Yamanaka Shinya’s research history

This failed orthopedic surgeon was finally recruited by Thomas Innerarity of the Gladstone Institute in California (Figure 1). Thomas' laboratory studies blood lipid regulation, which is somewhat related to Shinya's work during his period. Shinya's new topic is to study the editing protein ApoBEC1 of ApoB mRNA.

ApoB is the main component of low-density lipoprotein. ApoB mRNA can be deaminated by the editing enzyme ApoBEC1 to prematurely terminate translation, forming two proteins of different sizes: full-length ApoB100 and approximately half-length ApoB48. Edited ApoB48 is rapidly cleared from plasma. Thomas predicts that if ApoBEC1 is overexpressed in the liver, blood lipids may be reduced; if this model is feasible, gene therapy may be able to help some obese patients reduce blood lipids in the future.

Shinya worked diligently seven days a week and spent six months making the transgenic mice. One morning, the technician who helped him maintain the mice told him: Shinya, many of your mice are pregnant, but the mice are male. Shinya says you are not kidding me. When he went to the rat room, he found that many male rats looked pregnant. He killed a few of them and found that the mice had liver cancer, and their livers were enlarged and their bellies were enlarged.

After ApoBEC1 overexpression, low-density lipoprotein decreased, but high-density lipoprotein increased. At the same time, liver cancer was also diagnosed. This transaction is not worthwhile. Shinya summarized the lessons learned in a lecture: first, science is unpredictable; second, do not try new gene treatments on patients; third, and perhaps most importantly, do not believe your mentor’s hypothesis.

Thomas was disappointed that the results did not meet expectations, but this unexpected result aroused Shinya's curiosity: What is the mechanism that causes mice to develop tumors? Fortunately, Thomas was open-minded enough and allowed Shinya to deviate from the main direction of the laboratory and continue to explore the carcinogenic mechanism of ApoBEC1. It is conceivable that overexpression of ApoBEC1 may also edit other mRNAs besides ApoB. Finding these mRNAs may explain why ApoBEC1 can cause cancer.

Since ApoBEC1 is known to need to recognize the specific sequence of the substrate mRNA in order to edit it, Shinya designed primer amplification accordingly and found a new substrate of ApoBEC1 - Nat1, a gene that inhibits protein translation. After ApoBEC1 overexpression, Nat1 protein disappears. Logically, if editing Nat1 is an important molecule that causes ApoBEC1 to cause cancer, then Nat1 knockout mice would also develop cancer.

Gene knockout is more complicated than transgene, and requires the constructed plasmid to be integrated in situ into embryonic stem cells cultured in vitro. Isn't gene knockout technology the technology that Shinya dreamed of learning during his Ph.D. So Shinya found Robert Farese, an expert in gene knockout at the institute who was an assistant professor at the time, and learned every detail of this technology from his assistant Heather Myers, and successfully obtained Nat1 knockout heterozygous mice. Heather Myers is Shinya's lifelong friend; after Shinya discovered iPS, she also publicly expressed her gratitude to Heather Myers, because it was she who told Shinya that embryonic stem cells are not only a means of knocking out mice, but can also be very interesting in themselves. Research object.

While Shinya continued to ask about the function of Nat1 enthusiastically, his wife left him and returned to Japan with their daughter. Half a year later, he decided to discontinue his research and took three precious Nat1 heterozygous mice with him and returned to China with his family.

The Caterpillar Stage in Osaka-Nat1

With the four high-quality first-author papers he published during his postdoctoral period, Shinya found a position as an assistant professor at his alma mater, Osaka City University, in 1996 , continues his Nat1 research.

Once again, it deviated from the prediction: after Nat1 knockout, homozygous mice died early in embryonic development, and it was impossible to observe whether adult mice developed tumors. Shinya further studied and found that embryonic stem cells with Nat1 knocked out could not differentiate like normal stem cells in vitro. At this time, he remembered the words of Heather Myers: Embryonic stem cells are not only a tool for research, they can also be very interesting research objects in their own right. His focus began to shift to embryonic stem cells.

In the first few years after returning to Osaka, Shinya could only get a small amount of research funding because he was just starting out. He had to raise hundreds of mice by himself, and his life was very difficult. At the same time, the basic research at Osaka City University School of Medicine is very weak. People around her do not understand the significance of Shinya's research on the function of Nat1 in embryonic stem cells, and always persuade Shinya to do research closer to medical clinical aspects. Nat1’s research paper has been rejected after it was submitted to the magazine. Due to all kinds of pressure and frustration, Shinya contracted a disease called PAD (Post America Depression, self-invented joke) and almost gave up scientific research and returned to China to become an orthopedic surgeon.

