Scientists in stem cell and conservation biology are exploring how they might rescue endangered species, and perhaps even de-extinct some. From cell to genetically diverse population is a trek.

Sudan died in 2018. He was the last male northern white rhino on Earth. Credit: Ami Vitale/https://www.amivitale.com
Najin and her daughter Fatu might enjoy their grassy meals and frequent naps in the shade even more if they had company beyond the armed guards who watch over them 24/7 in the Ol Pejeta Conservancy in Kenya. They are the last of the world’s northern white rhinos, which is why stem cell biologists, developers of reproductive technologies, biobank directors and conservation biologists are exploring how to get Najin and Fatu some friends and family. They are working on other species, too1. One such project involves rendering coral species more resilient to warming seas. Others involve retrieving DNA from various types of specimens, such as those found in museums, to fortify their efforts to rescue extinct animals.
What these projects share is that researchers work out challenging science and apply it quickly. According to the International Union for Conservation of Nature (IUCN) and its Red List of Threatened Species, 26% of mammals and 44% of reef corals are threatened with extinction.
Much effort focuses on how best to generate induced pluripotent stem cells (iPSCs) from cells of endangered animals, such as fibroblasts. Next, one can differentiate the cells, derive mature egg and sperm, and perform in vitro fertilization in the lab, which, fingers crossed, leads to successful pregnancies and births of, say, northern white rhino calves. “We will be happy if we can have them reproduce in a zoo,” says Jeanne Loring from the Center for Regenerative Medicine at Scripps Research. She has long worked on stem cell biology and conservation and runs several ventures. When pregnancies are successful, she says, northern white rhinos could be set up in a sanctuary, say, in Texas, to hopefully thrive until a suitable, safe natural habitat is found for them.
Climate change is rapidly shaping the world, says Thomas Hildebrandt from the Leibniz Institute for Zoo and Wildlife Research, so “when you want to be successful by restoring a population, it should be genetically diverse.” He and others have set out to cautiously harvest DNA from museum samples to enrich endangered species with the genetic diversity the samples harbor.
Museum specimens have long been a source of ancient DNA, which is fragmented and must be pieced together. Such material “can tell us about genetic diversity that was present in the past,” says Beth Shapiro, chief scientific officer of Colossal Biosciences, who is on leave from her position as a Howard Hughes Medical Institute investigator and faculty member at the University of California Santa Cruz.
With genome engineering and living cells, it’s possible to tap the diversity in museum samples to help out an animal population. For instance, she says, an Asian elephant cell is already 99.5% identical to one from a mammoth. Comparing genomes between Asian elephants and mammoths means “we can use the tools of genome editing to change those parts that are different and important to making a mammoth a mammoth,” she says. Colossal has a number of efforts underway, including ‘de-extinction’ projects. Loring, however, prefers different, stem-cell-based approaches for rescuing endangered species.

Jeanne Loring and Oliver Ryder are friends and collaborate on the use of stem cells to rescue endangered species. Credit: (left) Courtesy of Jeanne Loring
Generating iPSCs
Loring and her then postdoctoral fellow Inbar Friedrich Ben-Nun were, says Loring, the first to derive and culture iPSCs from endangered species, which they did with cells from the northern white rhino and the drill, a primate2. Some reviewers, Loring recalls, saw no value in this research, a sentiment that has changed dramatically.
When the team first saw how the human reprogramming factors OCT4, SOX2, KLF4 and MYC, collectively called the Yamanaka factors3, can turn back the clock on fibroblasts to reprogram them into pluripotent stem cells, “We said, ‘oh, this is so cool, we’ve got to do it’,” Loring recalls. After some experiments with human iPSCs, they realized how they might use the cell lines, egg, sperm and embryos held in a large cryobank, the San Diego Zoo Wildlife Alliance’s Frozen Zoo, to work on endangered species. The Frozen Zoo has over 10,000 cell cultures, eggs, sperm and embryos from close to 1,000 taxa.
To ferry the factors into the animal cells, they hunted for vectors and built their own. She remembers the group’s surprise when both the rhino and primate cells made iPSCs from human reprogramming factors rather than species-specific ones. To get species-specific factors, they would have needed embryos or embryonic stem cells from the endangered animals, which was not possible. The project idea was born when her longtime friend and fellow scientist Oliver Ryder, who directs conservation genetics at the San Diego Zoo Wildlife Alliance, took the lab group on a day trip into the zoo’s Wild Animal Park. “It was our friendship that launched this,” she says.

