Back from the Brink: Can Stem Cells Rewind Extinction?

 

This story is part of our celebration of innovation in 2024.

 

by Dana D’Amico, Connect to Science

Dr. Jeanne Loring and the San Diego Zoo hope to revive the northern white rhinoceros and create a blueprint for stem cell-based conservation

 

Marisa Korody arrived at the lab one day in 2021 to find Angalifu’s heart cells beating for the first time since he’d died seven years before.

Pumping to the rhythm of their own electricity, here were the heart muscle cells of the second-to-last male northern white rhino to walk the Earth. That was Angalifu – careful, observant, or attentive in Swahili – who had spent 44 years of his life at the San Diego Zoo Safari Park and nearly a decade of post-life inside a sleek, cryogenic cylinder known as the “Frozen Zoo®.” Or, at least, his skin cells had.

How did we get here? And will another northern white rhino be born in our lifetimes?

Yes, if Dr. Jeanne Loring, the so-called “godmother of stem cells”, and colleagues like Korody at the San Diego Zoo have anything to say about it.

Loring and a suite of other researchers are working with stem cell-based technologies and a one-of-a-kind collection of cell samples from endangered and extinct species to undo the damage of human poaching, which has driven species like the northern white rhino to de facto extinction.

With only two left in the world – a pair of non-breeding females who are living out their senior years in a Kenyan reserve surrounded by 24/7 armed guards – the race is on to save these gentle giants from obscurity.

A safari, a serendipitous beginning

This story does not begin in a lab.

Loring believes that even scientists – especially scientists – need breaks and shared experiences beyond the bench. For this reason, in 2008, Loring rounded up her entire lab and took everyone on a field trip to one of her favorite places, the San Diego Zoo Safari Park.

It was also a bit of a quid pro quo arrangement. Loring’s old friend Dr. Oliver Ryder, then the Kleberg Endowed Director of Conservation Genetics at the San Diego Zoo Wildlife Alliance, had agreed to offer a tour in exchange for a scientific meeting-of-minds before they left.

Ryder was eager to pick his friend’s brain about stem cells, an exciting and fast-emerging topic on which Loring had become an international expert.

Just a few years prior, Shinya Yamanaka’s lab had shaken the world with the discovery that mature mammalian cells could be reprogrammed into immature cells capable of developing into all tissues of the body. They were the only non-cancer cells capable of living and dividing forever.

He called these resulting immature cells “induced pluripotent stem cells (iPSCs)”. Loring dove straight into exploring the possibilities of iPSCs for human health, joining the nearby Scripps Research Institute by 2007 to lead a new Center for Regenerative Medicine.

In soliciting Loring’s thoughts, Ryder had been imagining applications that might repair injuries for the zoo’s existing wildlife – a sprained leg, for example. But when she learned about the Frozen Zoo and its stockpile of living cell lines, something far bigger arose in Loring’s mind.

“As soon as I learned that the frozen specimens they had in the Frozen Zoo were fibroblasts, it just clicked. That’s what we were using to make human iPSC cells. I mean, it just made perfect sense,” said Loring.

They’d had great success with reprogramming human fibroblasts, a type of connective tissue cell found in skin and other parts of the body.

Why not animals too?

 

The dream that became the Frozen Zoo

Mark Twain famously wrote that no ideas are truly new. Old ideas are simply pieces of colored glass in a kaleidoscope of innovation, tumbling around into new combinations indefinitely. Loring was on the verge of something truly novel, but others before had laid the right pieces.

Predecessors like Kurt Benirschke, a German-American professor of pathology and reproductive medicine at the University of California, San Diego when a young Oliver Ryder – nearly done with his Ph.D. in molecular biology – approached him with an interest in conservation and genetics.

In what Ryder describes as a “life-changing” discussion, Benirschke offered Ryder a post-doctoral position in his lab at the medical school.

Among other things, Benirschke was studying the chromosomal evolution of different species. In lieu of blood sampling, he had been collecting skin samples for culture.

“If you had a skin sample and could grow fibroblast cells, you could freeze those down and return to them when you wanted to repeat a study or when a new technique became available for chromosome analysis,” said Ryder. “That’s important because when you’re working with zoo animals, you can’t easily go back to ask for another sample.”

In 1975, Ryder had lined up all of his postdoc funding and was finishing his last Ph.D. experiments in the lab when he received a call from Benirschke saying that he’d accepted a job to be director of research at the San Diego Zoo, and that he would like to take Ryder with him.

