{"id":2738,"date":"2024-03-29T19:11:39","date_gmt":"2024-03-29T19:11:39","guid":{"rendered":"https:\/\/www.thermofisher.com\/blog\/life-in-the-lab\/?p=2738"},"modified":"2024-03-29T19:19:02","modified_gmt":"2024-03-29T19:19:02","slug":"can-stem-cells-rewind-extinction","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/life-in-the-lab\/can-stem-cells-rewind-extinction\/","title":{"rendered":"Back from the Brink: Can Stem Cells Rewind Extinction?"},"content":{"rendered":"
\"One

One of the last northern white rhinos at a Kenyan reserve<\/em><\/p><\/div>\n

 <\/p>\n

\"For<\/p>\n

This story is part of our <\/em><\/strong>celebration of innovation<\/em><\/strong><\/a> in 2024.<\/em><\/strong><\/h3>\n

 <\/p>\n

by Dana D\u2019Amico, <\/em>Connect to Science<\/em><\/a><\/p>\n

Dr. Jeanne Loring and the San Diego Zoo hope to revive the northern white rhinoceros and create a blueprint for stem cell-based conservation<\/h2>\n

 <\/p>\n

Marisa Korody arrived at the lab one day in 2021 to find Angalifu\u2019s heart cells beating for the first time<\/a> since he\u2019d died seven years before.<\/a><\/p>\n

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 \u2013 careful, observant, or<\/em> attentive <\/em>in Swahili \u2013 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 \u201cFrozen Zoo\u00ae.\u201d Or, at least, his skin cells had.<\/p>\n

How did we get here? And will another northern white rhino be born in our lifetimes?<\/p>\n

Yes, if Dr. Jeanne Loring, the so-called \u201cgodmother of stem cells\u201d<\/a>, and colleagues like Korody<\/a> at the San Diego Zoo have anything to say about it.<\/p>\n

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.<\/p>\n

With only two left in the world \u2013 a pair of non-breeding females who are living out their senior years in a Kenyan reserve surrounded by 24\/7 armed guards \u2013 the race is on to save these gentle giants from obscurity.<\/p>\n

A safari, a serendipitous beginning <\/strong><\/h3>\n

This story does not begin in a lab.<\/p>\n

Loring believes that even scientists \u2013 especially scientists <\/em>\u2013 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<\/a>.<\/p>\n

It was also a bit of a quid pro quo arrangement. Loring\u2019s old friend Dr. Oliver Ryder<\/a>, 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.<\/p>\n

Ryder was eager to pick his friend\u2019s brain about stem cells, an exciting and fast-emerging topic on which Loring had become an international expert.<\/p>\n

Just a few years prior, Shinya Yamanaka\u2019s lab had shaken<\/a> 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.<\/p>\n

He called these resulting immature cells \u201cinduced pluripotent stem cells (iPSCs)<\/a>\u201d. 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<\/a>.<\/p>\n

In soliciting Loring\u2019s thoughts, Ryder had been imagining applications that might repair injuries for the zoo\u2019s existing wildlife \u2013 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\u2019s mind.<\/p>\n

\u201cAs soon as I learned that the frozen specimens they had in the Frozen Zoo were fibroblasts, it just clicked. That\u2019s what we were using to make human iPSC cells. I mean, it just made perfect sense,\u201d said Loring.<\/p>\n

They\u2019d had great success with reprogramming human fibroblasts, a type of connective tissue cell found in skin and other parts of the body.<\/p>\n

Why not animals too?<\/p>\n

\"Drs.

