CALIFORNIA: The San Diego Zoo and scientists at The Scripps Research Institute report significant progress in resurrecting a nearly extinct species of rhinoceros, perhaps within 10 to 20 years.
In a new study awaiting publication, the team said they have made artificial embryonic stem cells from the frozen tissue of four northern white rhinos, raising the total to five. Frozen tissue from seven other rhinos have yet to be converted into these artificial stem cells, which can produce nearly any cell in the body.
With these stem cells, the researchers plan to make gametes – sperm and egg cells – and then embryos by in vitro fertilization. These embryos are to be carried to term by a closely related species, the southern white rhino. Six of these potential surrogate mothers now live at the Zoo’s Safari Park.
The Safari Park has no northern white rhinos. Its last one, Nola, died in 2015. Her tissue was stored with that of other rhinos, and many other species, in the Frozen Zoo; a preserve established decades ago in the hope that one day science would be able to restore extinct and endangered species.
While there’s no guarantee of success, this strategy is the only chance for the northern white rhino. Only three are left in the world. All are past the age of reproduction, rendering the species functionally extinct.
Cloning vs sexual reproduction
While cloning the rhinos is possible, sexual reproduction, with its mixing of genes, produces more genetic diversity. Gametes from the same two parents can yield different genomes in the progeny, while cloning simply produces the same genomes.
Out of the total of 12 lines of frozen rhino cells, about 10 of them will eventually yield stem cells and then gametes, said Jeanne Loring, a Scripps Research stem cell scientist who is working with the zoo.
“Apparently we have enough genetic diversity in the freezer to reboot the species,” Loring said.
While Scripps researchers are tackling the cell end of the equation, the zoo’s experts focus on the reproductive issues, much of which requires original research, said Rick Schwartz, ambassador for San Diego Zoo Global.
This includes training the surrogate mothers-to-be to accept intimate rhino-gynecological exams and other work that has never been done before in the species.
All this work is going on in parallel, Schwartz said. At some point, this work may converge in production of a new northern white rhino.
The surrogate mother tactic has worked in other animals. In 2009, Scripps Research scientists reported producing mice from gametes made from the artificial embryonic stem cells. So there is precedent that it is possible to create an embryo and get an animal to accept the implantation and produce a healthy baby.
“But very little of that information can be applied to rhinos, because their reproductive tract is so unique,” Schwartz said.
“And if you look at it from an animal care side, how do you get a southern white rhino female to accept all of this without sedation? Because sedation of these animals can be very tricky as well,” he said. “So we’ve chosen to go from a behavioral standpoint.”
To do that, the zoo has assembled a rhino behavior team that works with the southern white rhino females every day. This includes examination of their reproductive tract with ultrasound, and measuring rhino hormones to determine when to implant the embryos.
“And they accept being touched and examined for this procedure. It is working shoulder-deep, literally,” Schwartz said.
The first embryo implantation will be a trial run, using a southern white rhino embryo. That’s so the method’s success can be evaluated without using any of the limited supply of northern white rhino cells.
The new study, which has not yet been peer-reviewed, is available at j.mp/norwhiterhino.
The stem cell project was sparked by a chance conversation with the zoo’s Oliver Ryder, Loring said. Ryder, a study co-author along with Loring, is the Kleberg Endowed Director of Conservation Genetics at San Diego Zoo Global, the zoo’s conservation arm.
“When I met Oliver Ryder we just started talking, and then we realized that he had cells and I had technology,” Loring said. “So we just went ahead and did it for the first paper in 2011, and since then the zoo has been supporting the work.”
In the process, Loring, a big fan of zoos since her childhood, jumped at the chance to meet southern white rhinos. These don’t have the nasty disposition often associated with rhinos.
“You can pet them. They’re quite responsive. You don’t want to get in a cage with them because they’re kind of big and clumsy,” Loring said. “But they’re very sweet animals and they know their names. They sort of remind me of cats.”
The new study uses an improved method of making the artificial embryonic stem cells so they don’t contain genetic alterations, Loring said.
These cells, scientifically called induced pluripotent stem cells, are made from normal adult cells, such as skin cells. These are reprogrammed with genes to revert back to an embryonic-like state. From these cells, scientists can produce nearly any kind of cell in the body, including sperm and egg cells.
The genes are carried into the cells by modified viruses. In the original version of the technology, the viruses integrated into the nucleus, altering the genome. The viruses and genes remain in the genome, even after they have done their job and are no longer needed. This risks harm if the virus lodges in the wrong place, disrupting essential functions.
In the improved version, the viruses remain outside the nucleus, and are eventually eliminated as the stem cells multiply. The genome itself is untouched.
“We want to disrupt the genome of the cells as little as possible because we actually want to make rhinos out of these cells,” Loring said. “It’s not just something we’re making to stay in culture. We actually want to make gametes from these cells and then make rhinos from the gametes. So we want to keep their genomes intact if we can.”