|hayashi katsuhiko||Last modified date：2018.07.13|
Professor / Department of Stem Cell Biology and Medicine / Faculty of Medical Sciences
|hayashi katsuhiko||Last modified date：2018.07.13|
|1.||K. Hayashi, Reconstitution of female germ line in a dish., Gordon Research Conference (Fertilization & Activation of Development) , 2015.07.|
|2.||K. Hayashi , Making oocyte and babies from stem cells., The congress of the Asia Pacific Initiative on Reproduction, 2014.04.|
|3.||Hayashi K, Prospects of gamete production from pluripotent stem cells, The 45th meeting of the Dutch Society of Obstetrics and Gynaecology, 2014.05.|
|4.||Katsuhiko Hayashi, Generation of eggs from mouse embryonic stem cells , Society of Reproduction and Fertility, 2014.09, Artificial gametes that are produced in culture from pluripotent stem cells would have a huge impact on not only infertility treatment but also understanding of molecular mechanism underlying germ cell development. Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are able to differentiate into all cell lineages of the embryo proper, including germ cells. We recently established a culture system that induces functional mouse primordial germ cells (PGCs) from ESCs/iPSCs. PGCs produced from ESCs/iPSCs are fully potent, since they differentiate into oocytes, which in turn give rise to healthy individuals. There are, however, many isues to be overcome for the robust generation of mature gametes or for application of the culture system to other species, including humans and livestock. In the meeting, I will discuss recent data and perspectives of germ cell production in vitro..|
|5.||Katsuhiko Hayashi, Germ cell differentiation from stem cells in mice, 17th World Congress on IVF, 2013.09.|
|6.||Katsuhiko Hayashi, Generation of eggs and spermatozoa from pluripotent stem cells in mice, The 3rd International Congress on Controversies in Cryopreservation of Stem Cells, Reproductive Cells, Tissue & Organs (CRYO) , 2013.03, Generation of gametes from pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), in culture is a key goal in developmental/reproductive biology. More practically, gametes from ESCs/iPSCs can be an indefinite source for reproduction and preservation of animals. In mice, a model of mammals, generation of gametes from ESCs/iPSCs in culture has been attempted for a decade. However, despite a lot of effort, a culture system that reconstitutes the entire process of germ cell development has not been established yet.
Toward establishing such culture system, we recently succeeded in producing a robust number of cells whose potential is equivalent to nascent primordial germ cells (PGCs), origin of eggs and spermatozoa. In the culture system, ESCs/iPSCs first differentiate into epiblast-like cells (EpiLCs) and then induce PGC-like cells (PGCLCs). The manner of the step-wise differentiation from ESCs/iPSCs to PGCLCs reproduces that of PGC specification in vivo. Importantly, PGCLCs produced from ESCs/iPSCs are capable of differentiating into eggs and spermatozoa, when transplanted into ovary and testis, respectively. Furthermore, PGCLC-derived eggs and spermatozoa are fully potent, as they give rise to healthy and fertile offspring.
Successful production of functional PGCLCs from ESCs/iPSCs may lead to an idea of applications to other mammalian species including human. There are, however, technical obstacles to apply immediately our culture system to other species. It would be necessary to understand similarity/difference between mice and other species, especially in nature of pluripotent stem cells and PGC development. Nevertheless, our study opens a possibility that pluripotent stem cells are a practical source for reproduction and preservation of animals.
|7.||Katsuhiko Hayashi, Producing sperms and eggs from iPSC, The International Conference on Preservation of Fertility in Cancer Patient , 2013.01, The generation of properly functioning gametes in vitro is a key goal in developmental/reproductive biology. Indeed, such attempts have been made in mice using embryonic stem cells (mESCs) and induced pluripotent stem cells (iPSCs). Despite a lot of effort, a culture system that reconstitutes an entire process of germ cell development has not been established yet. Toward establishing such culture system, we recently succeeded in producing a robust number of cells equivalent to nascent primordial germ cells (PGCs), origin of all germ cell lineage including oocyte and sperm.
In mice, PGCs arise from pluripotent epiblast cells in response to growth factors secreted from an adjacent extraembryonic tissue. The manner of PGC specification includes biological significance, such as re-acquisition of potential pluripotency and genome-wide epigenetic reprogramming. Since defective PGC specification may cause sterility and developmental disorder, understanding PGC specification would serve important information for not only basic biology but also applicable medicine. Moreover, reconstitution of PGC specification using mESCs/iPSCs would serve a robust number of cell materials for producing gametes in vitro.
In the culture system we developed, mESCs/iPSCs first differentiate into epiblast-like cells (EpiLCs) and then induce PGC-like cells (PGCLCs). The manner of the step-wise differentiation from mESCs/iPSCs to PGCLCs reproduces that of PGC specification in vivo. The characteristics of PGCLCs are highly similar, if not identical, to those of PGCs in vivo, based on the criteria of gene expression and epigenetic status. Importantly, PGCLCs are capable of differentiating into spermatozoa that give rise to healthy and fertile individuals through in vitro fertilization. These findings demonstrate that PGCLCs induced from mESCs/iPSCs are comparable to PGCs in their function as male germ.
More recently, we succeeded in reconstitution of female germ-cell development. PGCLCs produced from female mESCs/iPSCs, when aggregated with female gonadal somatic cells, followed by transplantation under ovarian bursa of adult mice, developed into germinal vesicle-stage oocytes. Through in vitro maturation, fertilization and transplantation into the surrogate mother, eggs derived from PGCLCs gave rise to healthy and fertile individuals. Collectively, we demonstrate that the culture system produces fully potent PGCLCs from mESCs/iPSCs in both male and female.