Drs. Jeanne Loring (left) and Oliver Ryder (right) at the San Diego Zoo Safari Park in 2023.<\/em><\/p><\/div>\n

 <\/p>\n

The dream that became the Frozen Zoo <\/strong><\/h3>\n

Mark Twain famously wrote<\/a> 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.<\/p>\n

Predecessors like Kurt Benirschke,<\/a> a German-American professor of pathology and reproductive medicine at the University of California, San Diego when a young Oliver Ryder \u2013 nearly done with his Ph.D. in molecular biology \u2013 approached him with an interest in conservation and genetics.<\/p>\n

In what Ryder describes as a \u201clife-changing\u201d discussion, Benirschke offered Ryder a post-doctoral position in his lab at the medical school.<\/p>\n

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.<\/p>\n

\u201cIf 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,\u201d said Ryder. \u201cThat\u2019s important because when you\u2019re working with zoo animals, you can\u2019t easily go back to ask for another sample.\u201d<\/p>\n

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\u2019d accepted a job to be director of research at the San Diego Zoo, and that he would like to take Ryder with him.<\/p>\n

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 \u2013 the culmination of years of quiet work in chromosomal evolution.<\/p>\n

In the decade of the Apollo space missions and the famous Blue Marble photograph<\/a> 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.<\/p>\n

He and Ryder often discussed the possibilities, present and future, of such a unique resource.<\/p>\n

\u201cBanked cells were a great source of DNA, and there were many studies we could imagine within the field of chromosome research,\u201d said Ryder. \u201cBut I never imagined the possibility of making pluripotent stem cells from them.\u201d<\/p>\n

Benirschke may not have envisioned pluripotent cells, but he had a fierce vision for the future that few others shared.<\/p>\n

\u201cYou must collect things for reasons you don\u2019t yet understand,\u201d he would often say, quoting<\/a> the historian Daniel Boorstin.<\/p>\n

He was right.<\/p>\n

“Dr. Benirschke opined that someday he thought the collection might be used for producing animals,\u201d Ryder said. \u201cI thought that was a little far-fetched, but I’m happy to eat my words.\u201d<\/p>\n

In the span of a single discussion, Ryder and Loring had reached into the past and plucked Benirschke\u2019s 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.<\/p>\n

 <\/p>\n

Witnessing extinction in real time <\/strong><\/h3>\n

As for which animal to start with, Ryder knew it had to be Nola. She was one of the park\u2019s female northern white rhinos, and one of the last in the world.<\/p>\n

Rhino conservation is deeply entwined with the San Diego Zoo\u2019s 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.<\/p>\n

 <\/p>\n

\"Nola,

Nola, one of the last female northern white rhinos, drinking from a water hole at the San Diego Zoo Safari Park.<\/em><\/p><\/div>\n

 <\/p>\n

\u201cThe zoo started to study the genetic variability between northern and southern white rhinos using technology of that day, mitochondrial DNA sequence analysis,\u201d said Ryder. \u201cThe two subspecies were easily, diagnostically differentiated. They were not the same population.”<\/p>\n

The zoo collected further samples from the world\u2019s largest captive northern white rhino population in the Dv\u016fr Kr\u00e1lov\u00e9 Zoo in Czechoslovakia.<\/p>\n

\u201cWe 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,\u201d said Ryder. \u201cI remember thinking that these cells may soon be the only thing that’s left of this animal.\u201d<\/p>\n

\u201cWhen 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,\u201d he said.<\/p>\n

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.<\/p>\n

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.”<\/p>\n

She drew a big fan club, no surprise to Ryder and the other wildlife specialists who cared for her that became \u201cthoroughly enchanted\u201d by her gentle nature.<\/p>\n

White rhinos are social and curious creatures; the females live in groups and in captivity, they are very comfortable around humans.<\/p>\n

\u201cThe notion of ferociousness came from colonialist big game hunters,\u201d said Ryder.<\/p>\n

Instead of charging, you could find Nola learning how to paint<\/a> by rubbing her horn (unusually downturned, another quirk special to Nola) across canvas.<\/p>\n

“Everyone that encountered her was moved by the kind of plight that she faced,\u201d said Ryder.<\/p>\n

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\u2019s wild analogs.<\/p>\n

And so it was settled. Just like that, scientific history was underway.<\/p>\n

From casual discussion to cover story \u00a0<\/strong><\/h3>\n

Back at the lab post-safari, Loring gathered her post-docs and asked the million-dollar question: \u201cis anyone willing to take a chance on a project like this?\u201d <\/em><\/p>\n

