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OUTLOOK
29 September 2021

Why stem cells might save the northern white rhino

Biologist Jeanne Loring explains how her work could bring endangered animal species back from the brink.

Julianna Photopoulos

Julianna Photopoulos

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Stem-cell researcher Jeanne Loring in her laboratory at Scripps Research.Credit: Nelvin C. Cepeda/SDU-T/Zuma/eyevine

Up to one million plant and animal species face extinction, many within decades, because of human activities. One of these is the northern white rhinoceros (Ceratotherium simum cottoni). Only two individuals remain, both of them female, making the subspecies functionally extinct. Jeanne Loring, a stem-cell biologist and founding director of the Center for Regenerative Medicine at Scripps Research in La Jolla, California, spoke to Nature about how collecting and reprogramming stem cells could save this species and others from extinction.What does stem-cell research have to do with saving endangered animals?Induced pluripotent stem (iPS) cells, which closely resemble embryonic stem cells, can develop into any tissue in the body, including sperm and eggs. The hope is to generate these reproductive cells from the reprogrammed stem cells of endangered animals, and use them in assisted captive-breeding programmes to rescue the species.How did you get involved in this work?My laboratory set out to make iPS cells from endangered animals in 2008, after we visited the San Diego Zoo Safari Park in California. The previous year, a team led by Shinya Yamanaka, who won a Nobel prize for the work, had become the first to make human iPS cells from skin cells called fibroblasts1, and we had immediately started making them too, to treat neurological diseases. The San Diego Zoo’s Institute for Conservation Research had been collecting and freezing fibroblasts from animals since the 1970s. The institute’s director of conservation genetics, Oliver Ryder, was thinking of using stem cells to try to treat musculoskeletal disorders, but nobody had created iPS cells from endangered species before.
Part of Nature Outlook: Stem cells
In 2011, my postdoctoral fellow Inbar Friedrich Ben-Nun was the first to reprogramme stem cells in two animals from endangered species: the northern white rhino and the drill monkey (Mandrillus leucophaeus)2. We’re now focused on saving the northern white rhino — Ryder’s favourite animal — but the techniques we are working on are going to become a standard way of rescuing species from extinction.When did this become a serious venture?Our endangered-species project mostly remained a hobby until 2015, when scientists and conservationists from around the world met in Vienna to explore how cell technologies might aid conservation. We seriously discussed the idea of using stem cells to rescue endangered species, and later published a rescue plan for the northern white rhino3. To begin with, embryos will be created from sperm and egg cells that were collected and stored. They’ll then be implanted into a surrogate mother, a southern white rhino (Ceratotherium simum simum). But we want to be able to create more sperm and eggs from iPS cells and implant them, too — and that’s where our team comes in.After the Vienna meeting, the San Diego Zoo invested in this idea. Staff there built a stem-cell lab and the Rhino Rescue Center, where they brought in six southern white rhinos from Africa, specifically to serve as surrogate mothers for embryos made from northern white rhinos’ cells. The animals should be compatible because southern white and northern white rhinos are closely related, and so have similar reproductive physiologies. A team of reproductive biologists led by Barbara Durrant is now working to perfect the techniques to fertilize eggs in vitro and transfer viable embryos into the southern white rhinos.What progress have you made in creating northern white rhinoceros iPS cells?When we first set out to make the cells from endangered animals, we assumed that human versions of the reprogramming genes would not work in a rhino. So we tried reprogramming the rhino’s fibroblasts with horse genes — the horse is one of the closest relatives of the rhino — but this failed. Surprisingly, the corresponding human genes did work, and we were able to generate pluripotent cells. However, we had used viral vectors to reprogramme the cells, and this has been shown to lead to tumours in mice, so it could not be used for reproduction purposes.After three years of tweaking the technique, we were able to perform the reprogramming without any genetic modification. It’s all trial and error — you just have to keep testing different combinations of variables. Earlier this year, we celebrated a milestone in our efforts to rescue the rhino: Marisa Korody’s lab at the San Diego Zoo was able to reprogramme frozen cells from nine northern white rhinos and two southern white females to become iPS cells4.

Najin (right) and her daughter Fatu are the world’s only remaining northern white rhinos.Credit: Tony Karumba/AFP via Getty

