ENDANGERED SPECIES:

Tissues in 'Frozen Zoo' hold hope for wildlife

Oliver Ryder has been saving skin tissue from endangered animals in camp coolers filled with liquid nitrogen at -390 degrees Fahrenheit for a long time. The collection of frozen tissue is supposed to be a reservoir of knowledge, ready to be harvested by scientists long after their donors have died.

The outlier goal, the stuff of "Jurassic Park"-esque narratives, is to restore endangered populations by assisted breeding, and one day, cloning.

Now, thanks to advances in stem cell research, scientists are a step closer. They have transformed adult skin cells into a parent stem cell state, and they have done so not in mice or human cells, but in cells derived from the endangered white rhinoceros and a primate called the drill. The findings were published in the journal Nature Methods.

It is the first time scientists are using cells from endangered animals to make all-purpose stem cells that can transform into cells belonging to any part of the body. The researchers hope this method could be used to turn bits of skin from highly threatened species into stem cell therapeutics or, in the wildest-case scenario, clones of these animals long after they have become extinct.

"If this technique could apply across species, we would have the world's largest repository of stem cells because we have the 'Frozen Zoo,'" said Ryder, a geneticist at the San Diego Zoo Institute for Conservation Research and a co-author of the paper.

The story of this transformation begins in Kenya, where Fatu, a 10-year-old female white rhinoceros donated a piece of skin connective tissue in a biopsy. There are seven white rhinos alive today, and they have very limited reproductive potential. Her cells were frozen and added last year to a collection of cells from more than 8,600 individuals from 800 species at the Frozen Zoo.

By then, researcher Shinya Yamanaka of Kyoto University, Japan, had figured out how to engineer normal human and mice skin cells into progenitor cells, the equivalent of human embryos. Ryder wanted to see if a similar thing could happen with other, more endangered animals. Armed with a collection of Futu's frozen skin cells, he approached his collaborator Jeanne Loring, a professor of developmental neurobiology at Scripps Research Institute, based in La Jolla, Calif.

How is it done? Skin to sperm

The feat of transforming normal cells into stem cells revolutionized research when Yamanaka discovered it in 2006 by ending moral debates about harvesting human embryos for research. Yamanaka went about it by introducing four genes into skin cells that triggered the change.

Building on Yamanaka's methods, Inbar Friedrich Ben-Nun, a researcher at Loring's lab, started experimenting with an endangered African monkey called the drill. She needed genes to transform the adult cells and decided to use ones from the rhesus monkey. For Fatu the rhino's cells, she used genes from a horse.

Like most animals out there, the genomes of the drill and the northern white rhinoceros have not been sequenced. Ben-Nun was working blindly, hoping that the close relatedness of the species would mean that the transformation would happen. A year passed with little success.

Then, on a hunch, Ben-Nun decided to introduce genes derived from humans into the Fatu's cells. She was not expecting much given how unrelated humans are from rhinos, but amazingly, the cells transformed. When she looked at the plate, she saw very tiny cells tightly packed together into colonies. "It was definitely stem cells," she said. "We didn't think it would work but it did."

"With drill monkey, you expect it because they are close to humans. But when you go to the rhino, it is outstanding," said Martha Gomez, a senior scientist at the Audubon Center for Research of Endangered Species who works on cloning and is not related to the study. The fact that human genes worked confirms that there is little separating us from rhinos at the very early stages of our lives, she said.

Whether these stem cells are similar to cells in the embryos of drill and the rhino remains to be seen since it is difficult to obtain embryos of threatened animals to make the comparison. If they are the same, converting the embryonic stem cells into sperm or egg cells is a short hop away.

"The fact that the scientists are proposing to produce sperm outside is not crazy," Gomez said. Researchers have already done this with mice and human cells, she noted. "It is not going to be tomorrow but it will be possible."

Sperm to animal

This roundabout way for making stem cells is necessary because there are few ways for Ryder to collect stem cells from the animals in the first place. Short of terminating a highly valuable rhino pregnancy to extract embryos, or doing a bone marrow extraction from large beasts, skin cells are the best Ryder can get. The tissue is taken from the inside of the legs of the animals.

The stem cells could be used for stem cell therapeutics, Ryder said. Drills are highly endangered, and they are not reproducing very well because the individuals are often diabetic. One of the potential applications of stem cell technology in humans was in offering relief to people who suffer from diabetes. "I thought this was another reason to recommend the drill," Ryder said. He thinks stem cell therapeutics could be designed for the drill, helping revive their numbers in the wild.

And sperm cells, if generated from the stem cells, can be used for introducing greater genetic diversity into threatened populations by a technique analogous to in-vitro fertilization. In such programs, a sperm fertilizes an egg outside the animal, and the fertilized egg is then transferred into the mother.

The goal of such assisted breeding programs is not to produce hordes of clones, but to keep the gene pool of threatened species as broad as possible. Cloning would be a lot tougher.

"It is difficult because cloning is not a straightforward process," said Gomez, who is currently working on cats. It has advanced since 1997, when researchers announced in the journal Nature the birth of Dolly the sheep, the first mammal derived from an adult cell. In 2001, scientists announced in the journal Cloning that the first endangered animal, a guar bull named Noah, had been cloned. He died in 48 hours. Other than viability, other problems exist, such as who would be the mother of an extinct species.

"If there are not many elephants, who is going to carry the pregnancy of an elephant?" Gomez said.

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