A new step
Induced pluripotent cells will help save the rhinoceros
Evgeniya Kostina, PCR.news
The international consortium BioRescue is one step closer to saving the northern white rhino, which is threatened with extinction. A team from the Max Delbruck Center for Molecular Medicine, together with partners from Munich, the Netherlands and Japan, obtained pluripotent stem cells from rhinoceros skin fibroblasts in the naive stage.
The Northern white rhinoceros Ceratotherium simum cottoni — one of two subspecies of the white rhinoceros — is considered endangered, possibly extinct in the wild. Fatu and Najin are apparently the last two northern white rhinos in the world, and they are both females. Thus, this subspecies can no longer reproduce naturally, and extinction seems inevitable. However, the BioRescue consortium is trying to prevent it. To do this, scientists are improving assisted reproductive technologies and, in particular, plan to create induced pluripotent stem cells (iPSCs) from the skin cells of the northern white rhinoceros, which can eventually develop into egg progenitor cells (oocytes). These eggs can be fertilized with the sperm of another subspecies and transplanted to a surrogate mother, thus it will be possible to restore the number of the threatened species faster.
But first it is necessary to solve technical problems. The generation of gametes in cell culture from iPSCs has already been developed for several mammalian species, but the effectiveness varies for different species, and the protocol needs to be adapted anew for each species.
For the rescue of the northern white rhino, BioRescue received funding in the amount of 4 million euros from the German Federal Ministry of Education and Research (BMBF). A team from the Max Delbrück Center for Molecular Medicine of the Helmholtz Association, together with collaborators from Munich, the Netherlands and Japan, published an article in Scientific Reports on the creation of the IPSC of the northern white rhinoceros. Professor Katsuhiko Hayashi from Kyushu University participated in the work. In 2016, Hayashi and colleagues obtained eggs from the skin of mice, artificially fertilized these cells and implanted them into females. Mice conceived by this method were healthy and fertile.
iPSCs can be in two states: naive (the basic state of pluripotence) and primed — "less" pluripotent. Experiments with stem cells obtained from mice have shown that naive iPSCs are particularly successful in turning into germ cells, which is why the researchers sought to obtain them.
The IPSC lines were created from a skin sample taken from Nabire, a female white rhino who lived in the zoo in Dvur Kralove (Czech Republic) and died in 2015 at the age of 31. We used a mini-intron plasmid (MIP) protocol based on two vectors, coMIP247 and pCXLE-hMLN, which support reprogramming of various cell types, including adult blood cells. The CoMiP design carries codon-optimized sequences of canonical reprogramming factors (Oct4, Klf4, Sox2 and c-Myc) and a short hairpin RNA for p53 suppression, pCXLE works on the same principle, only with other factors: Oct, Sox2, NANOG, and LIN28.
After reprogramming the cells, three independent colonies were multiplied and cryopreserved. Approximately 15% of the colonies in the mTeSR1 medium showed stable growth and morphology of undifferentiated cells that strikingly resembled primed human IPS.
Next, the scientists tested whether it was possible to change the states to naive using two protocols based on the RSeT and N2B27 environments. However, when scientists first tried to transfer rhino cells into a naively-like state, the cells died. Therefore, they injected the BCL2 gene into rhino cells, which prevents their death, and successfully obtained naive iPSCs.
Primed and naive cells were distinguished by transcriptome analysis, however, since there is no complete data on the genome of the northern white rhinoceros, a partially annotated genome of the closely related southern white rhinoceros (Ceratotherium simum simum) was used as a reference. The possibility of differentiation of cells into germ sheets was tested by the method of immuno-coloring.
However, scientists are not yet able to move on to the next stage of the rhino's return. The resulting cells are unsuitable for the production of gametes, since the reprogramming vectors have integrated into their genome. However, the authors of the work suggest that pluripotency is maintained independently of the expression of transgenes, and is controlled by the expression of endogenous factors, which makes it possible to study the regulation of pluripotency and differentiation of rhino stem cells. "For example, we can study why the pregnancy period in a rhinoceros is 16 months, and in a mouse is only 21 days, or how organs develop in different species," explains Vera Zyvica, the first author of the article from the Max Delbruck Center for Molecular Medicine.
Article by Zywitza et al. Naïve‑like pluripotency to pave the way for saving the northern white rhinoceros from extinction is published in the journal Scientific Reports.
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