Within the year, Benirschke had established the Frozen Zoo as a first-of-its-kind cryo-preserved tissue bank of rare and highly endangered species – the culmination of years of quiet work in chromosomal evolution.

In the decade of the Apollo space missions and the famous Blue Marble photograph that would kickstart an environmental movement, the Frozen Zoo was the rare idea still far ahead of its time. Humans were making their mark on space, but Benirschke saw new frontiers yet to be explored here on Earth.

He and Ryder often discussed the possibilities, present and future, of such a unique resource.

“Banked cells were a great source of DNA, and there were many studies we could imagine within the field of chromosome research,” said Ryder. “But I never imagined the possibility of making pluripotent stem cells from them.”

Benirschke may not have envisioned pluripotent cells, but he had a fierce vision for the future that few others shared.

“You must collect things for reasons you don’t yet understand,” he would often say, quoting the historian Daniel Boorstin.

He was right.

“Dr. Benirschke opined that someday he thought the collection might be used for producing animals,” Ryder said. “I thought that was a little far-fetched, but I’m happy to eat my words.”

In the span of a single discussion, Ryder and Loring had reached into the past and plucked Benirschke’s wildest dreams straight into the new millennium. What they saw was the possibility of reprogramming non-human fibroblasts into pluripotent stem cells. And from there? A clear path towards re-building an animal nearly from scratch. Towards a true de-extinction.

 

Witnessing extinction in real time

As for which animal to start with, Ryder knew it had to be Nola. She was one of the park’s female northern white rhinos, and one of the last in the world.

Rhino conservation is deeply entwined with the San Diego Zoo’s history. When the Safari Park first opened in 1972, the zoo imported two dozen southern white rhinos into its expansive habitat. The animals thrived there and in the wild, but zoo researchers soon learned about a northern white rhino variety that was in decline in Africa.

 

 

“The zoo started to study the genetic variability between northern and southern white rhinos using technology of that day, mitochondrial DNA sequence analysis,” said Ryder. “The two subspecies were easily, diagnostically differentiated. They were not the same population.”

The zoo collected further samples from the world’s largest captive northern white rhino population in the Dvůr Králové Zoo in Czechoslovakia.

“We started banking the sample cells and all the while, I was keeping track of the status of the wild population that was continuing to decline,” said Ryder. “I remember thinking that these cells may soon be the only thing that’s left of this animal.”

“When Jeanne asked me about an animal for stem cell work, the northern white rhino immediately came to mind because I knew that it might take the intervention of science or the development of some kind of new technology to prevent the extinction of this form of rhinoceros,” he said.

Nola was one of the animals that the zoo would bring over from Czechoslovakia for permanent exhibit. Her presence offered a different type of lesson for visitors.

According to Ryder, the visiting public would have a chance to see her and imagine in no uncertain terms: “this is what extinction looks like.”

She drew a big fan club, no surprise to Ryder and the other wildlife specialists who cared for her that became “thoroughly enchanted” by her gentle nature.

White rhinos are social and curious creatures; the females live in groups and in captivity, they are very comfortable around humans.

“The notion of ferociousness came from colonialist big game hunters,” said Ryder.

Instead of charging, you could find Nola learning how to paint by rubbing her horn (unusually downturned, another quirk special to Nola) across canvas.

“Everyone that encountered her was moved by the kind of plight that she faced,” said Ryder.

Loring too. She has said that she feels a collective human responsibility to right the wrong of horn poaching, which have decimated the numbers of Nola’s wild analogs.

And so it was settled. Just like that, scientific history was underway.

From casual discussion to cover story  

Back at the lab post-safari, Loring gathered her post-docs and asked the million-dollar question: “is anyone willing to take a chance on a project like this?”

For early career scientists, developing a novel method in a novel field is a professional risk. But one post-doc, a researcher named Inbar Friedrich Ben-Nun who had just arrived in the lab from Israel, raised her hand with enthusiasm.

To hedge their bets, Loring’s group also decided to try and reprogram iPSCs from the endangered drill, a baboon-like primate more closely related to humans than a rhino.

“We only had human reprogramming factors,” said Loring. “We thought, ‘what are the chances that we can reprogram a rhino with human genes?’”

It took some tinkering.

“There were a lot of challenges because many of the antibody markers that people often use for this kind of work didn’t work in the rhino model. [Ben-Nun] had to do a lot of things with the viruses that were used to deliver the transgenes, the Yamanaka factors, but she got it to work,” said Loring.