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.<\/p>\n

To hedge their bets, Loring\u2019s group also decided to try and reprogram iPSCs from the endangered drill, a baboon-like primate more closely related to humans than a rhino.<\/p>\n

\u201cWe only had human reprogramming factors,\u201d said Loring. \u201cWe thought, \u2018what are the chances that we can reprogram a rhino with human genes?\u2019\u201d<\/p>\n

It took some tinkering.<\/p>\n

\u201cThere were a lot of challenges because many of the antibody markers that people often use for this kind of work didn\u2019t 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,\u201d said Loring.<\/p>\n

In the end, it was a new marker developed by Loring\u2019s lab<\/a> in collaboration with Shinya Yamanaka\u2019s lab that did the trick and Ben-Nun\u2019s reprogramming efforts paid off for both species.<\/p>\n

\u201cIt was a whole lab experience; everyone was just thrilled,\u201d said Loring.<\/p>\n

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

\"Rhino

Induced pluripotent stem cells (iPSCs) from a rhino<\/em><\/p><\/div>\n


\nGo big or go home<\/strong><\/h3>\n

Ben-Nun\u2019s work was the first and most crucial step in the long path towards de-extinction of the northern white rhino.<\/p>\n

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.<\/p>\n

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.<\/p>\n

\u201cYou have to develop new technologies just to do things like the implantation of an artificial embryo,\u201d said Loring. \u201cYou can\u2019t easily perform an ultrasound<\/a> because rhinos are just so big around. There have been a lot of steps requiring innovation because you\u2019re working with such a large animal.\u201d<\/p>\n

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<\/a> and Future.<\/p>\n

This year, another group of researchers also successfully completed the world\u2019s first in vitro fertilization pregnancy<\/a> in a rhino, the final intermediary step before another northern white rhino can be born.<\/p>\n

It\u2019s 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<\/a> or their approximations via whole genome sequencing and genetic manipulation.<\/p>\n

Only iPSCs present a viable method for facilitating the reintroduction of a healthy, genetically diverse breeding population.<\/p>\n

\u201cYou can take two animals and create sexual reproduction so that you get a mixture of genes,\u201d said Loring.<\/p>\n

The team has developed iPSC lines from multiple individual rhinos to ensure adequate genetic diversity in the future.<\/p>\n

\"Loring

Loring and a southern white rhino<\/em><\/p><\/div>\n

 <\/p>\n

Roadmap to the next northern white rhino<\/strong><\/h3>\n

All of this, of course, is carefully laid out in a collective plan with origins much broader than Loring\u2019s lab and the San Diego Zoo.<\/p>\n

In 2015, Ryder, Loring, and colleagues gathered in Vienna with potential collaborators from all around the world.<\/p>\n

\u201cAt 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,\u201d said Loring. \u201cWe laid out a plan and we\u2019ve basically been following that plan.\u201d<\/p>\n

It was a global, bootstrapped effort inching slowly but surely towards progress.<\/p>\n

\u201cIt\u2019s taken longer than I would like. The funding has been very slim,\u201d said Loring, acknowledging that funding for ecological projects can be scarce relative to funding for human health applications.<\/p>\n

\u201cInitial financial support was crucial to starting the project,\u201d said Ryder. \u201cStill, funding the full range of research and activities to achieve the project\u2019s goals is a major challenge.\u201d<\/p>\n

Loring spread the word of the project through the research talk circuit, collecting interest and collaborators along the way.<\/p>\n

\u201cI 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,\u201d said Loring.<\/p>\n

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\u2019s talks, articles, publications, or blogs over the years.<\/p>\n

\u201cThere were attendees from all over the world \u2013 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,\u201d said Loring.<\/p>\n

Discoveries past, present, and future<\/strong><\/h3>\n

Today, the Frozen Zoo<\/a>\u2019s archive contains over 11,000<\/a> 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.<\/p>\n

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.<\/p>\n

It has become the blueprint for other emerging biodiversity tissue collections in regions across the world.<\/p>\n