At his lowest point, two things saved him from PAD. One is James Thomson (Yu Junying’s mentor, who announced the creation of human iPS almost at the same time as Shinya in 2007). In 1998, he announced that he had collected and established embryonic stem cell lines from human blastocysts: these stem cells were cultured in vitro for several months. Later, it can also differentiate into cells of different germ layers, such as intestinal epithelial cells, chondrocytes, neuroepithelial cells, etc. This gave Shinya great encouragement, and he became more convinced that embryonic stem cell research was meaningful and would one day be used clinically. The second thing was that the Nara Graduate School of Advanced Science and Technology, which had better conditions, took a fancy to his expertise, recruited him to establish a facility for gene knockout mice, and offered him the position of associate professor.

Nara’s pupa stage-Fbx15

After shedding several layers of skin after all the hard work, Shinya finally has her own independent laboratory. It’s so cool to be able to recruit helpers for the first time. But the problem arises again: the source of graduate students is limited, and students will tend to choose laboratories with older qualifications and better conditions, rather than necessarily laboratories that are just starting up; you want to recruit but they don't come. In order to attract students to his laboratory, Shinya thought hard for a while and proposed an ambitious plan, claiming that the long-term goal of the laboratory is to study how to transform terminally differentiated adult cells back into pluripotent stem cells.

The mainstream in the scientific community at that time was to study how to differentiate embryonic pluripotent stem cells into cells of various tissues, in the hope of using these differentiated functional cells to replace damaged or diseased tissue cells. Shinya believes that his laboratory does not have the strength to compete with these giants, so it is better to do the opposite and study how to reverse differentiated cells into pluripotent stem cells.

The mainstream view in the scientific community at the time was that cell differentiation during mammalian embryonic development was unidirectional, as if time was irreversible. This view is not without its flaws. For example, plant tissues are pluripotent. Some plants' stems will grow into a new plant when inserted into the soil. That is, differentiated stem cells can change their fate and differentiate into new root, stem, and leaf cells. As early as 1962, the year Shinya was born, Sir John Gurdon of the United Kingdom (who shared the Nobel Prize with Shinya) reported his amazing discovery: transplanting tadpole intestinal cell nuclei into enucleated frog eggs. , new cells can develop into tadpoles. If the heterozygous cells are developed to the blastocyst stage and the nuclei of the blastocyst stage cells are used for nuclear transplantation, adult frogs that can be reproduced can be developed.

Furthermore, in order to convince people to accept that terminally differentiated cell nuclei also have pluripotency, he cultured cells from different tissues of adult frogs in vitro and found that after nuclear transplantation, hybrid cells from different sources can develop to the tadpole stage. In 1997, based on the same principle, Ian Wilmut and Keith Campbell transplanted sheep mammary cell nuclei into enucleated sheep eggs and successfully created the cloned sheep Dolly. In 2001, scientists discovered that thymocyte nuclei were reprogrammed to a large extent through fusion with stem cells.

The first step of Shinya's plan is to find as many factors as possible that are similar to Nat1 and are involved in maintaining stem cell function (maintenance factors mean that these factors are necessary for embryonic stem cells to maintain pluripotency in vitro culture ). He boldly speculated that overexpression of these maintenance factors might allow terminally differentiated cells to revert to pluripotent stem cells. Once successful, induced pluripotent stem cells will have advantages that embryonic stem cells do not have: not only can they circumvent the ethical issues caused by embryonic stem cells, but when the patient's own induced stem cells are modified and re-implanted into the patient, since they are their own cells, they will There will be no problem of immune rejection.

Inspired by this great prospect, Shinya indeed "fooled" three students to join his laboratory. Soon, they identified a series of genes specifically expressed in embryonic stem cells. One of these genes is Fbx15. Shinya's student Yoshimi Tokuzawa discovered that in addition to being specifically expressed in embryonic stem cells, Fbx15 can also be directly regulated by two other embryonic stem cell maintenance factors, Oct3/4 and Sox2. Shinya told Yoshimi: Fbx15 should be involved in maintaining stem cell pluripotency and embryonic development. I guess you can't get Fbx15 knockout homozygous mice. Yoshimi constructed plasmids to create knockout mice, replacing the Fbx15 gene on the chromosome with the gene neo that resists the G418 drug through homologous recombination.

Complex life once again fooled Shinya: Fbx15 knockout homozygous mice lived healthy and had no obvious phenotype. Shinya challenged his students again: Well, Fbx15 may not be required for mouse embryonic development, but it should be required for the maintenance of embryonic stem cells in vitro. I bet you can't completely knock out this gene in embryonic stem cells. The diligent Yoshimi used a higher concentration of G418 to screen out homozygous knockout strains from stem cells, which were still alive and well and had no phenotype. Shinya later joked when recalling: The mice were very happy, and the cells were also very happy. The only one who was not happy was the poor student Yoshimi.

