March 2014
Vol.
3

Center for iPS Cell Research and Application, Kyoto University

CiRA
Newsletter

Shinya Yamanaka

New study section

Uehiro ethical research department established

Following receipt of a generous donation from the Uehiro Foundation on Ethics and Education, the Uehiro Research Division for iPS Cell of Ethics was launched at CiRA on April 1, 2013.

Now that medical applications of induced pluripotent stem cells or iPS cells appear about to begin in earnest, CiRA recognizes the need to gauge public awareness of and attitudes to iPS cell research and to respond to the various related concerns people have. It is in this context that the new ethical research department was established with the task of identifying social needs, proposing solutions to the ethical issues associated with iPS cell technology, and carrying out relevant publicity in Japan and overseas.

The staff of the new research department are Associate Professor Misao Fujita, Associate Professor Yoshimi Yashiro, researcher Mika Suzuki, and secretary Emi Kuwabara.

“I believe it is the duty of Japanese researchers to investigate the ethical, social, and legal issues connected with the clinical application of iPS cell technologies that originate in Japan and to publicize their findings in international forums,” said Fujita, who heads the new department.

The concept of research into ethics may be a little unfamiliar to most people. In the context of iPS cells, the management of genetic information is one of the issues. The creation of an iPS cell stock for use in regenerative medicine requires the production and storage of high-quality iPS cells, which are generated from somatic cells provided by healthy donors. As the donors’ genetic information is contained in these iPS cells, it needs to be demonstrated that the donors are given proper information and that the genetic information is appropriately stored and managed. There are also questions as to how far we should go in allowing research into the production of human organs in animal bodies using iPS cells and embryonic stem cells or infertility research using germ cells derived from iPS/ES cells. Such issues should be widely discussed among the public, with consideration given to framing appropriate ethical regulation.

After carrying out surveys and analyses using questionnaires, interviews, and other techniques to gauge public awareness and attitudes, the researchers of the Uehiro ethical research group will propose potential response strategies to scientists and the public. In addition, to raise awareness of issues in medical applications of new scientific methods and technologies, the department will also engage in educational activity aimed at researchers, young people, and members of the general public.

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iPS cells through the window of ethics

Therapeutic misconception

When I worked in a hospital many years ago, a patient once said to me: “It looks like I’m going to be taking part in a research trial for a new drug! That means I’ll be able to try out the latest treatment! I hope it works!” When I saw the smile on the patient’s face, I joined in with the celebratory mood. But having studied bioethics in the meantime, it seem to me now that this was a case of a patient mistaking research for treatment or therapeutic misconception.

By a “therapeutic misconception,” I mean that patients who take part in research trials are confused about the different objectives of research and treatment. When participating in research, it is only natural that people want it to be effective and it may give them great hope. However, if this expectation is too great, with patients thinking “it’s bound to work” or “this it the treatment I needed,” they end up having a therapeutic misconception. In the field of bioethics, this is a phenomenon which has been well known since the 1980s.

Normally, the medical treatment that patients receive in the research context is not “curative” treatment. Essentially, it is not yet known whether it will be effective or sometimes indeed entirely safe. This is because research is something that is performed to find these things out for the benefit of future patients. On the other hand, if we catch a cold and receive “treatment” from a doctor, we expect to be given safe medicine that has a reliable effect. That is because the prime objective of what we call “treatment” is to work for the benefit of the current patient.

If these two objectives become confused, the patient may make the mistake of overestimating the benefit from the research trial or underestimating the risk. Participation in research trials based on this kind of misunderstanding should be avoided as much as possible. When facing the important decision as to whether to participate in a research trial, patients must be fully satisfied that they have made the right decision, and one they will not regret later, based on a correct understanding of the research content.

iPS cell-based research is at a crucial turning point where clinical application is about to begin in earnest. What is important now is to maintain a level-headed approach that distinguishes clearly between the differing objectives of “research” and “treatment,” and, based on this understanding, to look forward with hope to progressing this new medical treatment from the “research” stage where it still is now to the “treatment” stage as soon as possible. Understanding these two points is important not only for patients and their families, but for society as a whole including researchers, the media, and the general public.

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New research

iPS cells used to create the world’s first model replicating muscular disease pathology

The U.S. online scientific journal PLOS ONE has published a paper by the research group of Hidetoshi Sakurai, lecturer in the Department of Clinical Application at CiRA, on their work to create a model that reproduces the pathological conditions of Miyoshi muscular dystrophy. Dr. Sakurai talked with us about his team’s research findings.

What are the characteristics of Miyoshi muscular dystrophy?

Muscular dystrophy is a progressive muscular degenerative disease in which the muscles gradually waste away as muscle cells degenerate and are destroyed. Miyoshi muscular dystrophy, which is one form of muscular dystrophy, was discovered in 1965. In 1998, it was established that the disease is caused by the dysferlin gene, which is present in the cellular membrane of muscle cells. It is thought that abnormalities in the dysferlin gene mean that, when the cellular membrane of the muscle cell is damaged, the membrane repair function does not work properly. At present, no radical therapy is available.

