BioTechniques
11/02/2016 Jyoti Madhusoodanan
The northern white rhinoceros is extinct in the wild, and only three animals still live in captivity. In a race to save the species, an international team turned to stem cells for solutions.
In sunny San Diego, puffs of sub-zero vapor escape massive steel tanks in 400-square-foot room. Pipes and electric cables snake across beige walls and a concrete floor. It’s a far cry from anyone’s idea of a sanctuary—much less one that houses 1000 different species, including a ~2-ton species of rhinoceros. But this otherwise unremarkable cell storage room—better known as the Frozen Zoo—is the northern white rhino’s best chance at life.
Once native to the vast African grasslands, this sub-species of rhinoceros is now extinct in the wild. Only three of these animals survive in protected reserves. A 42-year-old male, along with his daughter and grand-daughter—the last surviving females—live at the Ol Pejeta Conservancy in Kenya. All three suffer from illnesses that render them incapable of natural mating or carrying a pregnancy to term.
When researcher Thomas Hildebrandt of the Leibniz Institute for Zoo and Wildlife Research in Berlin assessed the odds of the species surviving in 2009, the situation seemed hopeless. “Looking at the state of the surviving animals, it made it impossible to be optimistic about saving this creature,” he recalled.
But in 2011, researchers created pluripotent stem cells from two endangered species, the drill (a short-tailed primate) and the northern white rhino, from cells preserved at the Frozen Zoo (1). “That was the turning point for the northern white rhinoceros,” Hildebrandt said. “We suddenly saw a future for this entire species.”
Last year, researchers from around the world gathered in Vienna to discuss how cellular technologies could be applied to conservation, and created a roadmap for saving the northern white rhino (2). In addition to recovering sperm and egg cells from the last surviving individuals, the researchers also focused on using samples from the Frozen Zoo to create artificial gametes.
Assisted reproductive technologies such as in vitro fertilization (IVF) are well-studied in humans and model organisms such as mice. Studies of cattle and horses have extended these methods to other animals. But little is known about creating gametes in the lab and using them to propagate a species.
“The calamitous decline of rhino populations in recent years suggested that this technology may be crucial in preventing their extinction,” said geneticist Oliver Ryder of the San Diego Zoo Institute for Conservation Research, who co-authored the 2011 study. “The only way to prevent the extinction of the northern white rhinoceros is going to be by using advanced stem cell and reproductive technologies.”
Starting With Stem Cells
While on a lab trip to the San Diego zoo safari in 2007, developmental biologist Jeanne Loring of The Scripps Research Institute and Ryder began to discuss the possibility of creating stem cells from the cells of endangered species.
Although Loring had extensive experience dealing with human and mouse stem cells, rhinos were a first to her and her team. Nonetheless, their cells proved surprisingly easy to manipulate. “It turns out that rhinos are a lot more like humans than they are like mice,” she said. “There are some assays that work differently because the genes are a little different, but early mammalian development is very similar in all species.”
Working with preserved rhino fibroblasts, the team engineered the cells to carry three genes necessary to convert them to pluripotent stem cells. The induced stem cells differentiated to form embryonic layers in a dish, but what the team could do with these cells next was unclear.
In October 2016, another collaborator, reproductive biologist Katsuhiko Hayashi of Kyushu University in Japan, and his colleagues reported that induced pluripotent stem cells from mice could be successfully transformed into functional oocytes (3). When fertilized with sperm collected from animals, the eggs formed embryos and, eventually, fertile adults.
“That work has been the biggest step forward so far,” Loring said. “We don’t know how difficult it will be to make gametes from the rhino iPS cells, but the methods are going to build from the mouse studies.”
Studying the northern white rhino’s genome is key to successfully producing germ cells such as oocytes. In addition to identifying key regulatory genes and their targets, the researchers will need to find and verify genetic markers that can be used to track cells as they differentiate. Once these steps are successful—and lab-grown oocytes are available for further work—the team will then use frozen sperm from deceased animals to fertilize the eggs in vitro.