How do you hope to create gametes from iPS cells?The major effort now is to make eggs that can be fertilized with sperm collected from adult males. We’re following in the footsteps of other researchers who have had success, mainly with mice so far. For example, in 2016, Katsuhiko Hayashi and his team at Kyushu University in Fukuoka, Japan, artificially engineered egg cells from reprogrammed mouse skin cells, entirely in a dish, and these were used to birth pups that were healthy and fertile5.That technique required ovarian tissue to be co-cultured with the developing eggs to get them to mature, and it’s impossible to get that kind of tissue from rhinos without putting them at risk. But in July, the same team showed that it could make both egg cells and ovarian tissue from iPS cells, which was a huge improvement6.We are now trying to find an efficient way to make the precursors of gametes, known as primordial germ cells, from the iPS cells of northern white rhinos. We know it’s possible — we’ve seen it happen spontaneously in cultures of these iPS cells — but we need to learn how to generate more of them. And then we have to turn those germ cells into eggs and sperm — or at least, something like sperm. Typically, the process of in vitro fertilization (IVF) involves knocking the tail off a sperm cell and injecting the small head directly into the egg, so we might not need to make sperm with tails. The IVF process itself will need to be adapted, however, to the southern white rhino surrogates — we don’t know for sure that it will work as it does in humans, because it’s never been done before.What advantage is there to using stem-cell technology over other approaches, such as cloning?The San Diego Zoo has frozen fibroblasts from 12 northern white rhinos. We didn’t want to clone those animals, because we would still have only the same 12 individuals. But if we make gametes from them instead — sperm from males, eggs from females and, in theory, sperm from females — then we could make various combinations through IVF to get a new, genetically diverse pool of animals that will help the species to survive. We have found that there is sufficient diversity in combining that group of 12 to exceed the diversity of the current population of southern white rhinos.
More from Nature Outlooks
Another group, at the Leibniz Institute for Zoo and Wildlife Research in Berlin, is instead harvesting eggs from the two living animals in the hope that they can fertilize them and get new animals that way. I’m perfectly happy if that works, but the challenge is getting enough diversity in the population if you have eggs from only one or two animals.Have you encountered opposition to your iPS-cell-mediated approach?If I were doing this with humans there’d be a lot of debate, but with animals there is less. One criticism is that resources for conservation should be invested differently, for example in restoring natural habitats and educating people. One argument we hear is that there’s no purpose in rescuing a species that will be confined to zoos because of poaching. I don’t know how to stop people from hunting rhinos for their horns, but I will do what I can to try to save an animal that humans have forced into extinction.Are you confident that your work will help to save the northern white rhino?It saddens me that as we’ve made progress in the lab, these animals have been dying out. When we started this project there were 8 of them alive, and now there are only 2: Najin, aged 32, and her daughter Fatu, aged 21, who live in a protected park in Kenya. It’s possible that these last two survivors will be gone by the time we succeed. I hope that’s not the case, but we’re working with cells that have been harvested and frozen, so we can try to bring the species back to life if necessary.I can’t predict how long it will take to get there — things have happened much more slowly than I’d like. But I do hope that our efforts will pay off over the next 10 to 20 years. I want to see a new northern white rhino in my lifetime — before I become ‘extinct’!

Nature 597, S18-S19 (2021)
doi: https://doi.org/10.1038/d41586-021-02626-zThis interview has been edited for length and clarity.This article is part of Nature Outlook: Stem cells, an editorially independent supplement produced with the financial support of third parties. About this content.

