Rescuing the northern white rhino

[Music] Welcome to Science Sessions, the podcast of the proceedings of the National Academy of Sciences, where we connect you with Academy members, researchers, and policymakers. Join us as we explore the stories behind the science. I'm Paul Gabrielson. The population of the Northern White Rhinoceros is down to two individuals, and they're both female. So saving the species requires an assist from scientists. There may be a way. Northern white rhino skin cells can be reprogrammed as stem cells, or more precisely, induced pluripotent stem cells. They may be able to then be differentiated into reproductive cells, but carrying the process out successfully requires that the cells' genomes remain mostly intact. In a recent PNAS study, Franz Josef Mueller, of University Hospital Schleswig-Holstein in Germany, and colleagues, produced a reference genome for the Northern White Rhinoceros that can be used to evaluate the genomic integrity of induced pluripotent stem cells. Franz Josef, tell us about the precarious state of the Northern White Rhinoceros. So the Northern White Rhinoceros is a charismatic species, which is, and this is now a technical term, functionally extinct. Functionally extinct means there are two animals left. They are guarded and kept in Africa, and because both of female, they can't reproduce by themselves any longer. The last male individual from the species died in 2018, I think. So the status of this subspecies is pretty dire, and they will be gone, for sure, in the next few years. Why are induced pluripotent stem cells a potential tool in assisting the species? So I was working as a postdoc from 2003 to 2005 in a lab in San Diego with my, at that time, PI, gene loring. Gene was one of those who derived embryonic stem cells in the 90s, and we both were present when induced pre-potent stem cells were developed as a tool. These stem cells, you take a, for example, skin sample from a human or a mammal, and then there's a biotechnological way of reprogramming them, so they are really nearly identical to embryonic stem cells. And she proposed that you could do that for species which are extinct, and where there are still some tissue samples left in some freezer. In the late 2010s, we were taking it back that there are these IPS cells, but you can't really work with them because you don't know if they're still okay. And that gave us the idea that we could start looking at the cells with genomic methods, and first of all, build a reference genome. You really want to check human stem cells all the time that they're still okay, because we know that human stem cells can acquire deleterious mutations, and you don't want to transplant a cancer. There's a study by a group from Berlin and Japan where they show that methods to move cells towards the direction of becoming oocyte, female germ cells, is actually feasible. So this is pretty plausible that oocyte as well as sperm cells can be derived, and then you would do in vitro fertilization of these. The next big step is to use the other sub species of the white rhinoceros, which is the southern white rhinoceros, as a surrogate mother for getting towards northern white calves. And that's sort of the ultimate goal we worked with San Diego Zoo and my colleagues at the Max Planck Institute for Molecular Genetics towards assembling a reference genome, and applying it to stem cell cultures. And this is what we report in our study. Why are there genomic risks with using induced pleuropodent stem cells? So, culturing cells is very stressful for the cells. And because you've got a lot of cell division going on, there's something that some scientists call evolution in a dish, meaning there's a selection for cells that grow faster. And through the cell division process, sometimes cells acquire genomic scars. Most of them are not relevant, but some of them, the cell loses large stretches of its genome, and by losing, for example, cell genes which are protecting from cancer, they get an advantage and grow faster. Having this reference genome, what we propose is that you can do very inexpensive genome sequencing. And with this inexpensive genome sequencing, you can actually detect if a cell is deviating from the reference genome. How did this study come together? We started the idea in 2018. My colleague, Björn Brandl, flew to the San Diego Zoo and did the sequencing. And then we had a very big project that we wanted to start it and it was February 2020, and then the Corona pandemic had. And this study was done mainly with our free time and additional funds we could put into there. There was no funding agency doing this. It was people coming from different disciplines, from stem cell signs, from conversation biology, a lot of mathematics, because assembling a genome is a lot of mathematics. They came in their free time together because they wanted to do something that's good, positive and important. And this is United Them. And for me personally, that journey of people coming together from diverse fields in their free time, because having an important thing to do that might better this world, that was the key in the study. And that's what's still staying with me, how this amazing group of people got this done against all odds. And this is how I actually want to remember this work. What did you do in this study to sequence the Northern White Rhinoceros genome? And how did it compare to other species genomes? The first step is to generate the reference genome and sequencing technologies have evolved massively over the last seven years. They were different when we started the study. For the Northern White Rhinoceros, we used four complementary methods. One of them is nanopore sequencing, long-read sequencing, which has become much better now. What we've been doing for years for the Northern White Rhinoceros, we can do with individual human patients that we're sequencing within two weeks. In the human case, we do have a reference genome and we can always check. If you don't have a reference genome, you really need to go step by step and you need to have a local assembly where you know, okay, this gene, this region is correct, but then you need to put it into larger contacts. And this is where we had these different sequencing technologies. The Rhinoceros are interestingly all kind of very similar on their number of chromosomes. We show that the Northern White is really close to the Southern White. We take that a signal that using Southern White cows is possible as a surrogate for bringing up Northern White coughs. What did your results tell you about the integrity of induced pluripotent stem cells? We've seen now that Rhinoceros stem cells can lose large chunks of their genome during this evolution in culture. Two of the lines we tested had major issues, which would prevent these specific lines from being used to move them to germ cells. This does not mean that there are no good IPS cells from the Northern White Rhinoceros. It just tells us we need to generate in a first step a pool of cells of different clones and then we need to screen them with a reference genome and then pick the ones which have no genomic scars. Doing this quality control will ensure that the big project of bringing Northern White Rhinoceros back is possible. What are the caviarter limitations of the study? So the current genome build is of great quality. It is on par to the human reference genome that's called GCRH38. For the practical application, I do think that the current reference genome is fit for purpose. I do not think that for the stem cell scientists having every centromere, which is a very repetitive region in every chromosome or every telomere, which is sort of the end of a chromosome assembled in a highly accurate fashion. I don't think this matters for the stem cell scientists at this stage. In the recent years, new technologies have become available that allow telomere to telomere assemblies, particularly for regions which are highly repetitive. We have I think three chromosomes which are almost telomere to telomere or centromere to telomere. Now the telomere to telomere quality for the genome is the next step to look at these dark regions as they are called. And all Also, the genome of the sun and white rhinoceros is of much less equality, and it would be, I think, a very important next step to also assemble this one, because then we can be sure how these animals relate to each other. I think there's a lot of interesting signs to be done to understand the differences but also similarities to a much deeper level between the two sub species. Thanks for tuning in the science sessions. You can subscribe to science sessions on iTunes, Spotify, or wherever you get your podcasts. If you like this episode, please consider leaving a review and helping us spread the word.