In the end, it was a new marker developed by Loring’s lab in collaboration with Shinya Yamanaka’s lab that did the trick and Ben-Nun’s reprogramming efforts paid off for both species.

“It was a whole lab experience; everyone was just thrilled,” said Loring.

The protocol made the cover story of Nature Methods in 2011. A second publication led by Korody in Stem Cells and Development detailed the reprogramming of cells from nine different white rhinos. It featured Nola on the cover, calmly sipping from the water hole.

Go big or go home

Ben-Nun’s work was the first and most crucial step in the long path towards de-extinction of the northern white rhino.

Using her methods, the team was now on its way towards reprogramming fibroblasts into the sperm and egg cells needed to create a complete artificial embryo.

There were still many kinks to work out first, many of which have little to do with stem cells themselves. For example, another key step in the process of restoration involves perfecting in vitro fertilization methods in an animal as large as an adult rhino.

“You have to develop new technologies just to do things like the implantation of an artificial embryo,” said Loring. “You can’t easily perform an ultrasound because rhinos are just so big around. There have been a lot of steps requiring innovation because you’re working with such a large animal.”

The San Diego Zoo has already completed artificial insemination in two southern white rhinos, close cousin to the near-extinct northern subspecies, that Loring and colleagues hope can serve as surrogates for artificial embryos. The result so far has been two baby rhinos, Edward and Future.

This year, another group of researchers also successfully completed the world’s first in vitro fertilization pregnancy in a rhino, the final intermediary step before another northern white rhino can be born.

It’s key to note that this stem cell de-extinction process is distinct from both cloning and other recent efforts to de-extinct animals like the woolly mammoth or their approximations via whole genome sequencing and genetic manipulation.

Only iPSCs present a viable method for facilitating the reintroduction of a healthy, genetically diverse breeding population.

“You can take two animals and create sexual reproduction so that you get a mixture of genes,” said Loring.

The team has developed iPSC lines from multiple individual rhinos to ensure adequate genetic diversity in the future.

 

Roadmap to the next northern white rhino

All of this, of course, is carefully laid out in a collective plan with origins much broader than Loring’s lab and the San Diego Zoo.

In 2015, Ryder, Loring, and colleagues gathered in Vienna with potential collaborators from all around the world.

“At the Vienna Zoo, we had a discussion among all the people who were working on the northern white rhino or developing any kind of genetic tricks to try to rescue or clone large animals,” said Loring. “We laid out a plan and we’ve basically been following that plan.”

It was a global, bootstrapped effort inching slowly but surely towards progress.

“It’s taken longer than I would like. The funding has been very slim,” said Loring, acknowledging that funding for ecological projects can be scarce relative to funding for human health applications.

“Initial financial support was crucial to starting the project,” said Ryder. “Still, funding the full range of research and activities to achieve the project’s goals is a major challenge.”

Loring spread the word of the project through the research talk circuit, collecting interest and collaborators along the way.

“I feel that one of my jobs has been to let people know that this is going on. I go out and give talks about this from a stem cell perspective, because my collaborators at the zoo do not have access to that entire audience,” said Loring.

Last year, she helped to organize the first international meeting focused on iPS cells from endangered animals. It was a resounding success. Many attendees had been introduced to the topic by way of Loring’s talks, articles, publications, or blogs over the years.

“There were attendees from all over the world – Japan, Europe, Australia, and all around the US. We all had one goal in mind, which was to figure out how to rescue endangered species with stem cell biology,” said Loring.

Discoveries past, present, and future

Today, the Frozen Zoo’s archive contains over 11,000 irreplaceable living cell cultures, egg and sperm cells, and embryos representing more than 1,250 species and subspecies. It is the largest and most diverse collection like it in the world.

Aside from stem cell research, the collection has proven useful for the regular identification of illegal primate specimens in the bushmeat trade and in the whole genome sequencing projects of countless animals.

It has become the blueprint for other emerging biodiversity tissue collections in regions across the world.

What’s next for stem cell innovation? A lot.

“Anything you can think of, stem cells can touch,” said Loring.

That anything includes cultured meat and exploration of pluripotency in plant cells, corals, and birds, as well as mammals.

“The stem cells we’ve made from the northern white rhinos are made with the CTS™ CytoTune™ reprogramming kits. But not all species are easily transformed into iPSCs with the current technology, in large part because there are not many people working on [conservation applications],” said Ryder. “An expanded effort in looking at the ability to induce pluripotency across the tree of life is not only of interest to basic science, but to understanding human health and development and to the conservation of endangered species.”

