Naka K.O. Nobel Prize of Shinya Yamanaka as a reason to rethink role...

Nobel Prize of Shinya Yamanaka as a reason to rethink role of science and mission of scientist in Japan

K.O. Naka

The article features the discovery and personality of Shinya Yamanaka, a Nobel Prize winner in Physiology or Medicine. It is based on Yamanaka's speeches and information about the discoveries made by Japanese researchers in various fields of medicine by use of induced pluripotent stem cells (iPS). The breakthrough research of Yamanaka not only laid the foundation of a new area in regenerative medicine, pharmacology and transplantology but also highlighted serious problems in the position of science and researchers in contemporary Japan.

Keywords: Nobel Prize, induced pluripotent stem cells (iPS)/artificial multifunctional stem cells, discovery, regenerative medicine, therapy.

Wonder Cells: Medical Revolution

In 2006, a 44-year-old professor of Kyoto University made a discovery in the area considered unpromising by many scientists - he managed to create induced pluripotent stem cells from skin cells of a mouse and, a year later, of a man [1].

Stem cells, a universal building material for all types of human cells at the fetal development stage, have been known to scientists for a rather long time. While an embryo develops, they can transform into prototypes of all the 220 specialized cells in a human body [2] - cells of the brain, bones, the cardiac muscle or the retina. Embryonic stem cells (ES or ESC) were isolated from mouse embryos in 1981 and from a man in 1998. Stem cell injections became popular in the West in the early 2000s as a way of rejuvenation. Experiments were staged to transform those cells into specialized tissue cells. Yet the research encountered a very serious or even insurmountable impediment: it was possible to isolate those cells only from a human embryo. Two major problems occurred: sources of the material were limited and morality of using cells isolated from a human embryo, one's own kind. There were also fears that the supply of living material for surgeries may become an attractive niche for criminals.

The discovery made by Yamanaka did away with all those problems. The cells he created were called artificial multifunctional stem cells. The term of induced pluripotent stem cells (iPS) has also been widely used. An artificial stem cell was produced in a laboratory from a skin cell of an adult.

It was hard to overestimate the vistas opened up for researchers. It is not accidental that the creation of stem cells by a relatively simple method, without moral reservations and from accessible material was called a medicinal revolution. In fact, it paved mankind's way to replacing ‘broken' organs not with donor material but with stem cells isolated from skin cells of the patient oneself. In the foreseeable future, artificial multifunctional stem cells (iPS) may resolve the problem of shortage of donor organs and replenish blood and plasma banks. The only problem that has yet to be solved is the transformation of some iPS cells into cancer.

As Prof. Yamanaka has put it, "iPS cells are a time machine, which can carry us back to the time when a person was healthy. We will be able to explore the mechanism of a disease and to change its course. We can create iPS cells by taking some skin cells from a patient and adding four genes. For instance, we can make cardiac muscle cells, which will be almost the way they were when the person was born. I am 52-years-old, and my cells are 52-years-old. But iPS cells are zero-years-old. We will cancel out the disease and reverse it to the point when the patient was still healthy" [3].

Doctor loser or typical Japanese scientist?

Yamanaka began his career as a surgeon, what's more a loser. In his lecture delivered to Kyoto high school and university students in 2010, ‘New Medicine Creating iPS cells' [4], he recalled with pain and shame that colleagues asked him to stay away from the operating table and nicknamed him ‘ Jamanaka' after ‘jama' (a nuisance).

Yamanaka recalled "graduating from the faculty of medicine, which he had been dreaming about since childhood, and starting his career of a surgeon 20 years ago. I was happy that I could start my career in the brand-new hospital with state-of-the-art equipment in the city of Osaka. I was an intern for two years and all that time my supervisor never called me by name, Yamanaka. He called me ‘Jamanaka'. It is the job of a surgeon to perform operations. We were allowed having some practice, but I always failed. It took me two hours to perform a simple 20-minute surgery" [5].