What are the difficulties involved in research into muscular degenerative disease?

So far, there have been attempts to generate muscle cells from pluripotent stem cells, i.e. ES cells and iPS cells, but there have been issues with low success rate and poor reproducibility. Although we may be able to take skin, blood, or other cells from patients and use them to produce iPS cells, unless we can overcome these two issues, it will be very difficult to get these cells to differentiate into muscle cells that can be used to reproduce the pathological conditions of the disease. We have therefore been working to find a method of generating muscle cells with good reproducibility.

Can you describe this latest research?

First, we were able to generate skeletal muscle cells with a success rate of around 90% by inducing the expression of the known transcription factor MyoD1 in human iPS cells. We then used this method to generate muscle cells from two iPS cell lines taken from a single Miyoshi muscular dystrophy patient and confirmed in vitro that the cells displayed the characteristic abnormality in the cellular membrane repair function. When skeletal muscle cells were generated under conditions of overexpression of the dysferlin which is deficient in patients with the disease, the abnormality in the cellular membrane repair function was mitigated.

What is the significance of this latest research?

Until now, there was no reliable method of inducing iPS cells to differentiate into skeletal muscle cells, so it was difficult to reproduce the pathology of muscular disease. The method we developed has allowed us to successfully replicate the pathology of Miyoshi muscular dystrophy in vitro. This is the world’s first successful reproduction of the pathological conditions of muscular disease using iPS cells.

Looking ahead, we aim to develop this method for use in the screening of new drugs for Miyoshi muscular dystrophy. We also hope to use this method of inducing skeletal muscle cells to benefit research into other intractable muscular diseases, leading to the development of new therapies and the discovery of new drugs for many muscular diseases.

Fig. Miyoshi Myopathy by patient derived-human iPS cells

A hole was opened at one point on the membrane of skeletal muscle cells by laser irradiation(triangle).
As skeletal muscle cells derived from Miyoshi myopathy patients cannot repair the membrane of the cells, green fluorescent substance were introduced into the cells(white allow). In contrast, when the gene in Miyoshi myopathy iPS cells was over expressed, the membrane was repaired, which resulted in introducing less green fluorescent substance.

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New research

Factors inhibiting iPS cell generation

The research group led by Shinji Masui, lecturer in the Department of Reprogramming Science at CiRA, has published a paper in the U.S. scientific journal Proceedings of the National Academy of Sciences in which they report that factors which inhibit (interfere with) the reprogramming of cells into iPS cells also have a promoting effect on induced differentiation. Dr. Masui talked about the details of the study.

Can you tell us about the background to the research?

The properties of a cell are determined by the combination of genes active inside it, but which of the genes become active is regulated by transcription factors. By inserting reprogramming factors, we can convert somatic cells into iPS cells, but the details of the mechanism involved are unclear. We began our research from the hypothesis that there was a core set of transcription factors which determine cell type, and that these factors not only decide what cell type the differentiation process results in, but also inhibit reprogramming into iPS cells.

What were the findings of your research?

We carried out an experiment in which we inserted into nervous system cells not only reprogramming factors but also one of the factors characteristic to nervous system cells (Figure 1). If this factor was one with almost no effect on reprogramming factors, the nerve cells would follow the regular path of conversion into iPS cells under the action of the reprogramming factors. However, if it was a core factor with a determining influence on the characteristics of nervous system cells, it would cancel out the effect of the reprogramming factors and reduce the cell’s likelihood of being converted into an iPS cell.

Using this technique, we identified six factors that strongly inhibit the reprogramming of nerve cells. When these factors were transduced into mouse fibroblasts and liver cells, the cell morphology changed to resemble that of a nerve cell (Figure 2). Moreover, the expression of nestin, a gene characteristic to nerve cells, was also confirmed. The same experiment was carried out in the liver cells. These findings indicate that factors that inhibit reprogramming are important factors in determining the direction of cell differentiation. The method developed in our study was named the iPS interference method.

What is the significance of these research findings?

We established that the reprogramming mechanism and the mechanism that controls the direction of differentiation are connected.

The iPS interference method can be applied in the same way to cell types other than neurons, and we believe that it may become a useful tool in elucidating cell reprogramming and differentiation mechanisms. For instance, if it promotes an understanding of systems for producing iPS cells from differentiated cells, it will lead to the development of technology to create iPS cells of higher quality.

Additionally, clarifying the action of the genes that determine the characteristics of differentiated cells should lead to the development of technology for the efficient generation of cells of all types. By developing methods of generating high-quality iPS cells and highly efficient induction methods in this way, we hope to use iPS cells to contribute to the discovery of new drugs and the development of regenerative medicine.

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New research

iPS cells used to model congenital diseases that cause platelet depletion

A Department of Clinical Application research team led by Professor Koji Eto has undertaken an analysis of the pathology of congenital amegakaryocytic thrombocytopenia (CAMT) by generating iPS cells from patients and inducing them to differentiate into blood cells ex vivo. The research findings were published in the U.S. scientific journal The Journal of Clinical Investigation. We asked Prof. Eto to outline the research.