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Insects and bamboosFive internodes (length: mean ± SD = 44.8 ± 1.1 cm, n = 5; diameter in the middle part of internodes: 21.4 ± 0.8 mm, n = 5) of five living mature culms of P. simonii bamboo were sampled at Kawaminami, Miyazaki Prefecture, Japan [32°9′ N, 131°29′ E] on 6 June, 2019. Per internode, four semi-cylindrical strips (ca. 15 × 2 cm) were made and stored at − 25 °C until use.To obtain fungus-free larvae of D. bucculenta, we sampled five beetle eggs from P. simonii bamboo obtained at Toyota, Aichi Prefecture, Japan [35°9′ N, 137°13′ E] on 9 May, 2019 in the laboratory from ovipositing females collected at Kawaminami on 10 and 11 April, 2019. The eggs were immersed in 99.5% ethanol for 10 s followed by 70% ethanol for 10 s for surface sterilization and then individually placed on potato dextrose agar (PDA) (Difco, Detroit, MI, USA) plates. The plates were incubated at 25 °C in the dark until 30 days after larval hatching to confirm the absence of the formation of yeast or other microbial colonies. Consequently, all five larvae hatched successfully and aseptically.The bamboo used in this study was morphologically identified using the literature29. This is native to the study areas and no other host bamboo species are distributed there29. Therefore, no voucher specimen of this bamboo has been deposited in a publicly available herbarium. No specific permits were required for the described field studies. The location is not privately-owned or protected in any way. The field studies did not involve endangered or protected species. All applicable international, national, and/or institutional guidelines for the care and use of animals and plants were followed. This study is reported in accordance with ARRIVE guidelines.Component analyses of bamboo tissuesFor YP and LP, the yeast W. anomalus originating from D. bucculenta in Kawaminami (strain: DBL05Kawaminami) was cultured on a 9-cm PDA plate to obtain enough biomass for further experiments. Afterwards, yeast cells were suspended in ca. 10 mL of sterilized water, and were inoculated on the inner surface of the autoclaved internode strips using an autoclaved tissue paper immersed with the yeast suspension. For LP, additionally, the fungus-free 2nd instar larvae (weight: mean ± SD = 2.4 ± 0.4 mg, n = 5) were individually placed on the yeast-inoculated strips. Each of these yeast-inoculated and yeast-and-larva-inoculated strips was then put in a sterilized test tube (3.0 cm in diameter and 20 cm tall) with moistened cotton placed at the bottom. Each of the test tubes was covered with a sterilized polypropylene cap, sealed with Parafilm Sealing Film (Pechiney Plastic Packaging, Chicago, IL, USA) on which three small holes were made using a fire-sterilized insect pin to avoid oxygen shortage, and individually put in a plastic zipper bag. These yeasts and insects were incubated at 25 °C in the dark for 47 days for YP (n = 5), and 47 (n = 4) and 73 (n = 1) days until these larvae reached adulthood for LP (adult elytral length: mean ± SD = 9.2 ± 0.4 mm, n = 5). Microbial contamination was invisible to the naked eye.For FP, YP and LP, the inner surface (up to 0.3 mm in thickness, dry weight: 336 to 935 mg) of a strip was sampled using a small U-shaped gouge. In the case of FX, first, the pith of a strip was completely removed, and then xylem tissue (up to 0.5 mm in thickness, dry weight: 729 to 872 mg) was sampled using a small U-shaped gouge. These tissues were individually sampled from five strips derived from five different internodes for each tissue type.Samples were extracted by aqueous ethanol and hydrolyzed by sulfuric acid with reference to the literature30,31,32 as follows. Four types of samples were freeze-dried and pulverized using a rotor-speed mill (Fritsch, PULVERISETTE 14, 0.2 mm mesh). About 80 mg of powdered sample was extracted using 5-mL 80% ethanol aqueous solution (aq.) at 63 °C three times. The volume of the extracts was adjusted to 25 mL, filtered, and analyzed using ion exchange chromatography measurements (extractable sugar analysis). Their extracted residues were hydrolyzed using sulfuric acid as follows: 50-mg samples were immersed in 1.64-g 72% sulfuric acid aq. at 30 °C for 2 h, boiled in 39.4-g 3% sulfuric acid aq. for 4 h, and filtered to collect sulfuric acid residues as sulfuric acid lignin fractions. The volumes of the filtrates were fixed to 100 mL, passed through a sulfuric acid-removing filter (DIONEX OnGuard IIA), and submitted to ion exchange chromatography measurements (structural sugar analysis). For the uronic acid measurements, the sulfuric acid-removing filter was not used.Ion exchange chromatography measurements were conducted using a DIONEX ICS-3000 apparatus. The measurement conditions were as follows: column, CarboPac PA-1 (2.0 mm I.D. × 250 mm L, Dionex corp.); flow rate, 0.3 mL min−1; column temperature, 30 °C; injection volume, 25 µL; eluent, H2O (solvent A), 100 mM NaOHaq. (solvent B), aqueous solution containing 100 mM NaOH and 1.0 M CH3COONa (solvent C), and aqueous solution containing 100 mM NaOH and 150 mM CH3COONa (solvent D). The gradient conditions for monomers, dimers, and uronic acids were as follows: for monomers, with a gradient of B 0.5% C 0% 45 min, C 100% 10 min, B 100% 10 min, B 0.5% C 0% 20 min; for dimers, with a gradient of B 50% C 0% 50 min, C 100% 10 min, B 100% 10 min, B 50% C 0% 15 min; for uronic acids, with a gradient of D 100% 10 min. These extraction, hydrolysis, and measurement procedures were conducted using n = 5 samples. For the structural sugars, their yield was calculated as the dehydrated state. The values of other extractives % were calculated by the subtraction of total extractable sugars % from total extractives %.Elemental analysis (carbon, hydrogen, nitrogen) was conducted by 2400 CHNS Organic Elemental Analyzer (PerkinElmer Japan, Yokohama, Japan). About 1-mg dried samples were burned completely and the produced CO2, H2O, and N2 (after reduction of NOx species) gasses were quantified by a thermal conductivity detector.Means of components of bamboo tissues were compared among tissue types using the Steel–Dwass test after the Kruskal–Wallis test. Calculations were performed using R 3.5.133.Carbon assimilation testThe yeast W. anomalus (DBL05Kawaminami) was cultured aerobically in 20 mL of yeast nitrogen base (YNB) (Difco) containing 0.5% glucose at 25 °C in the dark for 2 days with shaking at 85 rpm. The culture media were centrifuged and cell pellets were suspended in sterile water, in which the OD600 was adjusted to 0.10. Fifty μL of the cell suspension was added into a tube (2 mL) with 1 mL of each of 14 different media containing YNB and one of the following carbon sources: d-glucose, d-galactose, d-mannose, d-xylose, l-arabinose, d-fructose, d-galacturonic acid, d-glucuronic acid, sucrose, cellobiose, starch from corn, xylan from corn, carboxymethyl cellulose, and no carbon source (n = 5 to 6). The concentration of each carbon source was 0.5 g L−1, except for xylan at 1.5 g L−1. The tubes were shaken at 85 rpm and incubated at 25 °C in the dark for 7 days. Afterwards, the presence of visible pellets of yeasts and OD600 were recorded to determine the growth of the strain. The degree of assimilation was scored according to the presence of the pellets and the difference in the turbidity increase (ΔOD600) between culture media containing no and a given carbon source as follows: no growth (without a pellet, ΔOD600  More

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