Yamanaka learned from his internship that he would not be a surgeon and decided to give himself a try in the field of research. Realizing limits existing in the modern surgery profession was another reason why Yamanaka chose to be a researcher: "even the most talented surgeons can do nothing about a fractured spine or bone cancer which makes young men, athletes permanently bedridden" [6].

He decided to study the causes of incurable diseases. Yamanaka entered a graduate school and understood from the very first experiments staged in the new capacity that he had finally found his way. This complex period of life taught Yamanaka to be grateful for failures - "the worst events in one's life become the beginning of wonderful changes".

That experience was reflected in the wish he extended to students: "I wish you to make mistakes and to suffer defeats. Nine in ten attempts have to be a failure so the tenth is a success. This is what has happened to me" [7].

The time of his graduation coincided with first stem cells discoveries. Gene change experiments were gaining momentum in the United States in the early 1990s. Japan had nothing of the sort. Yamanaka was fired with enthusiasm for cells and gene change and wrote about 50 letters, responding to ads in the Nature and Science magazines. The response was positive to one of his letters and he went to Gladstone Institutes in San Francisco.

Yamanaka admitted that he was finally able to wrap up in science in America. He recalled that period of his life and ideal conditions for life and work with gratitude and nostalgia. Judging by his comments, he decided to leave the United States only after his wife had to return to Japan for the sake of their daughter's education.

The return to Japan was shocking for Yamanaka. In his words, Japanese scientists returning from the United States are prone to the ‘post-America depression' syndrome. This disease is unknown to psychotherapists, but Japanese researchers are well familiar with it.

Funding was ample, trained personnel was taking care of laboratory animals, and Yamanaka and his colleagues were eagerly studying cells and gene change in Gladstone Institutes, but the atmosphere was completely different in Japan.

"In Japan, a scientist has to take care of laboratory animals. Everyone knows how quickly mice propagate. There will be 20 of them in a month, 100 in three months, and 500 in a year. Soon enough my entire lab was packed with caged mice. I had to take care of them, feed them, change their water, clean their cages and dispose of their faeces. Every time I was cleaning cages bedraggled with mice faeces I was wondering who I was. Was I a scientist or a janitor cleaning mice cages? [8]" Yamanaka said, reflecting on his life in 2010.

He said he was astonished by the contrast in researchers' position in America and in Japan, first of all, in terms of stability and financing. Most Japanese scientists, at least those doing applied life science research, have a two-year contract. This means the contract may not be extended unless tangible results are achieved within two years. Yet a serious discovery may take decades. The system forces scientists to pursuit one-time studies or otherwise they will face total uncertainty and the search for a new job at the end of the contract.

Financing of research also left much to be desired; it was beyond comparison with the research budget in the United States. Besides, the Japanese institute he was working with had a medical focus and colleagues clearly indicated that it would be good to change the theme from stem cells of mice to something more akin to medicine. There was a moment when Yamanaka was ready to abandon research and to resume his medical practice.

Yet, shortly before he decided to break up with science, two events happened and changed the course of his career crowned by Nobel Prize. Stem cells were isolated from a human embryo in America in 1988, 17 years after the isolation of stem cells from a mouse embryo. The discovery gave a boost to regenerative medicine because it opened up prospects for the creation of healthy cells for various human organs from embryo stem cells.

"For me, it was a pivotal moment. I decided that my research could be useful for medicine and treatment of such incurable diseases as spinal cord and vertebral column damage. One big question remained: it was medicine for humans but did we have the right to use human embryos for that noble cause?" [9] Yamanaka asked.

He was not hoping for a positive outcome, but replied to an ad in a science magazine and submitted his CV in 1999. He was expecting a denial and an end to his scientific career. But the answer was affirmative, and he took a job with Nara Institute of Science and Technology. It provided perfect conditions for scientists and funding, which was good for Japanese standards. For the first time in his life, Yamanaka had a chance to head a laboratory. He tasked the lab to create a stem cell from a regular skin cell by means of cell ‘reconfiguration', erasure and ‘nullification' of all data, and recording of new data.