In the medical space, drug discovery will continue to be a major iPSC application; Loring herself is working on personalized stem cell therapies for Parkinson’s disease. Other researchers are exploring the use of iPSCs as drug delivery vehicles in the body.

“I have closed my seminars by asking, ‘what do you dream of that stem cells could do?’ There are seemingly infinite applications,” said Loring.

As for what kind of projects capture Loring’s interest in particular? That’s a more difficult question.

“Oh, they all do,” said Loring. “Everything.”

Though you might say that Loring is drawn to uncharted territories most of all.

“In my college embryology class, I realized that there was so much unknown. It was like nobody knew anything about embryonic development – sort of amazing,” said Loring. “We were looking at these sectioned slides and identifying things, but there was no clear action in the field.”

“When I started graduate school, I joined a lab studying proteins in mitochondria. But I saw another lab down the hall that was studying neural crest stem cell development, and I just moved there. I wanted to discover something like that – not necessarily something big, but something nobody had ever seen before.

“What really sold me on the field of stem cells was the magic of a cell that held all that information somehow,” she said. “I couldn’t do anything else after that.”

Curiosity appears to be, so to speak, in Loring’s DNA. When she isn’t working on one of her many research goals spanning from ecology to medicine, she is travelling around the world in search of the next solar eclipse. She has seen 19 in all, 15 of which were total eclipses – an official log time of more than 45 minutes of total darkness.

It’s not just watching. A part of her has actually been to space; early this year, Loring’s own iPS cells were part of a space flight for research studying neurological disease processes, for which she is a research partner.

“I have a really strong desire to go to every country in the world, just because I haven’t been there before, you know?” said Loring. “It’s an interesting type of adventurism. I just love being able to see new things and try to figure them out.”

Ironically, when it comes to wildlife research, Loring eschews the Indiana Jones aspects of on-the-ground, hands-on execution. She prefers to tackle the big issues from the benchside and to advise the next generation of stem cell biologists as they push the field even further.

“I have advised many companies that are trying to develop clinical applications for stem cells,” she said. “I was the first to do it, so I’m really, really happy to advise them about how to not make the same mistakes I did.”

Nola and the endlings

There is a term for the last member of a species on Earth – an endling. There have been approximately five billion of them since life began, 99% of all that have ever lived on Earth and probably more still. More than we will ever know.

There was Martha, the last passenger pigeon who died in 1914. And Lonesome George, the last Pinta Island tortoise who left us in 2012 after 100 years. The last Kaua’i ʻōʻō bird, whose haunting mating song to no one was recorded in 1987. Or the last Pyrenean ibex, Celia, taken out by a falling tree and then (semi)-successfully cloned in 2003 for several minutes before succumbing again to lung defects after birth –an endling twice over.

Nola* lived to be the San Diego Zoo’s last northern white rhino. She died in 2015, one year after her male counterpart Angalifu. In life, she rejected Angalifu as a mate for a long time. But perhaps there is hope yet for their future offspring, the founders of a new generation.

As Benirschke might argue, the future has a way of surpassing expectations.

Or, in the words of Loring: “it’s only limited by your imagination.”

Hear more from Dr. Jeanne Loring in the Biotech Innovators podcast »

 

*If you’d like to see her resurrected in another form, through the immersive plane of virtual reality, you can check out the documentary The Ark, which stars Nola, Loring, and other researchers and allows you a glimpse into the Frozen Zoo facility, the Kenyan reserve holding the last northern white rhinos, and more.

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Further Learning

• Saving Endangered Species with Stem Cells – Dr. Jeanne Loring at the 2023 World Stem Cell Summit (video; ~30 minute watch)
• “Stem cells and the frozen zoo” (Loring 2016, Genetic Engineering & Biotechnology News)
• “Induced pluripotent stem cells from highly endangered species” (Ben-Nun et al 2011, Nature Methods)
• “Rewinding extinction in the northern white rhinoceros: genetically diverse induced pluripotent stem cell bank for genetic rescue” (Korody et al 2021, Stem Cells and Development)
• Biotech Innovators podcast discussion with Dr. Jeanne Loring (1 hour)
• Induced Pluripotent Stem Cells (iPSCs) Learning Center (Thermo Fisher Scientific)
• San Diego Zoo Wildlife Alliance Science Blog
• Stem Cells to the Rescue: Saving the Northern White Rhino from Extinction (University of California Television)

 

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