Successful production of functional PGCLCs from mESCs/iPSCs may lead to an idea of applications to human reproductive medicine. Without mentioning the ethical issues, there are, however, technical obstacles to the direct application of our developed culture system to human PGC production from human ESCs/iPSCs. First, human and mouse ESCs/iPSCs are essentially different in responsiveness to growth factors. Second, mESCs can self-renew stably under a serum- and feeder-free condition, whereas such condition has not been firmly established in human ESC/iPSC culture. It is known that mESCs cultured with pharmacological inhibitors of MEK and GSK3β remain in the ground state, which allows the mESCs to differentiate homogenously in response to a set of growth factors. In contrast, a culture condition placing human ESCs/iPSCs in the ground state has not yet been identified. Finding such a culture condition may be necessary for producing a large number of PGCLCs from human ESCs/iPSCs. Most importantly, there is no tool to evaluate whether human PGCLCs from human ESCs/iPSCs are functional. This obstacle may be overcome in part by using primate ESCs/iPSCs. Nevertheless, further basic researches are required for derivation of bona fide PGCLCs from human ES cells.
|8.||Katsuhiko Hayashi, Sugako Ogushi, Mitinori Saitou, Production of fertile gametes from mouse pluripotent stem cells, SRD-NIAS the 2nd Japan-Czech Joint Symposium, 2012.09, The germ cell lineage ensures the creation of new individuals, thereby perpetuating the genomic and epigenetic information across the generations. In mice, primordial germ cells (PGCs), origin of the germ cell lineage, arise from epiblast cells in response to BMP4 secreted by adjacent extraembryonic ectoderm. The manner of PGC specification includes biological significance, such as re-acquisition of potential pluripotency and genome-wide epigenetic reprogramming. It is suggested that defective PGC specification causes sterility and developmental disorder. Thus, understanding PGC specification would serve important information for not only basic biology but also applicable medicine. Despite of the significance, the limited number of early PGCs, less than 100 cells per an embryo, has been an obstacle to research for PGC specification. We recently reconstituted the PGC specification in vitro using mouse embryonic stem cells (mESCs) as well as induced pluripotent stem cells (iPSCs). In the culture system, mESCs/iPSCs first differentiate into epiblast-like cells (EpiLCs) and then induce PGC-like cells from the EpiLCs. The manner of differentiation from mESCs to PGCs reproduces the manner of PGC specification in vivo. PGCs produced from mESCs, termed PGC-like cells (PGCLCs), are fully potent, since they differentiate into spermatozoa and in turn the fertilized eggs with the spermatozoa give rise to healthy individuals. The progress was made by accumulated knowledge of the manner of PGC specification in vivo, the nature of self-renewing pluripotent stem cells, and growth factors endowing EpiLC formation and PGCLC induction in vitro. We are developing further the culture system toward reconstitution of the entire process of germ cell development. We will show recent advance in reconstitution of germ cell development in vitro. .|
|9.||Katsuhiko Hayashi, Reconstitution of the mouse primordial germ cell specification by pluripotent stem cells, International Animal Biotechnology symposium, 2012.01, During embryogenesis, PGCs segregate from the somatic cell lineage at an early developmental stage (Hayashi et al., 2007). In mice, PGCs are derived from the post-implantation epiblast around embryonic day (E)6.25 in response to BMP4 secreted by the extraembryonic ectoderm, a tissue adjacent to the epiblast. Around PGC specification, about six cells of the posterior proximal epiblast at E6.25 start to express Blimp1/Prdm1, a zinc finger transcriptional repressor, and these Prdm1-positive cells are lineage-restricted to become PGCs (Ohinata et al., 2005). Soon after specification, PGCs undergo a unique program of gene expression that regulates epigenetic reprogramming. These successive steps during PGC specification have been thought to be important for establishment of toti-potency in germ cell lineage. However, study of PGC specification has been limited, mainly due to a small number of PGCs, for example less than 50 at E7.5. Therefore, an in vitro culture system reproducing PGC specification steps in vivo would be desired.
Embryonic stem cells (ESCs) are pluripotent stem cells that self-renew indefinitely while maintaining the capability of differentiating into various types of cells including germ cells. Therefore, ESCs are a suitable souse for developing the in vitro culture system reproducing PGC specification. There have been several attempts to produce PGCs, and gametes, from ESCs in vitro (Geijsen et al., 2004; Hubner et al., 2003; Toyooka et al., 2003). Despite these efforts, the generation of healthy individuals from PGCs derived from ESCs has not been achieved. This is partly due to the inappropriate differentiation of ESCs into cells that have an epiblast-like state. Differentiation of ESCs into epiblast has thus far been accomplished by either monolayer culture without LIF, a critical growth factor for self-renewal of ESCs, or formation of embryoid bodies that largely mimic early post-implantation development.
We have recently developed an in vitro culture system in which ESCs systematically differentiate into epiblast-like cells (EpiLCs) and then PGC-like cells (PGCLCs) (Hayashi et al., 2011). The manner of differentiation from ESCs to PGCLCs via EpiLCs reproduces the manner of PGC specification in vivo. Compared with developing PGCs in vivo, the identical patterns of the transcriptome and of epigenetic reprogramming are observed in PGCLCs. PGCLCs are fully potent, since they differentiate into spermatozoa and in turn the fertilized eggs with the spermatozoa give rise to healthy individuals. These results demonstrate that this in vitro culture system allows the production of a huge number of PGCLCs almost equivalent to nascent PGCs. This will make it possible to perform several types of experiments, for example biochemical analysis, which had been limited due to an insufficient number of PGCs in vivo. In the International Symposium on Animal Biotechnology, I would like to discuss how we developed the in vitro culture system, and show perspectives including possible applications.