"It was a very complex task but I was just about ready to quit science, so I decided to do it. I was prepared to work for decades, without seeing the fruit of my research. But thanks to my team of graduate students and employees, we managed to generate induced pluripotent stem cells, iPS. It appeared that four types of genes affecting a skin cell created - it was improbable but still happened - a multifunctional cell, which was very much alike a stem cell." [10]

Artificial stem cells were generated from mouse cells in 2006 in the Yamanaka laboratory at Kyoto University, where he started working in 2004. The success was renewed in 2007, after they had taken human skin cells for building material. Yamanaka was awarded Nobel Prize in Physiology or Medicine in 2012 for that discovery.

Applications for Discovery: "The World's First"

The academic community perceived the opportunities provided by iPS cells as a call for action. Scientists from various institutes in the country demonstrated impressive results in iPS cell experiments. The phase ‘the world's first' became common in reports released by Japanese researchers.

In February 2009, an academic team from Keio University led by Prof. Hideyuki Okano said they had restored the locomotive function in a mouse with a damaged spinal cord and paralyzed hind limbs by use of human iPS cells [11]. The scientists created an iPS cell from a human skin cell and grew nerve tissue cells from that material. The cells were implanted in mice with paralyzed hind limbs and the mice were walking again a month after the surgery. The therapeutic effect of human iPS cells was proven for the first time in the world.

In December 2009, an academic team from Tottori University led by Prof. Mitsuo Oshimura together with Yamanaka's team from Kyoto University reported cure of cells from people with muscular dystrophy [12]. The scientists ‘cured' defects in the gene of muscular dystrophy patients and grew iPS cells capable of forming healthy muscle tissue. The healthy muscle tissue was produced from the patients' cells.

Nara Medicine Institute grew the world's first bowel fragment from iPS cells of a mouse in March 2010. The pipe-shaped bowel fragment had a diameter of 2mm and a length of 5mm, peristaltic ability, just like a real bowel, and could propel a substance within its tube. It had a mucus membrane, muscles and nerve endings, or everything that made up the bowel structure. A 3D fragment of an internal organ was build from iPS cells for the first time in the world.

In December 2010, Prof. Okano told at a scientific conference, ‘Biochemistry and Molecular Biology 2010', about a new achievement: his team restored the walking ability of an almost fully paralyzed monkey with a damaged spinal cord by using human iPS cells (less than two years had passed since the successful experiment with mice). Nerve cells grown from iPS cells, which were produced from human skin cells, were implanted in the monkey with paralyzed limbs. Six weeks later, the monkey could walk and fully regained its ability to clench and unclench its paw.

In August 2011, Kyoto University researchers led by Prof. Saito Michinori achieved the first-ever birth of healthy offspring from a male mouse incapable of reproduction [13]. iPS cells were generated from its skin cells, and they produced primordial cells, which later developed into sperm and female eggs. Primordial cells were transplanted into testis of the infertile male and ten weeks later he produced sperm good enough for procreation. In contrast to their biological father, the offspring was able to procreate. On one hand, this achievement is the first step towards solving the infertility problem in humans but, on the other hand, it poses a new ethical problem - theoretically, it will be possible for just one parent to give birth to a child.

Next January scientists from the same university made impressive progress in curing Parkinson disease in primates by implanting nerve cells generated from human iPS cells into a monkey's brain. Six months later, the brain scan showed the implanted cells were producing dopamine - a substance, which is the key to curing this severe illness [14]. Modern medicine can alleviate symptoms of that disease but there is no drastic cure for now.

That summer scientists from Yokohama University grew the world's first liver in a mouse from human iPS cells [15]. A human iPS cell generated three types of cells needed to build liver tissue. They were used to create a liver cell prototype, which was implanted in the mouse. It turned into a regular liver cell in the mouse's organism. The liver which was only 5mm in size did not differ from a human liver; it produced proteins and broke down drugs. Its blood vessels were working perfectly.

In January 2013, Kyoto University reported the creation of fragments of human kidney tissue from iPS cells [16]. The researchers produced cells of five types characteristic of main kidney parts. For instance, they built a kidney tubule, which filters blood. In December that year, Kumamoto University grew the world's first human kidney glomerulus, removing harmful substances from the bloodstream, from iPS cells [17].