But the knockout mice that took so much effort to create cannot just be forgotten. Shinya once again used his brain to recycle waste. He found that because Fbx15 is only expressed in embryonic stem cells, the drug-resistant gene neo controlled by the Fbx15 promoter is not expressed in adult fibroblasts, so the cells are sensitive to the drug G418; however, embryonic stem cells obtained from knockout mice can react at very high concentrations. grown in G418. If terminally differentiated fibroblasts can be induced into embryonic stem cells, they will become resistant to G418. Even if fibroblasts acquire only some of the properties of embryonic stem cells, they should still be resistant to low concentrations of G418 (Figure 2). Fbx15 knockout mice actually provide a good system for screening induced stem cells! Thanks to his outstanding work in identifying embryonic stem cell maintenance factors, Shinya found a new position at the more prestigious Kyoto University in 2004. In addition to the Fbx15 knockout mouse screening system, Shinya also accumulated 24 maintenance factors that he identified and reported in the literature. Shinya is eager to try, he is ready to break out of the shell and flap his wings to become a butterfly!

Another student of Shinya, Kazutoshi Takahashi, has previously published a Nature article on the carcinogenicity of stem cells.

Shinya decided to let him take on the most daring subject - reverse differentiation of somatic cells, because he knew that with a Nature article as a guarantee, even if nothing was achieved in the next few years, his students would be able to bear it.

Even with a good screening system, this topic seemed very risky or even unfeasible at first. People at that time generally believed that adult cells had lost their pluripotency. Perhaps the adult cells themselves were irreversible and nothing you did would help. Even if the reversal of the nuclear fate of adult cells is achieved through nucleation technology, it is only the nucleus, not the entire cell. The chromosomes of embryonic cells and adult cells are the same, and the nucleus is totipotent, which is understandable. Moreover, to achieve the reversal of the nucleus, it needs to be transferred to the egg cell, so that the egg cytoplasm can help it reprogram, and there are countless proteins in the egg cytoplasm. To achieve reversal of the fate of the entire cell, all proteins in the cytoplasm need to be reshuffled. Even if cells can be reprogrammed, many proteins should be involved simultaneously. Shinya only had 24 factors in his hands back then. There may be hundreds or thousands of other factors that are missing, and reprogramming is impossible without even one of them. Using these 24 factors to achieve cell reprogramming in a whimsical way is logically impossible based on existing knowledge.

Kazutoshi, a stupid young man, didn't care about this. He infected fibroblasts one by one with viruses that overexpressed these factors. Of course, no cells resistant to G418 were screened out. Shinya knew how to keep students motivated. He pretended to be calm and said: "Look, this shows that our screening system is very good. There are no false positives."

After trying to no avail, Kazutoshi boldly proposed that he want to mix 24 viruses to infect cells at the same time. Shinya thinks this is a stupid idea: No one has ever done this, classmate, but it’s like treating a dead horse like a living horse. If you’re not tired, go ahead and try it.

After waiting for a few days, a miracle happened. There were more than a dozen anti-G418 cell clones sparsely appearing on the culture plate! An epoch-making discovery was made.

After the breakthrough in the key experiment, everything else fell into place. Kazutoshi removed one virus at a time and mixed the remaining 23 viruses to infect adult cells to see how many clones could be grown to identify which factors were necessary to induce stem cells. Finally, he identified four star factors: Oct3/4, Sox2, c-Myc, and Klf4. Overexpression of these four factors in fibroblasts is enough to reverse them into pluripotent stem cells!

Are the G418-resistant cell clones necessarily pluripotent stem cells? Through a series of indicators, such as gene expression profiles, differentiation potential, etc., they found that these cells are similar to embryonic stem cells to a considerable extent.

In 2006, Shinya reported induced stem cells in mice, causing a sensation in the scientific community [13]; in 2007, he also achieved reversal of cell fate in human cells, which caused a stir in the scientific community [14]. Looking back, how could Shinya be so lucky to succeed despite all the impossibilities? Through more research, we know that the maintenance of stem cell characteristics is coordinated by a gene network. By upregulating certain key genes, this network can be rebuilt and the fate of the cell reversed; the four finally identified by Yamanaka Shinya Factors are not necessary, and the same purpose can be achieved by combining factors other than the 24 factors. This is like a big net. As long as you can hold up a few of the fulcrums, you can hold up the entire net.

The discovery of iPS has unusual significance. First of all, it has updated people's concepts. From now on, people no longer believe that the fate of cells is irreversible. Not only can it be reversed, but cells can actually achieve transdifferentiation between different tissues. Secondly, iPS cells bypass the ethical dilemma of embryonic stem cells. Many laboratories can repeat this simple experiment to obtain iPS cells and conduct research on pluripotent stem cells.

Third, iPS cells have many advantages that embryonic stem cells do not have: if the patient's own iPS cells are manipulated in vitro and re-implanted into the patient, the immune response will be greatly reduced; if the patient's somatic cells are reversed into ips cells and differentiated in vitro By observing the problems that arise during this process, it is possible to simulate the occurrence of the disease to some extent in a petri dish; after disease-specific iPS are expanded and differentiated in vitro, they can also be used to screen drugs to treat the disease, or to Drug toxicity is tested.

But this is just a new beginning. Life science is so complex and unpredictable. There are still many difficulties in turning these visions into reality and making iPS truly benefit mankind. Shinya Yamanka, the darling of science, fearlessly embarked on a new journey with the original ideal of helping more patients.