Najin and her daughter Fatu are the world’s only northern white rhinos. Credit: (right) T. Karumba/Getty Images
Tomàs Marquès Bonet, an ICREA (Catalan Institution for Research and Advanced Studies) researcher at the University Pompeu Fabra, says he’s been inspired by work at San Diego’s Frozen Zoo. His team is committed to developing iPSCs for all samples in the Barcelona Cryozoo, which he directs. A number of institutions are involved, including his university and others. The cryozoo holds samples and cell lines with a focus on European and Mediterranean biodiversity. Generating iPSCs from non-model organisms is challenging, says Marquès Bonet, because standard protocols are more likely to work with species closely related to humans. Labs have to tinker with protocols, especially the transcription factors for establishing and maintaining pluripotency, he says.
The Barcelona Cryozoo holds characterized cell lines: the teams check iPSC pluripotency with gene markers, immunocytochemistry and PCR assays, perform whole-genome sequencing and transcriptomic analysis, and karyotype to verify genomic integrity. They confirm in spontaneous differentiation experiments that the cells can develop the three germ layers — endoderm, mesoderm and ectoderm — and validate that with lineage markers.
For quality control in such experiments, Loring likes to use optical genome mapping rather than karyotyping. An instrument from Bionano Genomics can be used to detect changes such as genetic rearrangements and structural variants at higher resolution than karyotyping, she says. At Bionano, Alex Hastie, the vice president of clinical and scientific affairs, organizes collaborations. He worked with Loring and others in a soon to be published reference genome of the northern white rhino. In this work, they used optical genome mapping.
The method doesn’t provide analysis at the single-base level, but it is a deeper analysis than sequencing — it finds structural variants at high sensitivity, says Hastie. “We can detect them, whereas they’re oftentimes missed with sequencing,” he says, and they can be detected in a very small fraction of the sample.
For this technique, DNA is set up in nanochannel arrays on a chip and imaged. Prep involves spreading fluorescent tags throughout the genome at particular motifs. Changes are detected when comparing the tested DNA to a reference. Karyotyping can detect changes at a level of five million base pairs or larger, says Hastie, and shows the general appearance of chromosomes, but optical genome mapping has “10,000-fold higher resolution,” he says. In practice, that means changes in 5 million bases are found with karyotyping, while optical genome mapping finds structural variants in shifts 500 bases in size. Once locations of interest are detected, sequencing can help to follow up on them, or one might shift experiments right away. He sees stem cell biologists use optical genome mapping to quickly check their cells to detect detrimental changes.
Loring worries that too many stem cell biologists avoid quality control methods, almost as if “they don’t want to know,” she says. But when working in this field, one must assure cells are as good as they can be. “And if they aren’t: do it again.”
Beyond quality control steps, diversity is needed for in stem-cell-based species rescue. Marquès Bonet says he and his colleagues plan to use museum samples to analyze genetic diversity in endangered animals and track changes over time. This insight, he says, can feed into biodiversity conservation strategies. Other scientists use different types of samples to apply the diversity they hold.
Hello, mammoths
“We now have high-quality sequence over 25 genome sequences for mammoths,” says George Church, who is on the faculty of Harvard Medical School and Harvard University’s Wyss Institute for Biologically Inspired Engineering and is a co-founder of Colossal Biosciences. The company works on ‘de-extincting’ animals such as the woolly mammoth. Similar efforts in other species are underway.
“You can’t reconstruct a cell by just having the DNA,” says Loring. “It is, in fact, science fiction,” she says about the idea of de-extincting animals from specimens. Much information from that genome is missing, and it is unclear how to insert this DNA into a cell and have it function as nuclear DNA. She prefers rescue efforts that leverage existing cells, such as those in the Frozen Zoo. Perhaps one day, genome engineering approaches can be useful, she says, but for now, “we have to get the animals first.”
One cannot ‘make’ mammoths exactly as they were. Says Church, one can integrate the DNA diversity that mammoth samples hold into healthy cells by using synthetic DNA. “This is how we engineered the pigs, which has worked out well.” What makes Church optimistic about the potential in this area is that he and colleagues successfully edited 69 changes into the genomes of donors — miniature pigs — and transplanted pig kidneys into cynomolgus monkeys. This was work completed with academic labs, including his, and researchers at the company eGenesis4.