In July 2014, Kobe University and Kyoto University's Center for iPS Cell Research and Application (CiRA) became the world's first to grow cancer stem cells providing rapid regeneration and growth of malignant tumors. Up till now, studies of the cancer growth mechanism were contained by the deficit of live cancer stem cells obtained by researchers. The possibility of their artificial production opens up broad vistas for studying and searching for efficient cures.

September 2014, seven years after the creation of a human iPS cell and two years after Yamanaka was awarded Nobel Prize, saw an event that would definitely go down in history of regenerative medicine. Japan performed the world's first surgery using artificial multifunctional stem cells. Prof. Masae Takahashi from Kobe RIKEN Institute transplanted a retina grown from the patient's iPS cells to a 70-year-old woman suffering from a severe illness - age-related macular degeneration. Hence, clinical research in the field of treating humans by use of iPS cells has begun.

Scientists have been discovering new areas for use of artificial multifunctional cells. They can not only grow organs in vitro from the patient's cells but also experiment with the influence of new or well known drugs on various kinds of diseases. In 2014, Prof. Tsumaki Noriyuki from Kyoto University found a cure for the hitherto incurable disease - cartilage degeneration. Chondrodystrophy - symmetrical shortening of limbs in the normal development of the body - happens because of flawed division of cells in an embryo's cartilage cells. Patients demonstrate symptoms of nanism, they have short hands and legs and large heads. Up till now the only therapy was the method of Gavriil Abramovich Ilizarov, who mechanically ‘lengthened' bones through numerous surgeries and long periods of hospital treatment. Japanese scientists grew cartilage cells from tissues of a chondrodystrophy patient and treated the artificial cartilage cells with various medicines. They did not expect a drug, which had long been used for treating cardiovascular diseases, to trigger growth of cells and formation of the cartilage. Clinical tests may begin already in 2016.

Cosmetic industry professionals took a practical interest in iPS cells last fall. A center will open in Kobe to study methods of treating baldness by use of iPS cells. The first patients will be admitted in 2018. A skin fragment of about 5mm taken from the back of the patient's head will produce hair growing cells. The cells will be implanted in the patient. The method may become a drastic solution to the baldness problem.

After Nobel Prize

The academic community responded to Yamanaka's discovery with a series of new research programs and experiments, but the government appreciated it only after he had been awarded Nobel Prize. The prize uncovered problems with the financing of research and creation of an appropriate research environment in Japan. After Nobel Prize was awarded to the academician, the government finally realized that Japan had acquired a mechanism, which could make it a leader in the most advanced field of medicine. It was also surprised to discover that no conditions had been created for retaining that leadership and that the outstanding results were mostly achieved by enthusiasm and asceticism of scientists.

Here is an illustrative example. Six months before the prize was awarded, in March 2012, Yamanaka bet online that he would run a marathon for raising 10 million yens needed for the studies (approximately $100,000 as of that date). People wishing to support the professor's undertaking could transfer money to his website // justgiving.jp/ under a scheme, which later became popular in Japan. Practically the entire sum was collected within six months, and one day after he was awarded the prize it nearly doubled. Yamanaka is a sportsman who runs every morning and a marathon would not risk his health. But are they any other examples in the world of a Nobel Prize winner raising funds for his research by betting on his physical abilities?

The new Nobel Prize winner was asked at a press conference how he learned about the award. He simply said that his wife picked up the phone because he was repairing a washing machine. The press laughed at his answer, and it was a subject of discussion in the media for a long time. Several days after the press conference, government members felt ashamed for the position of science in Japan and decided to jointly buy a new washing machine for the laureate so that he could focus on his studies. The degree of the government's understanding of the importance of this research is demonstrated by the fact that only 3.8 billion yens ($47 million) were assigned for iPS studies in the package of economic measures worth 422.6 billion yens (approx. $5 billion, by the official rate) two weeks after Nobel Prize was awarded.

During three years, which have passed since the awarding of Nobel Prize, the importance of the discovery and its significance for various branches of science and the economy has been realized at various levels.

