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Researchers for the future

Direct reprogramming of skin cells into hepatocytes

Dr. Atsushi Suzuki

Dr. Suzuki established a technique for generating "iHep cells"(induced Hepatocyte-like Cells) that give functions equal to those of hypatocytes from skin fibroblasts. This technique is called "direct reprogramming" of cells into another type of cells without intermediary of iPS cells. In this interview, he will talk about how he was involved in generation of iHep cells and what are the advantages of the direct reprogramming technique.

Photo:Dr. Atsushi Suzuki

Dr. Atsushi Suzuki

Interviewer :
How did you begin to study hepatocytes?

Suzuki :
I was interested in immunology, and when I was a graduate student at University of Tsukuba I wanted to study transplantation immunity in the clinical context. Meanwhile, Prof. Hiromitsu Nakauchi (currently Professor at the Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo) who was the boss of our lab published his studies on differentiation and self replication of heamatopoietic stem cells. How is it in other types of cells? In this context, he proposed me a subject of study to find liver stem cells in mice. It was very different from immunology and I was shocked at that moment. However, I began to study mouse fetuses for development of the liver, and found something like stem cells. Then, in making many trials and errors, I found little by little my research interesting.

Interviewer :
The liver is said to be 80% made up of hepatocytes. What are the characteristics of these cells?

Suzuki :
The liver has important functions of metabolism, detoxification, etc. Hepatocytes are "naughty", and if the liver lacks a part of it, for example by excision, the remaining cells begin to proliferate vigorously to regenerate the lacking part. They seem to have the nature of growing. One important characteristic is that they grow with their functions of metabolism and detoxification, and we do not know yet why they can do it.

Interviewer :
You went to study at Salk Institute in USA, where you learned their way of doing research, and came back to Japan in 2005. At that time, before iPS cells were generated, was there somebody who was studying direct reprogramming?

Suzuki :
There was some related study. It was reported 25 years ago that fibroblasts, in which certain genes are introduced, became muscle cells. I myself found, when I was a graduate student, that liver stem cells were able to become pancreatic or intestinal cells, and when I was in USA, found that ES cells that differentiated to some degree were able to return to the state of ES cells. What was interesting is that these processes took place without introduction of genes but only with modifications of culture conditions or transplantation sites. Similarly, hepatocytes are normally derived from liver stem cells, but it is known that in rare cases mesenchymal stem cells contained in pancreatic exocrine cells or in bone marrow differentiate into hepatocytes. And it is known that after bone marrow transplantation, blood cells sometimes fuse with hepatocytes and serve as hepatocytes to form liver tissues. Reviewing these findings, I could imagine that some changes in cell environment triggered the functions of certain factors inducing reprogramming, and that it would be difficult to identify such factors.
In the meantime, I attended the "Keystone Symposium" in March 2006, where Prof. Shinya Yamanaka gave a presentation on iPS cells. I listened to his presentation that inspired me in two points. One was that no doubt there exist some factors deciding the cell fates and inducing reprogramming. Another was that there is not only one factor but also two or more factors that induce(s) reprogramming. The generation of iPS cells led other researchers in the field to have an idea that if they would be able to reprogram differentiated cells into undifferentiated state, they would also be able to convert certain type of cells directly into another type of cells, and accelerated research on direct reprogramming.

Dr. Atsushi Suzuki

Interviewer :
You moved to Kyushu University in 2007 and also worked as researcher of PRESTO in 2008. I have heard that after that you started your work on development of techniques for generating iHep cells by direct reprogramming. Is that right?

Suzuki :
Yes. I had valuable experiences for my research when I was working at these two posts.
I was employed by the Medical Institute of Bioregulation, Kyushu University, as tenure track associate professor. At this post, we have to work hard, because we have to pass the examination for promotion to a tenure post, and if not we will be dismissed. On the other hand, we are less constrained and are allowed to devote ourselves on research.
In the JST's PRESTO "Understanding Life by iPS Cells Technology", I was one of the first year researchers, and it was only I who was studying something other than iPS cells. Prof. Shin-ichi Nishikawa, the project leader, asked me if I would really do it, and encouraged me. And I was acquainted with researchers of my generation, and discussed with them on the occasion of workshops. It was a very good experience.

Interviewer :
In such a research environment, you published your work on the generation of mouse iHep cells by direct reprogramming in June 2011 in Nature. How did you attain it?