From Cells to Creatures
Even with lab-grown gametes, the northern white rhino’s road to recovery is uncertain. The researchers still need to move from stem cells to living animals. In parallel with Loring’s stem cell studies, Hildebrandt and his colleagues in Europe are using oocytes retrieved from the southern white rhino—a close, less-endangered relative—to develop assisted reproductive technologies that can be applied to the northern white rhinoceros.
By attempting different collection techniques for cells, tweaking cell culture conditions, and refining methods such as intra-cytoplasmic sperm injections (ICSI), the team hopes to successfully produce a southern white rhino calf. Then, the methods could be extended to northern white rhinos. A fertilized egg must reach the blastocyst stage of development before it can be implanted in a surrogate mother to support a pregnancy.
In addition to the northern white rhino, the team is working with endangered Sumatran rhinos. Eventually, the methods could be applied to any of the 1000 species preserved in the Frozen Zoo. “This is not just a theoretical exercise,” said Hildebrand. “It’s a roadmap to saving many critically endangered species.”
References
1. Induced pluripotent stem cells from highly endangered species. Ben-Nun IF, Montague SC, Houck ML, Tran HT, Garitaonandia I, Leonardo TR, Wang Y, Charter SJ, Laurent LC, Ryder OA, Loring JF. Nature Methods 2011, doi:10.1038/nmeth.1706
2. Rewinding the Process of Mammalian Extinction. Saragusty J, Diecke S, Drukker M, Durrant B, Ben-Nun IF, Galli C, Goritz F, Hayashi K, Hermes R, Holtze S, Johnson S, Lazzari G, Loi P, Loring JF, Okita K, Renfree MB, Seet S, Voracek T, Stejskal J, Ryder OA, Hildebrandt TB. Zoo Biology 2016, doi: 10.1002/zoo.21284
3. Reconstitution in vitro of the entire cycle of the mouse female germ line. Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N, Shimamoto S, Imamura T, Nakashima N, Saitou M, Hayashi K. Nature 2016, doi:10.1038/nature20104
Keywords: stem cells endangered species extinction de-extinction white rhino assisted reproductive technology
11/02/2016 Jyoti Madhusoodanan
The northern white rhinoceros is extinct in the wild, and only three animals still live in captivity. In a race to save the species, an international team turned to stem cells for solutions.
In sunny San Diego, puffs of sub-zero vapor escape massive steel tanks in 400-square-foot room. Pipes and electric cables snake across beige walls and a concrete floor. It’s a far cry from anyone’s idea of a sanctuary—much less one that houses 1000 different species, including a ~2-ton species of rhinoceros. But this otherwise unremarkable cell storage room—better known as the Frozen Zoo—is the northern white rhino’s best chance at life.
Once native to the vast African grasslands, this sub-species of rhinoceros is now extinct in the wild. Only three of these animals survive in protected reserves. A 42-year-old male, along with his daughter and grand-daughter—the last surviving females—live at the Ol Pejeta Conservancy in Kenya. All three suffer from illnesses that render them incapable of natural mating or carrying a pregnancy to term.
When researcher Thomas Hildebrandt of the Leibniz Institute for Zoo and Wildlife Research in Berlin assessed the odds of the species surviving in 2009, the situation seemed hopeless. “Looking at the state of the surviving animals, it made it impossible to be optimistic about saving this creature,” he recalled.
But in 2011, researchers created pluripotent stem cells from two endangered species, the drill (a short-tailed primate) and the northern white rhino, from cells preserved at the Frozen Zoo (1). “That was the turning point for the northern white rhinoceros,” Hildebrandt said. “We suddenly saw a future for this entire species.”
Last year, researchers from around the world gathered in Vienna to discuss how cellular technologies could be applied to conservation, and created a roadmap for saving the northern white rhino (2). In addition to recovering sperm and egg cells from the last surviving individuals, the researchers also focused on using samples from the Frozen Zoo to create artificial gametes.
Assisted reproductive technologies such as in vitro fertilization (IVF) are well-studied in humans and model organisms such as mice. Studies of cattle and horses have extended these methods to other animals. But little is known about creating gametes in the lab and using them to propagate a species.