What\u2019s next for stem cell innovation? A lot.<\/p>\n

\u201cAnything you can think of, stem cells can touch,\u201d said Loring.<\/p>\n

That anything includes cultured meat<\/a> and exploration of pluripotency<\/a> in plant cells, corals, and birds, as well as mammals.<\/p>\n

\u201cThe stem cells we\u2019ve made from the northern white rhinos are made with the CTS\u2122 CytoTune\u2122<\/a> 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],\u201d said Ryder. \u201cAn 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.\u201d<\/p>\n

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\u2019s disease<\/a>. Other researchers are exploring the use of iPSCs as drug delivery vehicles<\/a> in the body.<\/p>\n

\u201cI have closed my seminars by asking, \u2018what do you dream of that stem cells could do?\u2019 There are seemingly infinite applications,\u201d said Loring.<\/p>\n

As for what kind of projects capture Loring\u2019s interest in particular? That\u2019s a more difficult question.<\/p>\n

\u201cOh, they all do,\u201d said Loring. \u201cEverything.\u201d<\/p>\n

Though you might say that Loring is drawn to uncharted territories most of all.<\/p>\n

\u201cIn my college embryology class, I realized that there was so much unknown. It was like nobody knew anything about embryonic development \u2013 sort of amazing,\u201d said Loring. \u201cWe were looking at these sectioned slides and identifying things, but there was no clear action in the field.\u201d<\/p>\n

\u201cWhen 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 \u2013 not necessarily something big, but something nobody had ever seen before.<\/p>\n

\u201cWhat really sold me on the field of stem cells was the magic of a cell that held all that information somehow,\u201d she said. \u201cI couldn\u2019t do anything else after that.\u201d<\/p>\n

Curiosity appears to be, so to speak, in Loring\u2019s DNA. When she isn\u2019t 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<\/a>, 15 of which were total eclipses \u2013 an official log time of more than 45 minutes of total darkness.<\/a><\/p>\n

It\u2019s not just watching. A part of her has actually been <\/em>to space; early this year, Loring\u2019s own iPS cells were part of a space flight for research<\/a> studying neurological disease processes, for which she is a research partner.<\/p>\n

\u201cI have a really strong desire to go to every country in the world, just because I haven\u2019t been there before, you know?\u201d said Loring. \u201cIt\u2019s an interesting type of adventurism. I just love being able to see new things and try to figure them out.\u201d<\/p>\n

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.<\/p>\n

\u201cI have advised many companies that are trying to develop clinical applications for stem cells,\u201d she said. \u201cI was the first to do it, so I\u2019m really, really happy to advise them about how to not make the same mistakes I did.\u201d<\/p>\n

Nola and the endlings <\/strong><\/h3>\n

There is a term for the last member of a species on Earth \u2013 an endling<\/em><\/a>. 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.<\/p>\n

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\u2019i \u02bb\u014d\u02bb\u014d bird, whose haunting mating song<\/a> to no one was recorded in 1987. Or the last Pyrenean ibex, Celia<\/a>, taken out by a falling tree and then (semi)-successfully cloned in 2003 for several minutes before succumbing again to lung defects after birth \u2013an endling twice over.<\/p>\n

Nola* lived to be the San Diego Zoo\u2019s last northern white rhino. She died in 2015<\/a>, 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.<\/p>\n

As Benirschke might argue, the future has a way of surpassing expectations.<\/p>\n

Or, in the words of Loring: \u201cit\u2019s only limited by your imagination.\u201d<\/p>\n

Hear more from Dr. Jeanne Loring in the Biotech Innovators podcast<\/strong><\/a> \u00bb<\/strong><\/h3>\n

 <\/p>\n

*If you\u2019d like to see her resurrected in another form, through the immersive plane of virtual reality, you can check out the <\/em>documentary The Ark<\/em><\/a>, <\/em>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.<\/em><\/p>\n

Want to read more articles like this? Subscribe to Connect to Science<\/a>.\u00a0<\/em><\/p>\n

Further Learning<\/strong><\/h3>\n