Ecology

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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This is an observational study with no intervention on flock and hatchery practices. None of the birds or eggs were exposed to experimental procedures. The study was based mainly on existing data provided by the hatchery company (DanHatch Denmark A/S) from five broiler breeder flocks in Denmark during the period from November 2018 to January 2019 when the breeders were 46 to 62 weeks of age, see details in Table 1. In addition, feed samples from the flock locations and eggs from grocery stores were acquired.Table 1 Flocks and production periods.Full size tableThe average age of breeders was 48–59 weeks (SD from 0.5 to 2.2) ranging from 46–50 weeks to 57–62 weeks (Table 1; Supplementary Fig. S1 online) with observation period ranging from 1.6 to 7.6 weeks in the five flocks. Average laying percent over observation days was 65% (SD = 5.4%) and average hatchability over deliveries was 79% (SD = 5.8%).Feed samplesTwenty-six feed samples were collected for analysis of glyphosate content, 3 to 10 feed samples per flock. The glyphosate concentration related to a given sampling date was assumed representative for the flock from this day and until next sampling. Average duration of the preceding samples were used as duration for the last sampling date within each flock. Glyphosate (N‐(phosphonomethyl) glycine) and the glyphosate degradation product, aminomethylphosphonic acid (AMPA) in feed samples were analysed by the method described by Nørskov et al.4.Production dataData on egg production and hatchability from periods following each feed sampling was obtained from the hatchery company. Daily information was available on laying percent (100% * number of eggs/number of breeders), breeder age (days) and egg weight. For the hatchability, this was calculated as the proportion of eggs placed in incubators from which a viable chicken hatched (but presented as a percentage, i.e. multiplied by 100%). Daily egg weight had been calculated as the average from approx. 30 randomly sampled eggs.Glyphosate concentration of the feed consumed by the breeders during the 10 days prior to laying was the explanatory variable of main interest. The weighted average of glyphosate concentrations across the 10 days of development from follicle to ovulation of egg was used with number of days each glyphosate sample is representative during these 10 days as weights. For hatchability, glyphosate concentrations were aggregated at the level of delivery by weighted averaging using number of hatch eggs as weights.Eggs from grocery storesNo eggs were obtained from the five flocks, however we acquired eight cartons of conventional as well as eight cartons of organic eggs from eight different grocery stores. Three eggs from each carton were selected and egg yolk were analysed for glyphosate by the microLC-MS/MS method as described by Nørskov et al.4 adjusted to the egg yolk matrix.Statistical analysisLaying percent and hatchability were analysed by linear mixed effects models, including a random effect of flock and a first order autoregressive correlation structure to account for the repeated measurements from each flock. Following two covariates were considered for both outcomes: average egg weight (g) and breeder age (decimal weeks). However, since egg weight and breeder age are highly correlated (Pearson’s correlation coefficient ranging from 0.73 to 0.95 in the five flocks; Supplementary Fig. S1 online), only breeder age was included in the models. An important reason for this choice being that average egg weight was missing for 24% and 43% of the days from flock 4 and 5, respectively. In the age range used for this study, laying percent decrease with breeder age (Supplementary Fig. S1 online) as substantiated by a correlation coefficient between − 0.38 and − 0.87. Hatchability also decrease with breeder age (Supplementary Fig. S1 online).In addition, storage time on farm until delivery (1 to 5 days) and storage time at hatchery until incubation starts (1 to 11 days) were included as covariates for hatchability. The incubation start date was determined as date of hatching minus 21 days. For hatchability, covariates obtained from flock production data were aggregated at the level of delivery by weighted averaging; using daily number of eggs as weights for the calculation of average egg weight, number of hatch eggs as weights for average storage time on farm, and current number of breeders as weights for average breeder age. Weighted average storage time on farm until delivery varied from 1.0 to 4.0 and was on average 2.1 days. For storage time at hatchery, deliveries had been split on one to four incubator start dates. Therefore, weighted average of storage days was calculated using number of delivered eggs as weights. Weighted average storage time at hatchery before incubation starts varied from 1.2 to 8.0 days and was on average 4.8 days.Final models were fitted with restricted maximum likelihood estimation using the lme function from the nlme package v. 3.1-152 in R version 4.0.45 and with a significance level of 0.05. Fixed effects were tested by χ2 likelihood ratio tests after maximum likelihood estimation. Model checking was carried out by examination of qq-plots for normality and scatter plots of residuals versus predicted values to look for uncovered trends and variance heterogeneity. More

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