Na\xefve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction

Vera Zywitza,Ejona Rusha,Jan Stejskal,Valentyna M Rishko ,Narasimha Swamy Telugu ,Thomas B Hildebrandt,Kozaburo Hayashi ,Sebastian Diecke,Giovanna Lazzari,Robert Hermes,Jichang Wang,Masafumi Hayashi,Jorge Ruiz-Orera,Cesare Galli,Geert Michel,Frank Göritz,Lars Wittler,Zsuzsanna Izsvák,Dmitry Shaposhnikov,Silvia Colleoni,Susanne Holtze,Micha Drukker

doi:10.1038/s41598-022-07059-w

Abstract

The northern white rhinoceros (NWR) is probably the earth’s most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Highlights

The northern white rhinoceros (NWR) is probably the earth’s most endangered mammal
Rhinocerotidae spp. are good candidates because (1) this family includes highly endangered as well as less threatened species, which could serve as surrogates, (2) important advancements have been made in assisted reproductive technology (ART)[6–9], and (3) induced pluripotent stem cells (iPSCs) have already been derived from specimen of the functionally extinct northern white rhinoceros (NWR, Ceratotherium simum cottoni) using standard techniques for reprogramming and maintaining human iPSCs10,11
The importance of advanced assisted reproduction technologies may go beyond supporting conventional conservation approaches such as habitat protection and breeding as they might in some cases be the last resort to rescue critically endangered species from extinction

Summary

Introduction

The northern white rhinoceros (NWR) is probably the earth’s most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. The seminal increase in understanding developmental biology in combination with technological advancements in stem cell biology create innovative opportunities to circumvent the inevitable extinction of diverse species of large mammals In this respect, female somatic cells could in principle be used as a source for induced pluripotent stem cells (iPSCs), which in turn could be differentiated into oocytes in vitro, fertilized and transferred to surrogates of same or related s pecies[4,5]. While mouse ESCs resemble the naïve state of their in vivo counterpart, human ESCs and iPSCs strongly resemble mouse epiblast stem cells (EpiSCs), which are derived from the post-implantation epiblast and reside in primed state pluripotency Through inhibition of both GSK3 and MEK signaling pathways in addition to LIF (2iL conditions), mouse ESCs can be maintained serum-free and stably in naïve state pluripotency. Typical primed state PSCs have little PGC competence, while cells at the transition from naïve to primed state readily generate PGCs15,16

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Conclusion