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Researchers for the future

To generate reproductive cells from mouse ES/iPS cells

Dr. Katsuhiko Hayashi

A research group including Dr. Katsuhiko Hayashi at Kyoto University succeeded in differentiating male mouse ES cells and iPS cells into primordial reproductive cells in vitro and induced sperms from the cells, to give healthy pups by in vitro fertilization in 2011. In 2012, they differentiated female mouse ES cells and iPS cells into primordial reproductive cells in vitro, grew them in culture with fetal mouse somatic cells that were to be ovaries, and transplant the cells into ovaries of other mice, to obtain unmatured ova. Then they grew the unmatured ova in vitro into mature ova, and give pups with these ova by in vitro fertilization. These studies on differentiation of ES or iPS cells into reproductive cells can contribute to developing reproductive medicine for humans in the future and therefore attracts attention in various related fields.

Photo:Dr. Katsuhiko Hayashi

Dr. Katsuhiko Hayashi

Interviewer :
You successfully generated sperms in 2011, and ova in 2012. Which are difficult to generate?

Hayashi :
Technically speaking, the critical stage is to generate primordial reproductive cells, and it is not so difficult to generate sperms or ova from these cells. More precisely, for sperms, after we get at least one sperm stem cell from primordial reproductive cells, we will be able to get sperms from the stem cell(s) as many as we want, while for ova, there is no ovum stem cell and we are allowed to obtain as many ova as the primordial reproductive cells that we have. For generating ova, it is crucial to generate good-quality primordial cells as many as possible. In this sense, we can say that the efficiency of producing ova is lower than that for sperms.

Interviewer :
To generate ova, you culture the primordial reproductive cells with future-ovary somatic cells collected from mouse fetuses and in mouse ovaries. Are the primordial cells not able to differentiate into functional ova when cultured solely in vitro?

Hayashi :
No, they are not. We do not know yet why they differentiate only when they are cultured with somatic cells, nor what is going on there. If we find the factors responsible for differentiation of the primordial cells into ova, perhaps we will be able to induce differentiation in vitro using these factors. It is very important for us to study these mechanisms that will give us a hint about how to do when we will try to get reproductive cells from human ES /iPS cells.

Interviewer :
Is it indispensable to take a process of transplanting the primordial cells cultured with somatic cells back into testes or ovaries?

Hayashi :
Yes. For generating both sperms and ova, we transplant the respective primordial cells back into testes and ovaries. It is to observe whether the primordial cells derived from ES or iPS cells differentiate into sperms or ova that we use testes or ova as optimal environments for their differentiation. How we obtain sperms and ova by a completely in vitro process is to be studied further.

large view
Process for generating ova from ES / iPS cells(large view(PDF))

Interviewer :
You used iPS cells generated from fetal mouse cells in your studies that we are talking about. Do you find any significance of using these iPS cells?

Hayashi :
No, I don't find any special meaning. Still, it is necessary to use female cells for generating ova. For generating ES or iPS cells, it is easier to use male mouse cells, and nobody had it in mind to use female cells. We were looking for female iPS cells of good quality, and found ones that were generated from fetal cells.

Interviewer :
To which point of basic research on development of reproductive cells will your studies contribute?

Hayashi :
In the field of research on reproduction or breeding, people tend to say OK if they successfully get progenitors, and have not been so active in doing basic studies from the viewpoint of embryology. The process of differentiation of ES or iPS cells into the primordial cells must be one that reproduces what is going on in fetuses where many genes are involved. In mice, we observe several primordial reproductive cells in embryos of 6 days of fertilization. In maternal bodies, there are prepared primordial reproductive cells that will serve to make their "grand children". However, it is difficult to collect many these cells from maternal bodies for experimental purposes. Our studies showed that we would be able to generate a large number of primordial reproductive cells from ES cells or iPS cells, and thus I can say that our studies offer a good experimental tool, and with this tool we will be able to study further gene functions and their processes.

Photos:Mouse offspring were given by in vitro fertilization of ova derived from primordial reproductive cells differentiated from mouse iPS cells.
Mouse offspring were given by in vitro fertilization of ova derived from primordial reproductive cells differentiated from mouse iPS cells.
Upper left : unmatured ova
Upper center : ova matured in in vitro culture
Upper right : mouse pups given by in vitro fertilization
Lower left : healthy grown animals derived from iPS cells
Lower right : iPS cell-derived adult animals delivered pups of the next generation.

Interviewer :
What is your next object?

