The title of a new article in Stem Cells and Development may seem audacious to those unfamiliar with the plight of the northern white rhino and the promise of induced pluripotent stem cells. But “Rewinding Extinction in the Northern White Rhinoceros” may turn out to be accurate for what a genetically diverse stem cell bank can do. The ultimate goal: nurture gametes from the stem cells as a starting point for assisted reproductive technologies to re-establish the species.
The researchers, from the San Diego Zoo Institute for Conservation Research and Scripps Research Institute, generated the induced pluripotent stem cells (iPSCs) from cells of nine northern white rhino (NWR). “The nine iPSC lines capture a genetic diversity that is not possible through cloning of individual animals. This is the first step to genetic rescue of a species through the use of reprogramming technology,” said team member Jeanne Loring, director of the Center for Regenerative Medicine at Scripps.
And Then There Were Two …
The northern white rhino population, with only two infertile females left, is “functionally extinct” – beyond conventional conservation efforts, but perhaps not creative cell biology interventions. Efforts are underway by various teams for attempts at de-extinction of other species as well including going way back to mammoths.
Sudan, the last male northern white rhino, died from an infection on March 19, 2018 at the Ol Pejeta Conservancy in Kenya. The 45-year-old left behind only Najin and her daughter Fatu, who are both members of The Nine, their genomes preserved in the iPSC lines at the San Diego Frozen Zoo. The facility houses more than 10,000 cell cultures, oocytes, sperm, and embryos representing nearly 1,000 species.
Of the five living species of rhinoceros, the Javan, black, and Sumatran are “critically endangered,” and the greater one-horned rhino “vulnerable.” The white rhino Ceratotherium simum includes the southern subspecies (C. simum simum), which is near-threatened, and the northern subspecies (C. simum cottoni).
The southern white rhino (SWR) population is now 20,000 strong, thanks mostly to conservation efforts after poachers reduced the number to fewer than 50 in the early 20th century. Most of these rhinos live in South Africa. The threat isn’t shrinking habitat, but poachers seeking the horns for supposed medicinal value, for knife handles, and perhaps for the hideous thrill of disfiguring a large mammal.
The northern white rhino once roamed South Sudan, the Democratic Republic of the Congo, Chad, the Central African Republic, and Uganda. The 1970s and 1980s brought poaching and war. By the 1990s and into the new millennium the remaining population of 30 rhinos, in Garamba National Park in the DRC, dwindled as human violence escalated. The last NWR was born into captivity in 2000.
By 2008, the NWR was extinct in the wild. Only four animals remained, at a zoo in the Czech Republic. Wildlife biologists moved them to the Ol Pejeta conservancy in Kenya, hoping to entice mating among the two cows and two bulls. The animals complied, but no calves resulted. Nor did introducing a stud from the south help – Najin and Fatu were infertile, but made oocytes. Even if they could have given birth, their genetic diversity would likely not have been enough to reboot a founding population, let alone save the subspecies.
Researchers from the San Diego group, University of London, Safari Park Dvůr Králové in the Czech Republic, and the University California at Santa Cruz, published a report on the genome sequences of nine NWR and four SWR from frozen fibroblasts, in 2018. The genome sequences in the north and south rhinos are similar enough that using southern rhinos as surrogate mothers seemed feasible.
The new paper in Stem Cells and Development describes the first step in fashioning sperm and oocytes from induced pluripotent stem cells from NWRs.
A Safer Viral Vector to make northern white rhino iPSCs
The skin fibroblasts used to derive iPSCs were frozen from rhinos from 1979 through 2016. An attempt in 2011 to reawaken fibroblasts from Fatu using lentivirus as the vector for transcription factor genes led to integration into chromosomes. The approach also had the risk of unpredictably reactivating the reprogramming factors, triggering tumor formation. Instead, the more predictable Sendai virus ferried the transcription factor genes into the new cohort of 9 NWR and 2 SWR cells.
The researchers also modified the culture conditions, optimizing cell density and using human fibroblast growth factor and human insulin, which apparently are great for growing rhino cells. And rhino iPSCs stay pluripotent without regular infusions of reprogramming factors.
Then a battery of markers highlighted the pluripotency and differentiation potential of the resulting iPSCs. The gold standard test of coaxing them to form embryoid bodies revealed the three primary germ layers. A little later cardiomyocytes beat, capturing the transition from differentiation to development. Best of all, the researchers detected markers of gene expression (bone morphogenic protein 4, podoplanin, SOX17 and others) that are early signs of the sculpting of primordial germ cells, a stop on the way to mature sperm and oocytes.
As in many areas, work in mice is paving the way for applications in the much larger mammal.
Katsuhiko Hayashi of Kyushu University in Japan and his group published in December 2020 in Naturederivation of fertilizable, oocyte-like cells from mouse pluripotent stem cells by activating the genes that encode eight transcription factors.
“I was initially in complete disbelief to see mouse stem cells so quickly and easily take the form of oocytes based on introducing just a handful of factors, but repeated experiments proved it was true. To find that eight transcription factors could lead to such big changes was quite astonishing,” said Nobuhiko Hamazaki, first author on the study, in a news release.
The technique isn’t simple, said Dr. Loring, requiring incubating the stem cells with tissue from ovaries – easier to get from a mouse than from a rhino, even if the two surviving NWRs had functioning ovaries. “But rhino researchers have been considering using mouse ovary tissue to incubate rhino iPSCs, or making the rhino equivalent of the supporting cells in the ovary out of the iPSCs,” she added. That approach seems to be working in mice.
“There’s still a lot of trial and error exploration that’s needed to ultimately make rhino gametes. But most research like this starts in mice, and the lessons learned are applied to the animal of interest, like endangered species,” said Dr. Loring.
However gametes are coaxed from rhino iPSCs, the next step would be IVF. Then, viable embryos could perhaps develop in white rhino surrogates from the south.
The work has relevance beyond Fatu and Najin. The northern white rhino iPSCs, also provide “a renewable resource for research and the potential recovery of endangered species. Furthermore, the information we and colleagues are obtaining about all aspects of rhinoceros reproduction will aid conservation of other species,” the researchers conclude.
An IVF Approach Is Promising Too
While the San Diego team is taking a stem cell approach to saving the NWR,
a team from the Leibniz Institute for Zoo and Wildlife Research, Safari Park Dvůr Králové, Kenya Wildlife Service and Ol Pejeta Conservancy created two blastocyst-stage embryos from frozen sperm of deceased NWR Suni and oocytes from Fatu in December 2020, despite COVID-related delays. The group had derived three embryos in 2019.
The frozen oocytes traveled from Africa to the Avantea laboratory in Cremona in Italy, for IVF and ICSI. The embryos were frozen as blastocysts on Christmas Eve. The researchers are already preparing for the next step: they’ve sterilized a northern white bull who had fathered many calves so that his mating behavior can be used to entice a female to become a surrogate for the embryos-on-ice. The five embryos await their future, frozen for now.
With the stem cell and IVF approaches moving forward, even during the pandemic, it seems that the global efforts to bring back the northern white rhinoceros may, eventually, work.