“The calamitous decline of rhino populations in recent years suggested that this technology may be crucial in preventing their extinction,” said geneticist Oliver Ryder of the San Diego Zoo Institute for Conservation Research, who co-authored the 2011 study. “The only way to prevent the extinction of the northern white rhinoceros is going to be by using advanced stem cell and reproductive technologies.”
Starting With Stem Cells
While on a lab trip to the San Diego zoo safari in 2007, developmental biologist Jeanne Loring of The Scripps Research Institute and Ryder began to discuss the possibility of creating stem cells from the cells of endangered species.
Although Loring had extensive experience dealing with human and mouse stem cells, rhinos were a first to her and her team. Nonetheless, their cells proved surprisingly easy to manipulate. “It turns out that rhinos are a lot more like humans than they are like mice,” she said. “There are some assays that work differently because the genes are a little different, but early mammalian development is very similar in all species.”
Working with preserved rhino fibroblasts, the team engineered the cells to carry three genes necessary to convert them to pluripotent stem cells. The induced stem cells differentiated to form embryonic layers in a dish, but what the team could do with these cells next was unclear.
In October 2016, another collaborator, reproductive biologist Katsuhiko Hayashi of Kyushu University in Japan, and his colleagues reported that induced pluripotent stem cells from mice could be successfully transformed into functional oocytes (3). When fertilized with sperm collected from animals, the eggs formed embryos and, eventually, fertile adults.
“That work has been the biggest step forward so far,” Loring said. “We don’t know how difficult it will be to make gametes from the rhino iPS cells, but the methods are going to build from the mouse studies.”
Studying the northern white rhino’s genome is key to successfully producing germ cells such as oocytes. In addition to identifying key regulatory genes and their targets, the researchers will need to find and verify genetic markers that can be used to track cells as they differentiate. Once these steps are successful—and lab-grown oocytes are available for further work—the team will then use frozen sperm from deceased animals to fertilize the eggs in vitro.
From Cells to Creatures
Even with lab-grown gametes, the northern white rhino’s road to recovery is uncertain. The researchers still need to move from stem cells to living animals. In parallel with Loring’s stem cell studies, Hildebrandt and his colleagues in Europe are using oocytes retrieved from the southern white rhino—a close, less-endangered relative—to develop assisted reproductive technologies that can be applied to the northern white rhinoceros.
By attempting different collection techniques for cells, tweaking cell culture conditions, and refining methods such as intra-cytoplasmic sperm injections (ICSI), the team hopes to successfully produce a southern white rhino calf. Then, the methods could be extended to northern white rhinos. A fertilized egg must reach the blastocyst stage of development before it can be implanted in a surrogate mother to support a pregnancy.
In addition to the northern white rhino, the team is working with endangered Sumatran rhinos. Eventually, the methods could be applied to any of the 1000 species preserved in the Frozen Zoo. “This is not just a theoretical exercise,” said Hildebrand. “It’s a roadmap to saving many critically endangered species.”
References
1. Induced pluripotent stem cells from highly endangered species. Ben-Nun IF, Montague SC, Houck ML, Tran HT, Garitaonandia I, Leonardo TR, Wang Y, Charter SJ, Laurent LC, Ryder OA, Loring JF. Nature Methods 2011, doi:10.1038/nmeth.1706
2. Rewinding the Process of Mammalian Extinction. Saragusty J, Diecke S, Drukker M, Durrant B, Ben-Nun IF, Galli C, Goritz F, Hayashi K, Hermes R, Holtze S, Johnson S, Lazzari G, Loi P, Loring JF, Okita K, Renfree MB, Seet S, Voracek T, Stejskal J, Ryder OA, Hildebrandt TB. Zoo Biology 2016, doi: 10.1002/zoo.21284
3. Reconstitution in vitro of the entire cycle of the mouse female germ line. Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N, Shimamoto S, Imamura T, Nakashima N, Saitou M, Hayashi K. Nature 2016, doi:10.1038/nature20104
Keywords: stem cells endangered species extinction de-extinction white rhino assisted reproductive technology