The Japanese government started to create an entity, which would search for brand new cures for 50 most serious and rare diseases by use of iPS cells. The new entity will operate under the aegis of the government and will pool efforts of five research centres which have achieved best results in iPS cell projects and seven private pharmaceutical companies. It will be formed by 2017 as the first joint project of scientists and industries supervised by the government [18]. Presumably, cell material for testing new medicines will be taken from cells of patients suffering from severe diseases.

In February 2013, Kyoto University's Centre for iPS Cell Research and Application (CiRA) initiated the creation of an iPS cell bank for growing necessary kinds of cells for transplantation. In fall 2015, the bank will start to provide research and medical centres with artificial multifunctional stem cells for clinical research. The bank of artificial multifunctional stem cells is being created from donor blood cells taken in the university hospital from healthy people with a combination of tissue compatible anti-genes, which minimize a chance of being rejected in transplantation. In the opinion of scientists, the bank will meet 30-50% of the needs of the Japanese population by 2017. Since fall 2015, cell material from the bank will become available to ten major research and medical institutes of Japan.

Another landmark event happened in the history of iPS cell research in spring 2015: big business took an interest in using the cells. A biggest pharmaceutical company in Japan, Takeda Pharmaceutical Company Limited, and Kyoto University's Centre for iPS Cell Research and Application (CiRA) led by Yamanaka announced the launch of ten projects for the period of ten years for the joint development of medicines and treatment methods for severe diseases using iPS cells. The pharmaceutical company will invest 20 billion yens (over $160 million, by the current exchange rate) in the program. This project will be the first step towards the wide use of iPS cells in practical medicine.

Yamanaka gave a forecast for regenerative medicine in his interview with the television and radio broadcaster, NHK, on 8 October 2012, the day when he was awarded Nobel Prize. "I believe that clinical research would be the rapidest in retina treatments, for instance, macular degeneration [that forecast came true]. But there will be a difference between diseases. For instance, studies of the cure for spinal cord damage, Parkinson disease and heart diseases by use of iPS cells will begin within the next few years. The full-scale therapy will become available in 10 to 20 years."

Conclusion

The Nobel Prize in Physiology or Medicine awarded to Japanese Prof. Yamanaka in 2012 lit up all shadow sides of the current status of science and the attitude to science in Japan like a ball of lightening and, at the same time, brought on stage the man who soon earned the title of ‘a real Japanese scientist' and ‘a science samurai' in the press. It became known after the prize was awarded that he was personally maintaining contact with hundreds of people suffering from incurable and rare diseases. It also appeared that he was regularly inviting hundreds of patients to scientific symposia and giving them a chance to ask questions and to take part in the discussion. Prof. Yamanaka underscored in his post-Nobel speeches that the prize was not a goal in itself. His opinion expressed soon after the discovery of iPS cells in 2010 was unwavering even after the prize was awarded, "The creation of iPS cells is not the end of research. This is just the beginning of a long road. So far, iPS has not saved any lives. It is my task to carry on this research to the end, in the true sense of this word." [19]

The award drew public attention to the fact that the actual attitude to the scientific career and scientists in Japan was not as dedicated as it was believed to be, and discoveries and breakthroughs often happened only because of the will and talent of scientists, who were ready to put up with scanty financing and instability in order to pursue their dream.

The ‘discovery' of the so-called STAP cells - artificial cells that can allegedly be produced under the influence of a certain acid - proved that the practice of gaining quick results in science was flawed. The discovery was a fake, Haruko Obakata, a 30-years-old employee of RIKEN institute, was dismissed and her academic supervisor was unable to bear the shame and committed suicide.

There is no doubt that the discovery of Yamanaka will have a bright future. During eight years, which have passed since the discovery of human iPS cells, scientists have learned how to grow cells of the cardiac muscle, bowels, pancreas, retina, blood, skin, nerve cells, the female egg and sperm cells and kidney nephrons [20]. The first-ever retina transplantation surgery using iPS cells was successfully performed last year. The discovery made it possible to test drugs on organs grown from iPS cells. First results have been achieved in fighting limp paralysis, Parkinson disease and others.

