August 1, 2016 (Vol. 36, No. 14)

Jeanne F. Loring Ph.D. Director Center for Regenerative Medicine

Regenerative Properties of Stem Cells Are Being Used to Improve the Conservation of Endangered Animal Species

Pluripotent stem cells are having a tremendous impact on medical research, improving the biological relevance of drug screens and potentially providing effective cell-replacement therapies for degenerative diseases. But the regenerative properties of stem cells also make them useful for more unusual purposes, specifically, to improve conservation of endangered animal species.

This perspective explains how I got involved with conservation research and how the idea of stem cells being used to rescue animals under threat of extinction has caught the attention of a different sort of institution: zoos.

In late 2007, my lab moved from the Burnham Institute (now Sanford Burnham Prebys Medical Discovery Institute) to The Scripps Research Institute. It was a journey of only a couple of blocks, but nevertheless it had many moments of drama, and we were all tired. I decided to reward my lab members (and me) with a trip to one of my favorite places—the San Diego Zoo Safari Park, which is about 30 miles north of my lab in La Jolla.

I contacted Oliver Ryder, who directs the San Diego Zoo Institute for Conservation Research, and he offered us a safari—a long drive around the open spaces of the Safari Park that is evocative of a safari in Kenya. The free safari had a price: we were to spend some of the time talking about how stem cells might be used to help animals.

Oliver and I had talked about this idea for years, but we couldn’t ever come up with a practical plan. But this time, things fell into place. Oliver was in charge of The Frozen Zoo®, a large cryopreserved collection of fibroblast cultures and tissues from thousands of exotic animals. The collection was started in 1976, as an effort to preserve cells from as many species as possible in anticipation of a future in which technology could put the specimens to good use.

A few months before our safari, Shinya Yamanaka had reported a method for reprogramming human fibroblasts into induced pluripotent stem cells that had the same self-renewal and differentiation capabilities and human embryonic stem cells (Takahashi, et al., 2007), and we started reprogramming human cells shortly after the paper came out. It was obvious to us: why not try to reprogram fibroblasts from the Frozen Zoo?





Reprogramming Fibroblasts

When my group returned to the lab from the safari, I asked them: who would like to try to reprogram fibroblasts from an endangered species? It was far from a safe bet, but a young postdoctoral researcher who had recently joined my lab from Israel said that she’d love to give it a try. Inbar Friedrich Ben-Nun spent the next couple of years trying out methods in parallel on human cells and fibroblasts from the zoo. We chose fibroblasts from the drill because it is a primate, making it more likely that the technology used for humans would work.

Oliver chose the northern white rhino, a particular favorite of his, and one of the world’s most endangered mammals. Through hard work and insight, Inbar reprogrammed both species, and in 2011, we published the first report of making iPSCs from endangered species (Ben-Nun, et al., 2011). Nature Methods featured our work, with a cover illustration of an ark stuffed with endangered animals.

For decades, rhinos have been hunted relentlessly for their horns; the horns are made of keratin, like fingernails and hair, but in some East Asian cultures the rhino horn is believed to have miraculous medicinal qualities. A single rhino horn could bring in more than $250,000, most going to the middlemen, but still a huge windfall for the poachers. In 2011, there were only 8 northern white rhinos alive: Nola and Angalifu at the San Diego Zoo, Nesari and Nabire at the Zoo Dvur Králové in the Czech Republic, and Najin, Suni, Sudan, and Fatu, who were transferred from the Czech Republic in 2009 to a guarded sanctuary, the Ol Pejeta Conservancy in Kenya in the hope that they might breed.

Five years have passed, and my lab has focused on human iPSCs, investigating their genomics and epigenetics, creating an ethnically diverse collection of iPSCs, launching projects to treat Parkinson’s disease and multiple sclerosis, and studying iPSCs from people with Fragile X Syndrome, a genetic form of autism. But, there has always been something going on with endangered species. One lab member or another would reprogram another animal to add to our stem cell zoo. But without any funding, the endangered species project remained just a kind of hobby in the lab.

By late 2015, five more northern white rhinos had died. I felt like I’d lost a friend when Nola, the last of the San Diego northern whites, died just before Thanksgiving. Only Najin, Sudan, and Fatu remain.

Over the years, this has become personal for me; I wouldn’t be any use for guarding the three remaining rhinos in Africa, but I wanted to help save them. Remarkably, the idea of using stem cell technologies to rescue endangered species, which has been percolating within the conservation research community, finally came into serious discussion at a meeting at the Vienna Zoo in early December, 2015.