Suzuki :
In the beginning, I spent much time to establish an appropriate experimental system. How to seed fibroblasts, the starting cells? What are the medium conditions? How to prepare viruses? I tried to optimize these conditions to establish a system in which reprogramming of cells would be facilitated. When we generated iPS cells, we found that it was not easy to change the fate of cells. From this experience, I thought that I should do my utmost to find optimal conditions, and if I give up doing it halfway, I would regret. I wanted to be convinced that I would have done my utmost, regardless of the results. If an optimal system would be established, it would be useful for my research later.
After that stage, I did everything smoothly without spending much time. First, I selected 12 transcription factor candidates. There were published data on hepatocytes, and reviewing them, I had an idea of which important factors are. Next, I did the same thing as Dr. Kazutoshi Takahashi did when he found the transcription factors for inducing iPS cells. I introduced all of the twelve candidates into fibroblasts. Besides, I prepared 12 different sets of eleven out of the twelve candidates, that is to say, one factor was removed from the twelve in each set. And I introduced each set into fibroblasts. I found that when a factor called Hnf4α was removed, certain genes that are to be expressed in hepatocytes were not expressed, and thus Hnf4α was necessary. However, Hnf4α alone was not enough for inducing hepatocytes, and I made combinations of Hnf4α + another factor. I found that the combinations of Hnf4α plus Foxa1, Hnf4α plus Foxa2, and Hnf4α plus Foxa3 were effective for inducing hepatocytes.
The induced cells (iHep cells) looked like liver epithelial cells that I was familiar with for a long time. And analytical data showed that these cells had characteristics of hepatocytes. However, we know that hepatocytes in culture are not the same as those in vivo. I made an experiment in which I introduced iHep cells into mice to see how these cells behave in vivo for gene expressions, morphology and functions, and found that they came to behave like hepatocytes. I was sure that they were hepatocytes.

Interviewer :
What you think are advantages of direct reprogramming in generating hepatocytes, as compared to the techniques using ES or iPS cells?

Suzuki :
One advantage is that this technique gives a very high reproducibility. With this technique, under-graduate students who are not skilled in experiments will generate iHep cells successfully. In contrast, they will not easily generate hepatocytes from ES or iPS cells, since in this case slight differences in the experimental conditions and culture conditions can affect the results, and it is not easy to get the desired cells.
For carcinogenesis, we are able to avoid formation of teratomas that is one of the two paths by which ES and iPS cells will become cancer cells. On the other hand, we cannot avoid expression of cancer genes by retroviruses that we use for introduction of transcription factors. For iPS cells some techniques have been developed for preventing cancinogenesis, and if similar techniques will be available for direct reprogramming, it will be better.
We are now able to get iHep cells from fibroblasts in about one week, and the time required has been shortened. On the other hand, there are still many fibroblasts that die after introduction of transcription factors without becoming iHep cells, and I am still looking for optimal conditions.

Interviewer :
After mouse iHep cells, you will do it with human cells, won't you?

Suzuki :
Up to the present, with direct reprogramming of human cells, it is reported that neurons have been generated, but not other cells. If we do something successfully in mice, it does not mean that we will always be able to do the same thing in humans. I am trying to find newly the best conditions for human iHep cells.
We are one of the research groups that are in a position most close to the generation of human iHep cells by direct reprogramming. The target of this kind of research is clinical application of the cells obtained, and we will make efforts to study the generation of human iHep cells.

Interviewer :
I have an impression that direct reprogramming, that does not pass through the stage of iPS cells, will be more advantageous in generating target cells. What is the actual situation?

Suzuki :
It is not the question of one is better than another. There are certain cells that cannot be generated by direct reprogramming but through iPS cells. It will be ideal that we will have both and use one or another depending on the case.
What is more, there must be "the third method", not iPS cells nor direct reprogramming. It may be one that is to induce cell differentiation directly in vivo, or to regenerate the target organ in the body of the patient who received a "medicine". It is an ultimate method. Research will advance if we are working with a next idea and not only for refining the existing one.

Photo:Dr. Atsushi Suzuki and His Member

Interviewer :
Jiro Urushibara
Interview date : June 5, 2012

Dr. Atsushi Suzuki

Associate Professor, Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University

Born in Gunma Prefecture, Japan, in 1974. After graduated from the Faculty of Science, Tohoku University, he was a graduate student at the Faculty of Medicine, University of Tsukuba, from 1998. He studied at the Salk Institute for Biological Studies (USA) from the third year of his doctorate studies, and received a doctor's degree in medicine. He was back to Japan in 2005, and appointed as tenure-track associate professor at the Medical Institute of Bioregulation, Kyushu University, in October 2007, after working as research fellow at RIKEN Center for Developmental Biology. He was appointed also as researcher of PRESTO in June 2008. He is associate professor at the Medical Institute of Bioregulation, Kyushu University, since April 2011, and leader of CREST since December 2011.

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