In this instance, the scientists used fibroblasts, but, says Church, the cells “kept going senescent, and we had to frequently rejuvenate via SCNT cloning, which would have been much easier if we had immortal-yet-normal stem cells,” he says. In SCNT, or somatic cell nuclear transfer, a nucleus from a body cell is transferred to an egg from which the nucleus has been removed. Speaking more generally about this type of work, he says, “every step seems to be getting easier.”
Among other aspects, inducing pluripotency requires an epigenetic reset in which the Yamanaka factors are delivered to reprogram somatic cells into iPSCs. Transcription factors are a big challenge, says Church. He and his team spend much time on developing new types of transcription factors. To enhance iPSC generation, he and colleagues engineered SOX2-17 (ref. 5). This ‘super-SOX factor’ harnesses the reprogramming ability of two development regulators, SOX2 and SOX17. Part of this work involved generating human iPSC lines and a construct against the tumor suppressor TP53. Although this tumor suppressor is knocked down, the overall process probably cuts into iPSC quality.
What makes generating iPSCs hard is not the endangered species status itself, says Church, although it is challenging to get high-quality primary cells and one must avoid stressing the last couple of members of a species. “It is more a matter of the idiosyncrasies of the biology of the species,” he says. For instance, the Asian elephant has a “a crazy number of P53 genes.” One also needs stem cells to make ovarian tissue, given that coculturing stem-cell-derived eggs with ovarian support cells improves how well stem-cell-derived eggs mature6.

Thomas Hildebrandt (middle) and team remove eggs from the anesthetized Fatu, a northern white rhino. Credit: Ami Vitale/https://www.amivitale.com
Into the jungle
At the Sumatran Rhino Sanctuary in Jakarta, Thomas Hildebrandt from the Leibniz Institute for Zoo and Wildlife Research sits outside during our online chat. He looks up and laughs about monkeys scampering about in the nearby trees. When a thunderstorm rolls in, our connection is lost, but we reconnect quickly and he talks about how Sumatran rhinos have been losing their habitat, which has led to them becoming critically endangered. In Indonesia, he works with the nonprofit BioRescue and colleagues at IPB University. In 2013, the Sumatran Rhino Crisis Summit estimated there were around 300 Sumatran rhinos, but now the number is maybe 40, he says. In Malaysia, the Malaysian Sumatran rhino population is gone, so Indonesia is the sole country with these animals, which are smaller than northern white rhinos. In the sanctuary, the Sumatran rhinos are successfully reproducing, but they need to be more genetically diverse. The plan is to use IVF and museum specimens to extend the gene pool.
Hildebrandt’s science connects fieldwork, in vitro fertilization and stem cell biology. In Indonesia he and his colleagues have been anesthetizing Sumatran rhinos to harvest eggs and sperm. They do so with a method he developed for the northern white rhino. It works as in people: an ultrasound-guided needle removes an egg from the ovary. “In rhinos, the ovaries are so far inside the abdominal cavity,” he says. To reach and aspirate ovarian follicles, a needle is inserted at an angle into the anesthetized animal’s sanitized rectum.
The team used this method with the northern white rhino Fatu. In the lab, her egg was fertilized with sperm from a deceased northern white rhino held in the Leibniz Institute’s biobank. The embryo was transferred to a surrogate of a different subspecies, a southern white rhino, because Fatu has health problems. Alas, 70 days into the 16-month pregnancy, the surrogate died of an infection. Post-mortem analysis revealed a male fetus that had been developing well. The Leibniz group has 30 northern white rhinos embryos in the biobank and is trying for another pregnancy. From ovum pickup to transport, in vitro maturation of the eggs, fertilization, embryo culture and embryo transfer, “We have the entire protocol in place to produce live offspring in the northern white rhinos,” he says. “We hope, in two years’ time, we have the first calf on the ground,” he says. These calves will be introduced to Najin and Fatu to learn the social behavior of their species.
The years of methods development with northern white rhinos are now traveling to the Indonesian teams at IPB University to help build capacity for rescuing Sumatran rhinos. Hildebrandt and others in the West can provide technology and expertise, but “it is not self-sustaining,” he says. To apply the methods to this species, they all work and learn together.