Mammalian Extinction

This meeting (Conservation by Cellular Technologies) was organized by representatives from several zoos, and brought together scientists from four continents to plan a rescue plan for the northern white rhino. A report in the journal Zoo Biology, “Rewinding the Process of Mammalian Extinction”, authored by all 21 attendees at the meeting, outlines the roadmap we developed for rescue of this nearly extinct species (Zoo Biol. published online May 2, 2016).

The reason that this meeting and the publication are so remarkable is illustrated by the diversity of the scientific expertise in the room. Inbar Friedrich Ben-Nun and I represented one end of the spectrum of knowledge; our part of the plan is to reprogram and characterize all 12 individual animals whose fibroblasts are stored in The Frozen Zoo.

A scientist from Japan provided expertise for the next step in the roadmap, the generation of functional gametes from the iPSCs. This has been done with mouse PSCs, but the methods are still under development and haven’t yet been tested in any other species. The rest of the roadmap, culminating with the birth of a rhino calf, was filled in by scientists who clone large animals, experts in the reproductive physiology of rhinos, and the San Diego Zoo itself, which has already brought in 6 healthy females from the closely related southern white rhino species who will serve as surrogate mothers if the team’s work results in production of rhino embryos.

Does this plan have any chance of succeeding? I know it will be difficult, but I think it’s not impossible. Perhaps the most important advance is that such a diverse group agreed on a plan—it wasn’t just a stem cell biologist like me imagining how the cells might be used, but rather a whole chain of experts who can imagine how to accomplish each step.

This means that an aspiration of the group, that our strategy could be adopted for other species, might be feasible. I’m optimistic about this possibility. Just last week I was invited to a meeting at the San Diego Zoo to discuss possible options for a long-term rescue of the black-footed ferret, a native of the U.S. West. Other than Oliver Ryder and me, the group was a completely different assortment of scientists and policy experts. Although the animal and the challenges are different, the general roadmap that we developed for the rhino might apply to the ferret as well.

There are many arguments against launching a complex project like the rhino rescue program. Some say that the money is wasted unless it is used to restore the habitat that has been destroyed by humans. Others see no purpose in rescuing an animal that will be confined to zoos because of poaching. Finding funding for this rescue approach is a major challenge—my lab’s step is the simplest: now that we know we can make iPSCs, I can accurately gauge the costs. We will need to raise funding at every step, and the sources of such support are not clear.

We chose the northern white rhino because of our history and special fondness for these animals that humans are responsible for killing. But perhaps another animal would better serve as proof of principle. But we’ve taken the first step: a large change in direction like this requires that the people involved agree with the change. We’ve achieved that, and now we just need to interest many, many more people in this unprecedented plan to use stem cells to help “rewind” the process of extinction.

















































































































References

Ben-Nun, I.F., Montague, S.C., Houck, M.L., Tran, H.T., Garitaonandia, I., Leonardo, T.R., Wang, Y.C., Charter, S.J., Laurent, L.C., Ryder, O.A., et al. (2011). Induced pluripotent stem cells from highly endangered species. Nature methods 8, 829-831.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872.

Conservation by Cellular Technologies Meeting Attendees

Inbar Friedrich Ben-Nun. Cell Therapy Research and Technology, Lonza, Walkersville, Maryland

Joseph Saragusty, Thomas Hildebrandt, Robert Hermes, Susanne Holtze, Frank Göritz, Steven Seet. The Leibniz Institute for Zoo and Wildlife Research, Berlin

Sebastian Diecke. Max Delbrück Center for Molecular Medicine, Berlin

Micha Drukker. Institute of Stem Cell Research, German Research Center for Environmental Health, Helmholtz Center Munich, Neuherberg, Germany

Barbara Durrant and Oliver A. Ryder. San Diego Zoo Institute for Conservation Research, Escondido, California

Stacey Johnson, San Diego Zoo Global, San Diego

Cesare Galli. Laboratorio di Tecnologie della Riproduzione and Fondazione Avantea, Cremona, Italy; Dipartimento Scienze Mediche Veterinarie, Universita di Bologna, Ozzano dell’Emilia, Italy

Giovanna Lazzari. Laboratorio di Tecnologie della Riproduzione, Cremona, Italy,

Katsuhiko Hayashi. Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan

Pasqualino Loi. Faculty of Veterinary Medicine, Univeristy of Teramo, Campus Coste San Agostino, Teramo, Italy

Keisuke Okita, Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto, Japan

Marilyn B. Renfree, School of BioSciences, The University of Melbourne, Melbourne, Victoria, Australia

Thomas Voracek. Tiergarten Schoenbrunn, Vienna

Jan Stejskal, ZOO Dvur Králové, Dvur Králové nad Labem, Czech Republic

Jeanne F. Loring, Ph.D. (jloring@scripps.edu), is professor of developmental neurobiology and director of the center for regenerative medicine in the department of chemical physiology at The Scripps Research Institute.