scientists with BioRescue had been trying to achieve the same for some time, using skin cells taken from northern white rhinoceros to convert them into induced pluripotent stem cells. The ultimate aim is to develop these iPS cells into immature egg cells, or oocytes, which can later be fertilized and implanted into southern white rhino surrogates, the closest living relatives.

They’ve only recently been successful, however. Previously, all the iPS rhino cells eventually died. Stem cell researcher Professor Micha Drukker and his teams at Helmholtz Zentrum München and at Leiden Academic Center for Drug Research at Leiden University introduced foreign DNA molecules called “plasmids” into rhino skin cells. These foreign genes reprogram the skin cells into iPS cells, while also preventing cell death. The resulting rhino stem cells are remarkably similar to their human counterparts.

“Viewed under the microscope, they are barely distinguishable from human iPS cells,” Drukker said in a press release. “They also respond very similarly to external influences.”

However, the foreign genes came with their own problems because they introduce risks in the germline that could be pathologically altered. For this reason, scientists can’t make rhino oocytes out of such iPS cells. They’re still useful though, revealing the molecular mechanisms that take place in stem cells in greater detail.

To move their conservation project forward, the researchers used an alternative method to coax matured cells to turn into stem cells. Instead of foreign genes, Diecke’s team generated iPS cells using RNA viruses that introduce the required reprogramming factors without any risk of altering the basic genetic fabric of primordial germ cells.

The next big step involves turning these primordial germ cells into egg cells. But that can only happen when the cells are surrounded by ovarian tissue. That kind of tissue is impossible to source since there are only two northern white rhinos left, the females Fatu and Najin — both much too precious to embark on a risky tissue extraction with. This is why the researchers want to convert rhino iPS cells into ovarian tissue in the lab. Researchers in Japan, who successfully cultivated ovarian tissue from mouse stem cells, are lending a helping hand.

Another lifeline: assisted reproduction

The researchers are actually pursuing two separate lines of conservation for the endangered rhinos. Besides making oocytes from iPS rhino cells, the consortium is also making strides in assisted reproduction. After collecting oocytes directly from Fatu, scientists in Italy inseminated them with thawed sperm from a bull, now long dead. A total of 14 northern white embryos have been made so far this way, currently stored in liquid nitrogen until they are ready to be implanted in a southern white rhino surrogate.

The reason why researchers are struggling so much with the iPS approach is that these natural oocytes are too few to guarantee success. What’s more, only eggs from Fatu proved viable, which introduces problems related to poor genetic diversity.

“Due to age and reproductive tract issues, we were unable to collect any oocytes from Najin that could be developed into embryos, so all 14 embryos are from Fatu. We therefore urgently need a complementary strategy for creating gametes – eggs and sperm – from significantly more individuals,” said Professor Thomas Hildebrandt of  Leibniz Institute for Zoo and Wildlife Research (IZW), who leads the BioRescue research consortium.

This is why both lines of research are critical if we’re to rescue the northern white rhinoceros from near-certain doom. If successful, this ambitious project could serve as a model for the restoration of many other threatened species — and potentially already extinct ones too. Between the two of them, the Frozen Zoo at the Arnold and Mabel Beckman Center for Conservation Research in San Diego and IZW’s biobank in Berlin house 10,000 living cell cultures from more than 1,000 endangered species. This precious vault is the ultimate Noah’s ark, which may one day help us reverse the destruction humans have inflicted on wildlife. We just have to save the northern white rhinos first.