CAMT is a disease we do not hear of very much. What kind of a disease is it?

Patients are affected from birth by serious platelet deficiency (5-10% of the level of healthy subjects) and over the following years there is a gradual depletion of first red and then white blood cells leading to bone marrow dysfunction. This is a serious disease which requires treatment via bone marrow transplantation to achieve complete cure. It has been established that it is caused by the congenital absence of cell surface thrombopoietin receptors.

What were the difficulties involved in analyzing the pathology of CAMT?

To analyze the pathology of CAMT, a mouse model was created in which the thrombopoietin receptors had been rendered inactive from the start. However, although platelet count declined in these mice, red blood cell count did not decrease and the animals were able to live to a normal age, so this model did not manage to reproduce the pathology of human CAMT patients. Naturally we cannot take cells for analysis from the bone marrow of CAMT patients whose blood cell count is already in decline. This lack of an appropriate experimental model was a barrier to elucidation of the disease conditions.

What are the advantages of using iPS cells?

iPS cells retain the genetic information of the donors from whom they were created. Additionally, as iPS cells can be replicated almost limitlessly, it is possible to obtain large quantities of cells with patient genetic information which can be used in the development of therapies and new drugs.

What did the research involve?

With the cooperation of a CAMT patient (who has since received a bone marrow transplant and is now recovered), iPS cells were prepared from skin cells and induced ex vivo to differentiate into blood cells, whose behavior was then analyzed in detail. We succeeded in this way in replicating the pathology of CAMT, in which the production of platelets and red blood cells falls to very low levels compared to that of white blood cells. We additionally established that, in humans, thrombopoietin receptor signaling plays an important role in the maintenance of the multipotent hematopoietic progenitor cell population from which red blood cells, platelets, white blood cells, and other cell types develop, and in their differentiation into the megakaryocyte and red blood cell progenitor cells from which platelets develop. We thus demonstrated that this signaling is essential in the production of red blood cells.

What is the significance of this research?

The present findings suggest that the thrombopoietin-like drugs used hitherto to boost platelet count may also be effective in relieving anemia. Moreover, using iPS cell technology allowed us to not only analyze disease states, but also to investigate the hematopoietic mechanism, or how blood cells are produced. Going forward, iPS cells modeling this disease will be useful tools in research into the origin of human blood production and blood production pathways.

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New research

Altering cell fate with synthetic RNA switches

A research team led by Associate Professor Hirohide Saito of the Department of Reprogramming Science at CiRA has taken the ``RNA switch” technology which allows gene expression to be regulated flexibly in response to intracellular conditions and successfully extended it to allow the action of the switch to be inverted and adjusted in a versatile manner. The research findings were published in the British scientific journal Nature Communications. We spoke to Dr. Saito.

What is an RNA switch?

RNA stands for ribonucleic acid, which is one of the biomolecules contained in the cell. DNA genetic information is transcribed onto mRNA, and protein synthesis (translation) takes place based on the mRNA information. This process of protein synthesis from DNA genetic information is known as gene expression. The RNA switch is an mRNA molecule that detects intracellular substances and regulates gene expression in response. In other words, it can turn gene expression on and off in response to intracellular information.

What does the RNA switch allow you to do?

When inducing differentiation from an iPS cell to the target differentiated cell, for instance a nerve cell, there are sometimes undifferentiated cells left behind that have failed to develop into nerve cells. These cells which remain undifferentiated can form tumors if they are used in transplantation, and a method of selecting only successfully differentiated neurons is therefore needed. One such method would be for an RNA switch to react automatically in response to intracellular conditions, for instance by inducing cell death in undifferentiated cells only. This is one of the expected applications of the switch.

When inducing differentiated cells from iPS cells, a common technique is to introduce proteins, chemical substances, or other materials from the exterior in a number of separate steps. The RNA switch however should make possible a method that automatically selects for continued differentiation only those cells which have reached certain conditions of development.

What was the RNA switch you developed in the research?

The RNA (OFF) switches developed so far operate so that if the input substance A is present, the expression of the output gene is suppressed. By adding an RNA component known as an RNA inverter to this switch, we developed an RNA (ON) switch which operates so that if input substance A is present, the output gene is expressed (right-hand figure). With the previously developed technology, it was difficult to simultaneously regulate expression of a number of genes in response to a single input substance, meaning that individual switches needed to be created and their functions tuned separately, which was time- and resource-consuming. Simply by adding an RNA inverter to the RNA switch, the newly developed method has made it possible to switch from OFF to ON, so that a single input factor can be used to turn the expression of a number of genes on and off individually and simultaneously. We hope that this will lead on to the development of technology for regulating cell fate, for instance by inducing cell differentiation in response to intracellular conditions which are not visible from the outside.

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This newsletter is produced with the support of the Funding Program for the World-Leading Innovative R&D on Science and Technology (FIRST Program).

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