An international joint research group led by Project Associate Professor Toshihiro Kobayashi and Dr. Kenyu Iwatsuki (Research Fellow of the Japan Society for the Promotion of Science) in the Division of Mammalian Embryology of the Center for Stem Cell Biology and Regenerative Medicine at the Institute of Medical Science, The University of Tokyo (IMSUT), in collaboration with Associate Professor Masumi Hirabayashi of the National Institute for Physiological Sciences (NIPS), Professor Shinichi Hochi of the Faculty of Textile Science and Technology at Shinshu University, Nara Medical University, Kyoto University, and University of Cambridge in the UK, has successfully developed a new culture method that can efficiently and stably produce pluripotent stem cells called 'epiblast stem cells' from post-implantation fertilized eggs in the uterus using rats.
They also revealed that the epiblast stem cells can produce primordial germ cells, which are the origins of sperm and ovum, in a shorter period than that of conventional methods. The result is expected to contribute to reproductive medicine research and the livestock industry by clarifying the mechanism involved in the development of fertilized eggs at early pregnancy, especially before and after implantation, and facilitating the efficient production of gametes. The results were published on July 28, 2023, in the online version of the international scientific journal Cell Reports Methods.
Researchers have presumed that there are differences in the properties of pluripotent stem cells (e.g., embryonic stem cells and induced pluripotent stem (iPS) cells) between rodents (including mice and rats) and non-rodent animals (including humans). In general, rodent pluripotent stem cells have similar properties to pre-implantation fertilized eggs, whereas non-rodent pluripotent stem cells have properties similar to post-implantation fertilized eggs.
Using rats in which fertility can be evaluated, the research group developed a culture system for producing epiblast stem cells (i.e., pluripotent stem cells with properties similar to post-implantation fertilized eggs), clarified their characteristics, and further examined if germ cells can be produced.
A mass of pluripotent stem cells called an epiblast was first isolated from post-implantation fertilized eggs of rats. Then, low-molecular-weight compounds of a ROCK signal inhibitor (Y27632, which is known to suppress cell death) and Wnt signal inhibitors (IWP2 and XAV939, which suppress spontaneous differentiation) were continuously added to the culture medium. Consequently, the researchers successfully produced epiblast stem cells that maintained high viability and uniform undifferentiation during their culture.
When gene expression in the established rat epiblast stem cells was checked, they were verified to have characteristics closer to those of the post-implantation fertilized eggs from which they were derived than to those of pre-implantation fertilized eggs.
Subsequently, the researchers verified whether or not primordial germ cells could be produced from rat epiblast stem cells, and they were able to produce approximately 20% of primordial germ cells under specific culture conditions. When the produced primordial germ cells were transplanted to rat testes that cannot produce sperm, after two to three months of the transplantation, the formation of sperm and sperm cells was confirmed in part of the testes. Then, the formed sperm and sperm cells were microinseminated with rat unfertilized eggs, and they were able to develop as normal offspring. Based on these results, it was revealed that primordial germ cells produced from rat epiblast stem cells have normal functions that leads to healthy individuals.
Kobayashi expressed, "Since the developed method can produce a large amount of rat primordial germ cells more easily in a shorter period, it is possible to perform large-scale analyses with a large quantity of cells and is suitable for drug screening. We expect to make significant contributions to reproductive medical research."
Publication: Cell Reports Methods
Title: Rat post-implantation epiblast-derived pluripotent stem cells produce functional germ cells
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