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Researchers for the future

To obtain pancreatic or hepatic cells from ES or iPS cells

Dr. Nobuaki Shiraki

Dr. Nobuaki Shiraki, who is studying cell differentiation at the Institute of Molecular Embryology and Genetics, Kumamoto University, was a researcher in pharmacy. He was interested in the "magic" of drugs and studied pharmacy. Then he wanted to study regenerative medicine and asked Drs. Shoen Kume (professor) and Kazuhiko Kume (associate professor) at the Institute, who are well known in stem cell research, to work in their lab. He has been working on a technique for induction of differentiation of ES or iPS cells into pancreatic or hepatic cells, and obtained good results which may open the door to applications of such technique in clinical settings or in drug screenings. It was his encounter with a "scene" that led him to the world of ontogeny.

Interviewer :
You first entered the faculty of pharmacy at Kumamoto University, didn't you?

Photo:Dr. Nobuaki Shiraki

Dr. Nobuaki Shiraki

Shiraki :
I was interested in the world of drugs where fine granules or powders serve to cure diseases. My mother was a nurse and in her wake I thought to work in a drug-related field. With a picture in my mind that as a pharmacist I would hand drugs to patients, I was studying in a lab at the University Hospital where we are in contact with clinical staff members.
At that time, I was interested in the maternal body and not in the development of organisms. I removed livers or pancreas from mother rats at 10 days, 15 days and 20 days of gestation, and analyzed pharmacokinetics in the maternal organs. In this case, pregnant rats were dissected and there were fetuses in their uteri. I found that fetuses at 10 days and 15 days of gestation were different in terms of the stage of growth. "How dramatic is the growth of fetuses! How marvelous is the ontogeny". I was so surprised that I began to think to study how an organism develops.
I was then at the first year of the doctoral course. I thought to prepare my doctoral thesis on a study that interests me, and began to focus on embryology.

Interviewer :
When you moved to the lab of Prof. Shoen Kume at the Institute of Molecular Embryology and Genetics (at that time Center for Molecular Embryology and Genetics) to which you are currently belonging?

Shiraki :
Formally it was in 2003, when I was at the 3rd year of the doctoral course, but actually I was already there one year before. I had not met Prof. Kume before, but I thought that if I would like to study embryology, it should be in the Kume's lab, and came to the lab and told my intention to Drs. Shoen and Kazuhiko Kume (Professor and Associate Professor). I was a little nervous in front of them, but they said to me open-heartedly, "Come tomorrow if you want". Then I made arrangements for moving, and several months later started my study on ES cells

Interviewer :
At that time, ES cells were already generated. When did you note that ES cells would be useful?

Shiraki :
It was in 1999 when I was at the graduate school of pharmacy. While many people paid special attention to "pluripotent cells", somebody said that some day one would be able to use human ES cell derived hepatocytes for drug efficacy evaluation. At that time I had an impression that it would be difficult to generate complex hepatocytes from ES cells and use them for experiments.

Interviewer :
What was the research theme that you dealt with after moved to the Institute?

Shiraki :
It was to develop a technique for inducing pancreatic precursor cells from mouse ES cells. The technique was to culture mouse ES cells with fetal mouse pancreatic cells. I got expected results but my technique was complicated because of the use of mouse fetuses.
It was why I began, after granted a doctoral degree, to try to develop another technique using certain cells other than fetal mouse pancreatic cells for signalling differentiation of mouse ES cells into pancreatic precursor cells. Finally I established a technique using "M15", renal cells, cultured with ES cells for inducing pancreatic precursor cells. We make a layer of M15 cells on the culture dish surface and put ES cells on this layer. Then, the ES cells begin to differentiate into pancreatic precursor cells.

Interviewer :
How did you come up with M15 cells?

Shiraki :
In our lab, more than 20 candidate cell lines were kept. I screened these cell lines one by one for their ability of differentiation, and determined that this M15 was the most suitable among the final candidates.
Then, I analyzed the products of M15 cells and found that activin, fibroblast growth factor (FGF) and retinoic acid were expressed in large quantities. We knew then that these constitute humoral factors that function for differentiating ES cells into the precursor cells.
To improve the efficiency of differentiation of ES cells into the precursor cells, I studied the optimal levels of the factors to find the best combination. Thus I succeeded in improving the efficiency from 2% to 30%. I published an article titled "Induction of differentiation of ES cells into pancreatic precursor cells" in the journal "Stem Cell" in February 2008.

Interviewer :
In six months after this publication, you published another article on a technique for inducing differentiation of mouse and human ES cells into hepatocytes, didn't you

Shiraki :
Yes. When I induced pancreatic precursor cells, I got also a small number of hepatocytes. Both the pancreas and the liver are derived from the endoderm of the early embryo. I hypothesized that one would be able to induce differentiation of ES cells into hepatocytes if one would arrange conditions suitable for it, for example, adding specific humoral factors. I made experiments, and found that it was so.

Interviewer :
Is it also the case in iPS cells?

Shiraki :
Yes. I can say that all the results of my studies on ES cells are reproduced in my studies on iPS cells. I am using iPS cells as often as ES cells in my research.

Interviewer :
Generation of mouse iPS cells was first published in August 2006. That of human iPS was in November 2007. When did you hear about iPS cells for the first time?