Unlike the northern white rhino, which has a potential surrogate in the closely related southern white rhino, the Sumatran rhino has no obvious surrogacy candidate. Hildebrandt and colleagues have been developing chimeric embryos that would be transferred to an unrelated species. This builds on research from the 1980s in which a sheep carried to term and birthed a goat kid7.
“In the most cases, a critically endangered species is characterized by a lot of infertile females,” he says. A surrogate is needed, as well as ovarian tissue, which is often biopsied when follicles are removed. He is working with researchers at the University of Copenhagen to explore using these tissues to support maturing eggs in vitro.
To successfully restore a population, especially in light of rapid shifts due to climate change, it should be genetically diverse, he says. Diversity makes the population more capable of reacting more flexibly to new challenges. Setting up the Sumatran rhinos with such diversity is still a work in progress. Matters are, sadly, set with northern white rhinos, where “we have no natural mating anymore,” he says. Only the two females, Najin and Fatu, remain.
Diversity is the plan
Genetic diversity can help to instill resilience for a changing world. Some groups explore transplanting stem cells from one organism to another to give them resiliency. Others are working on ways to enrich the gene pool with genes from DNA assembled from samples taken from museum specimens. Along with Katsuhiko Hayashi and colleagues at Osaka University in Japan and stem cell biologists in Berlin, Hildebrandt and his team are working to generate iPSCs, produce sperm and eggs in vitro, and mature them8. This in vitro gametogenesis will be accompanied by an ovarian matrix.
Eggs and sperm result arise from primordial germ cells. The Leibniz Institute team, the Hayashi team and colleagues in Italy and the Czech Republic have developed an induction protocol to generate induced primordial germ-cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhino. The term ‘germ-cell-like’ indicates their origin. Says Hildebrandt, primordial germ-cell-like cells have the behavior and gene expression patterns of natural germ cells and but are not natural in origin. The scientists found SOX17 to be essential for PGCLC differentiation in southern white rhinos. With this insight, they are tinkering with the conditions to culture and mature northern white rhino eggs to be fertilized with banked sperm from deceased northern white rhinos. The in vitro system to produce northern white rhinos is “working quite well,” he says.
Next, the plan is to head to museums in the USA, the UK and Germany to find museum specimens of northern white rhinos. They represent the population before poaching of northern white rhinos began. “We utilize museums in a completely new way because we analyze the genetic pattern and find new haplotypes, which are no more in our living biomaterial,” says Hildebrandt. Colossal, he says, is supportive of this idea.
The scientists will sample northern white rhino specimens, extract DNA, assemble and analyze it. DNA will be extracted from the most ossified part of the animal, says Hildebrandt, the inner ear. They will enter the skull carefully to cause minimal damage. Next will be gene-editing to modify cells so they have the traits of animals from many years ago, and the process replenishes the gene pool. He likes how one can tap into the genetic makeup these animals once had and that “museums get a completely new task to help us to restore things which are long gone,” he says. The hope is that the process will lend the population diversity and resilience for the challenges ahead.

High heat in the Caribbean has led to increased coral bleaching. Some individuals survive, which hints at how one might foster resilience. Credit: Top left: N. Traylor Knowles
Resilient coral
Given how genetic diversity lends resilience, two scientists have an idea about stem-cell-based possibilities with coral. With funding from the non-profit Revive & Restore, the labs of Nikki Traylor-Knowles of the University of Miami and Benyamin Rosental from Ben-Gurion University of the Negev are building an ‘advanced coral toolkit’ for enhancing coral resilience and supporting coral restoration. The scientists met during their postdoctoral fellowships at Stanford’s Hopkins Marine Station and are collaborating to extract and transplant stem cells from coral.
Stony coral and sea anemones are cnidarian organisms in the class Hexacorallia. Corals are one of the most important ecological systems in the tropical ocean, says Traylor-Knowles. “They facilitate biodiversity in just their structure, but they also act as food.” The high heat levels of the past few Caribbean summers have wrought much coral bleaching. Coral restoration is underway, she says, but bleaching, which can kill coral, is taking place fast. Some coral can survive bleaching a bit longer than others, so the scientists wondered how to harness such resiliency.
In classic stem cell therapy, says Rosental, such as for children with severe immune deficiencies that can lead to death before their first birthday, transplanted bone marrow cells help. The cells of the hematopoietic system differentiate and replenish the blood and immune cells in these children. They bring this same concept to bear with corals, he says. The idea is transfer stem cells from coral that can withstand higher water temperatures to more temperature-sensitive coral. The resulting coral would be chimeric, with both genotypes.