Hayashi :
It is to extend the possible area of in vitro culture and reconstruct the whole process of development of reproductive cells. The first target is the late stage of development of primordial reproductive cells. We have many basic questions to solve, and it will take more time than my life to solve them.

Interviewer :
I have heard that many of the patients receiving fertility treatments saw the news about your studies and are expecting their further development. Will you be ready to meet these expectations?

Hayashi :
It is theoretically possible that we will offer a treatment to male patients who are not capable of producing sperms, by generating primordial reproductive cells from their own cells and transplanting them back into their own testes to produce sperms. Apart from ethical problems, to realize such a treatment, we have to deepen our understanding of the development of human reproductive cells, to reproduce it in vitro, and to ensure the safety of the treatment. We have many steps to clear and the barrier between mice and humans is not low. We cannot always extrapolate human cell events from the results obtained in mice, and have to study human cells from scratch.
Mouse ES cells and iPS cells grown in culture have been analyzed for their stages of development, and we know to some extent at what stages they are, but we have no similar data yet on human ES or iPS cells.

Interviewer :
What led you to study the development of reproductive cells?

Hayashi :
When I was an undergraduate student, I was in a lab of animal reproduction and was working on transgenic animals and clone animals. In doing that, I wanted to know how sperms and ova were produced, and I began to study the development of reproductive cells when I was a M1 student.

Interviewer :
Was there somebody who encouraged you to work as researcher?

Hayashi :
During the last year of my study in the master course, I found that the Research Institute for Biomedical Sciences / Graduate School of Biological Science at Tokyo University of Science was looking for a technician in developmental engineering. I came to interview with Prof. Tomio Tada, President of the Institute, who said to me, "you seem to do something and you would not be technician but assistant professor". I did not really understand at that time how important his advice was for me, but it was really the one that opened my way, and I were not researcher if he did not encourage me in this way.
At the Institute, there were many immunologists, and a very active atmosphere of starting something was prevailing. Immunology is to study invisible and very complex networks. I learned much from the ways of research in this field and it was a very good training for me to construct a theory for solving questions in an invisible world.

Interviewer :
You went to work at the Gurdon Institute, The University of Cambridge, of which Professor Gurdon is President, who was awarded the Nobel Prize 2012 in Physiology and Medicine jointly with Prof. Yamanaka. What did you learn during your stay at the Institute?

Hayashi :
The most important thing that I learned at the Gurdon Institute is to discuss with somebody. You may need much time to solve a question if you are working on it alone by trial and error, but you can soon find a solution if you discuss with your colleagues. In this sense it is important to discuss with somebody.
Another important thing is that I found Japanese scientists are working very well. After thorough discussion, the only thing to do is to do experiments. Japanese scientists, when they face a challenge, try to do their utmost to find a solution in a very meticulous manner, with much patience and in good faith. I found that it is a virtue of the Japanese scientists that we should be proud of. I hope young scientists will go to work in another country when they have accumulated some experiences necessary for working as researcher after receiving a doctorate degree. It will be a good opportunity for them to see virtues of Japanese scientists.

Interviewer :
Would you give advice to young scientists?

Hayashi :
Science is a world where everybody is equal. You have to tell others what your idea is. You do not have to be possessed with negative ideas like "there is no post", but I hope you do what you want to do. The situation will change. Do not be pessimist, and try.

Interviewer :
In ten years, what will be the situation in this field of research?

Hayashi :
I can only imagine the situation in five years at the longest. I am working in a way that I try to have an image of five years later for a subject and to complete it in four years. In five years, we will not yet be able to generate sperms from human ES cells, but will have significant data on genes and factors involved. Perhaps we will know how mouse ES cells begin to differentiate into ova. Studies on human cells will be advanced, ethical discussions will be deepened and social reactions will be matured. Ethical discussions will be developed in relation to technical maturation, and technical progress will play a key role there.


Interviewer :
Furugori Etsuko
Interview date : October 31, 2012

Dr. Katsuhiko Hayashi

Associate Professor, Graduate School of Medicine, Kyoto University

Born in Saitama Prefecture, Japan.
1994 Graduated from School of Agriculture, Meiji University. Doctor of Science (Tokyo University of Science).
1996 Assistant Professor at the Research Institute for Biomedical Sciences / Graduate School of Biological Science, Tokyo University of Science.
2002 Research fellow at Osaka Medical Center and Research Institute for Maternal and Child Health.
2005 Research fellow at the Gurdon Institute, The University of Cambridge, UK
2009 Lecturer at the Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University.
2012 Associate Professor at the same Department.

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