Shiraki :
It was when I joined the "4th Stem Cell Research Symposium" held in May 2006. Dr. Shinya Yamanaka gave a presentation titled "Identification of factors inducing ES-like cells from fibroblast cultures". At that time, the name "iPS cells" was not yet used, but it was a presentation on generation of ES-like cells that were to be called "iPS cells" from fibroblasts. I remember that the audience admired him for his presentation

Interviewer :
What are advantages of using iPS cells in your present research?

Shiraki :
One advantage is that, if we use ES cells, we have to care about ethical problems inherent in these cells, however, if we use iPS cells, we do not have to. Human ES cells are generated from human embryos, and if we want to use these cells, we have to take necessary procedures according to the government's guidelines, and design our studies without ethical infringement. Otherwise, we should not conduct studies using ES cells. In contrast, if we use iPS cells that are derived from human somatic cells, we do not have to care about such ethical problems.
Another advantage is that we get closer to realization of personalized medicine, if we use iPS cells. So far, we have been trying to induce hepatocytes from human ES cells and use the hepatocytes for pharmacokinetic assay. However, there are individual differences in liver functions, and if we use hepatocytes derived from human ES cells, the hepatocytes may reflect the individual characteristics of the ES cells used. On the other hand, in the case of iPS cells, we are able to generate these cells relatively easily from somatic cells collected from different donor individuals, and thus it will be possible that we induce hepatocytes from iPS cells derived from a patient and use these hepatocytes for testing the ability of the patient's liver to decompose a given drug. If a patient has a pancreatic or hepatic disorder caused by unknown factors, we will be able to study what is going on in the patient's pancreas or liver, using pancreatic or hepatic cells induced from the patient derived iPS cells.

Interviewer :
Later, you successfully induced not only endoderm but also mesoderm and ectoderm. In 2010, you published a study for inducing pancreatic cells from ES or iPS cells without using feeder cells.

Shiraki :
I thought before that M15 cells were needed as feeder cells for inducing differentiation of ES or iPS cells. I found then that if we use a synthetic material called sBM (synthesized Basement Membrane), we are able to induce differentiation of ES or iPS cells into pancreatic cells without using M15.
We noted that in experiments for inducing differentiation using M15 cells, a protein called laminin was expressed in large quantity. We asked Dr. Katsumi Mochidate at the National Institute for Environmental Studies to prepare sBM containing a high level of laminin, and used this sBM in experiments for inducing differentiation of ES or iPS cells into pancreatic cells. It worked well.
In regenerative medicine, it is said that it is not good to introduce exogenous cells other than ES or iPS cells into the patient's body. If we use sBM, we can avoid this risk. I can say that this is an improvement.

Dr. Nobuaki Shiraki

Interviewer :
What objectives do you have toward clinical applications of these cells?

Shiraki :
For regenerative medicine, my objective is to induce from iPS cells pancreatic cells that are resistant to diabetic treatment. These are pancreatic cells that secrete a moderate level of insulin, the hormone controlling blood sugar level.
On the other hand, I have in mind that I will prepare model cells that will be useful for testing drug toxicity or viral infections, and use these models in practice. At present, we are using hepatocytes imported from other countries for these tests, and in this case we have a problem of ethnic as well as individual differences in the liver. Another problem is that it is difficult to maintain the cells in culture for a long period. If human iPS cell derived hepatocytes are available for replacing the cells actually used, these problems will be cleared.

Interviewer :
How do you find your research environment at the Institute of Molecular Embryology and Genetics, Kumamoto University

Shiraki :
It is not a large institute, and we have close communications with each other, among all members including students and researchers of different labs. Here it is easy for us to have contact with colleagues of different fields.
On the other hand, research projects from different departments including the institute have been adopted as the Global COE Program "Cell Fate Regulation Research and Education Unit". In the framework of this program, the students and post-docs involved organize a study meeting every week and give presentations in English.

Interviewer :
To conclude, would you tell us what your research objectives are?

Shiraki :
I was studying pharmacy, and then came to this field of ES or iPS cell research. I thought it would be difficult to prepare livers from ES or iPS cells and use them, but I am now working on it. I am now at a stage where I will be able to use hepatocytes induced from ES or iPS cells for phamacokinetic assay, which I was studying when I was at the graduate school of pharmacy. I will continue my research, based on my knowledge accumulated in the course of my past career.


Interviewer :
Jiro Urushibara
Interview date : February8, 2011

Dr. Nobuaki Shiraki

Assistant Professor, Department of Stem Cell Biology, Division of Stem Cell Research, Institute of Molecular Embryology and Genetics, Kumamoto University

In 1977, he was born in Kumamoto Prefecture, Japan. In 1999, he graduated from School of Pharmacy, Kumamoto University, and entered the Graduate School of Pharmacy, Kumamoto University, where he continued his study until 2003. In 2003, he moved to the Department of Stem Cell Biology, Center for Molecular Embryology and Genetics, Faculty of Life Science of the same university (Shoen Kume's Lab). After he was granted a doctor's degree in 2006, he was working as a researcher. Since 2009, he is associate professor at the Division of Stem Cell Research, Institute of Molecular Embryology and Genetics, Kumamoto Univerisity.

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