In a proof-of-principle experiment9 with the sea anemone Nematostella vectensis, they found the transplanted cells were fully functional once transplanted. Says Rosental, they showed the transplanted cells proliferate, differentiate and function in the coral they were transplanted to. “We also found where, within the animal, exactly those cells are going.”
To track the cells after injection, they equipped a housekeeping gene in the transplanted cells with the fluorescent reporter mCherry. They isolated and sorted the cells for increased aldehyde hydrogenase (ALDH) activity, which is also used with human stem cells. At one point they thought they had cultured stem cells, says Traylor-Knowles. But what was actually in the dish was a marine protist. “When we were trying to isolate the stem cells, we were using markers that are not unique to the coral,” she says. ALDH is a marker for proliferating cells, which also include the many microorganisms that live in coral. They regrouped, and “now we have markers that we can use to exclude microorganisms,” she says. They are still validating their method but see that it excludes this one marine protist that cost them headaches and time. The approach will help them home in on stem cell populations, isolate those populations with confidence and start to transplant them, she says.
The sea anemone is small and fairly translucent, and they were able to use existing transgenic lines. This is not the case with stony coral, which they have set their sights on next, and “We have a lot of autofluorescence just naturally in the coral,” she says.

Nikki Traylor-Knowles and Benyamin Rosental extract and transplant stem cells to confer resilience on corals. Credit: (bottom left) U of Miami, (bottom right) O. Gershoni-Yahalom
To foster innovative approaches to mitigating biodiversity loss, projects need close collaboration between biomedicine and conservation, says Tomàs Marquès Bonet.
They are working on ways to track the transplanted stem cells in stony coral. It is work that will help with conservation and to gain a better understanding of coral more generally. It will also be useful when they begin working with organoids, says Traylor-Knowles, and as they build an in vitro experimental system in the lab for transplanting stem cells in live coral. Along the way, they keep learning about the immune system. Corals have innate but no adaptive immunity. “They don’t have immunological memories,” says Rosental. “In corals we’ve tested, we haven’t seen rejection.”
Overall, the tools for such research were non-existent, “so we needed to develop new tools just to be able even to ask the questions,” he says. They enjoy how they each bring different background and expertise to the project. “We’re creating a huge toolbox of things that we can use to help with coral resiliency,” says Traylor-Knowles. She wishes she could say it will save coral reefs, but only slowing climate change will do that for sure. Yet she is hopeful that this toolbox can help. “Especially here in Florida, we’re really all hands on deck,“ she says. “There’s a need to just try things.”
Many ideas, one goal
There is, says Loring, tension between the different approaches being brought to bear at the intersection of stem cell biology and conservation biology. Beyond such discussions, scientists in their own lab and biobank leaders working on stem cell projects, both in this area and more generally, need to be vigilant about stem cell quality, she says. For instance, p53 mutations can take over cells in culture, which in biobanks can be a treacherous development.
Quality control is key in stem cell science, including work with endangered species. Says Marquès Bonet, preserving biodiversity through viable cell lines isn’t a project for one individual institution. “Global networks are essential for coordination, exchange of biomaterials and expertise and for developing shared strategies,” he says. The International Union for Conservation of Nature has set up the Animal Biobanking for Conservation Specialist Group, and his biobank and many others are involved. Such initiatives are vital, he says, to foster a global perspective on the biodiversity crisis and how to advance collaborative solutions.
“The future of cryozoos and similar initiatives hinges on integrating disciplines that, unfortunately, have not always been recognized for their synergy,” he says. To foster innovative approaches to mitigating biodiversity loss, such projects need close collaboration between biomedicine and conservation. Biobanks should not have as their primary objective species resurrection. Rather, he says, biobanks “should serve as a last resort when all other efforts have failed — reflecting our societal shortcomings in safeguarding biodiversity in the wild.”
The Sumatran rhino and the Javan rhino are both at the top of complex ecosystems. Saving them also saves many other species — plants, insects — says Hildebrandt. One cannot readily model how the world will change, “but we know it will change.” Just as COVID-19 has, tragically, shown how interwoven the world is, what happens in the jungle in Indonesia has impact around the world. It’s worth doing all we can, he says, to protect this ecosystem and the organisms in it.