The national government does not conceal its plans to turn regenerative medicine into a Japanese brand and a branch of medical tourism, and to make Japan a world leader in the field of research and practical use of artificial multifunctional cells iPS in medicine. Time will show whether Japan will become a world regenerative medicine power and gain benefits for the economy, science and researchers.

[1] Jinkotanoseikan saibo no juritsu no seiko (Success in creating iPS cells) Kyoto Daigaku 21.11.2007 URL: http://www.kyoto-u.ac.jp/notice/ 05_news/documents/071121_11.htm (accessed 05.11.2015)

[2] Diano P. Human Anatomy Atlas M.: Bertelsman, 2007. S.P -1.

[3] Shinya Yamanaka. IPS saibo no tanze kara jitsue (IPS cells: from origin to practical use). Interview with the sponsor of Kyoto University Center for Applied iPS-Cell Research // Credit Season card. URL: http://www.saisoncard.co.jp/ support/ips/ (accessed 05.11.2015.)

[4] Shinya Yamanaka. Koko tokubetsu zuge (Lecture for Kyoto high school and university students)// URL: https://www.youtube.com/watch?v=ReKaknHHyTM&feature= youtu.be (accessed 05.11.2015)

[5] Ibid

[6] Ibid

[7] Ibid

[8] Ibid

[9] Ibid

[10] Ibid

[11] Sentan kagakugizutsu simpojiumu iPS saibo ga kirihiraku kongo-no ingakukenkyu (Advanced science and technology symposium ‘Research in medicine of the future opened up by iPS cells)'// Keio Daigaku URL: http://www.keio.ac.jp/ja/news/2008/kr7a 43000000nv2u.html (accessed 05.11.2015).

[12] Suji jisutorofi: kanja yurai no iPS saibo: ni okeru idensi syu: fuku ni seiko (Successful restoration of DNA in stem cells of a muscular dystrophy patient) // Tottori Daigaku URL: http://www.jst.go.jp/pr/announce/20091209/ (accessed 05.11.2015)

[13] Tanosei kansaibo de sakusei sita seiseku saibo ni yurai suru mausu no sansyutsu ni seiko - seiseku saibo keisei mekanizumu no kaimei, funinsho genin nado ni koken (Successful mouse reconstruction by means of embryo stem cells to help uncover causes of infertility and mechanism of germ cells formation). Research report dated 05.08.2011. //Kyoto Daigaku URL: http://www.kyoto-u.ac.jp/static/ja/news_data/h/h1/news6/2011/110805_1.htm (accessed 05.11.2015.)

[14] Ibid

[15] Yomiuri Shimbun 08.06. 2012.

[16] Hito iPS saibo o mochiite jinzo no ichibu kozo o saigen (Japanese scientists grow kidney fragment from human stem cells) // Kyoto Daigaku URL: http://www.kyoto-u.ac.jp/static/ja/news_data/h/h1/news6/2012/130123_1.htm (accessed 05.11.2015.).

[17] Sekai de hajimete hito iPS saibo: kara sanjigen jinzo: sochiki sakusei ni seiko: ~ jinzo: saisei ire: e no tobira o hiraku (World's first 3D kidney tissue grown from human stem cell to pave way for curing kidney diseases by regeneration)// Kumamoto Daigaku URL: http://www.kumamoto-u.ac.jp/whatsnew/seimei/20131213 (accessed 05.11.2015).

[18] Yomiuri Shimbun 03.05. 2013.

[19] Sentan kagakugizutsu simpojiumu iPS saibo ga kirihiraku kongo-no ingakukenkyu (Advanced science and technology symposium ‘Research in medicine of the future opened up by iPS cells)// Keio Daigaku URL: http://www.keio.ac.jp/ja/news/2008/kr7a 43000000nv2u.html (accessed 05.11.2015).

[20] iPS saibo: kihon jo:ho (Basic facts about stem cells)// Kyoto Daigaku URL: http://www.cira.kyoto-u.ac.jp/j/faq/faq2.html (accessed 05.11.2015)