Understanding cells

John B. Gurdon of the United Kingdom.-JUSTIN TALLIS/AFP

The Nobel Prize in Physiology or Medicine for 2012 goes to scientists John B. Gurdon and Shinya Yamanaka for their pioneering research on stem cells.

THE 2012 Nobel Prize in Physiology or Medicine has been awarded to two scientists for discoveries that have led to a paradigm shift in our understanding of how immature cells differentiate and become specialised cells in the body of an organism such as neurons, muscle and skin cellsand the plasticity of the differentiated state. John B. Gurdon, 79, of the Gurdon Institute, Cambridge, United Kingdom, and Shinya Yamanaka, 50, of Kyoto University, Japan, and Gladstone Institutes, San Francisco, California, United States, showed in different experiments that mature, differentiated cells can be reprogrammed to become pluripotent.

Gurdon discovered in 1962 that the specialisation of cells is reversible. Yamanaka, who was born coincidentally in the same year as Gurdons discovery, showed in 2006 how intact mature cells in mice could be reprogrammed by clever introduction of some genes to become immature stem cells. Their findings have revolutionised our understanding of how cells and organisms develop. We now know that differentiation is not a unidirectional process, as was believed earlier, and that mature cells need not remain locked forever in their specialised states. Gurdon and Yamanaka showed that, under some conditions, the clock of cellular development can be turned back.

During normal development, the egg cells and cells in the early embryo proceed from the initial undifferentiated state into a more specialised state by differentiation. In an adult organism, different kinds of differentiated cell types are needed to perform different specialised tasks. The fertilised egg and the cells of the embryo in the earliest stage of its development (the zygote) can give rise to all types of cells in the embryo, as well as extra-embryonic tissues such as the placenta, and hence they are termed totipotent. During the development process, cells at the blastocyst stage begin to be distinguished. The cells in the inner cell massthe embryonic stem (ES) cellsare pluripotent. That is, they can give rise to all somatic cells as well as to cells that become gametes (eggs and sperm).

Cells progressively become restricted in their differentiation potential and thus do not retain their pluripotency. This maturation of cells results in most cells becoming fully differentiated. The process also results in the formation of cells with limited and specialised potencythe somatic or adult stem cells. They remain in certain locations in the body such as bone marrow, intestine, and skin and act as the reservoir for cell maintenance and repair machinery. Differentiated cells are very stable and do not, as a rule, become cells of any other type or revert to the early undifferentiated state. The long-standing dogma in biology, therefore, was that somatic or adult cells were permanently locked in the specialised state of a specific part of the body and that the journey back to the undifferentiated, embryo-like, stem cell state was impossible. The specific pattern of expression of functional proteins in differentiated cells also suggested that these carried irreversible genetic modifications that rendered induction of pluripotency in them impossible.

But the idea that the specialised cells could somehow be made to de-differentiate never completely disappeared. Experimental strategies were attempted at various times to achieve this. Hans Spemann (Nobel Prize 1935) had conceived of what he had called a fantastic experiment in which nuclei from differentiated cells could be transferred into the cytoplasm of an immature cell and its development potential tested.

This basic idea came to be realised in the early 1950s by Robert Briggs and Thomas King, who developed a technique to transfer cell nuclei from both undifferentiated and differentiated cells to an enucleated (from which the nucleus had been removed) fertilised egg in the frog species Rana pipiens. Briggs and King found that the transfer of the embryonic undifferentiated cell nucleus led to the development of the enucleated egg cell into the tadpole stage and that this did not happen in the case of the nucleus of the differentiated cell. Therefore, they concluded that differentiated cells undergo irreversible changes in such a way that their capacity to support development was lost. This only confirmed the prevalent view.

Gurdons research

Enter Gurdon, who had trained in embryology at Oxford. In 1958, as a graduate student, he repeated the Briggs-King experiment but with a different frog family, Xenopus laevis. He enucleated the eggs by ultraviolet radiation and found that a few tadpoles were indeed created when the eggs were transplanted with nuclei from cells from the lining of tadpole intestines (Figure 1). Thus Gurdon succeeded where Briggs and King had failed. He also showed that the efficiency of the process could be greatly increased by performing serial transplantation, through which he could revert the status of a large proportion of all the epithelial cells of the tadpole intestine. This led him to conclude that differentiated somatic cell nuclei had the potential to revert to pluripotency.

So his discovery was not immediately accepted by the scientific community given the results of scientists of the calibre of Briggs and King. Indeed, Gurdon said in the post-prize announcement interview with the Nobel Foundation, there was quite a period after the early work when people did not believe the results. So it took nearly 10 years for the major result to be accepted.

Soon after his major finding Gurdon left his frogs, which he had grown by nuclear transfer, with his supervisor and moved to Caltech where he had taken a post-doctoral position and began to work in a completely unrelated field. The frogs were tended to by his supervisor and a technician. So by 1962, Gurdon recalls, they were adults and one could publish a paper to say that these animals, derived from nuclear transfer, really were absolutely normal. So it took a little time to get through. So its entirely reasonable for the skeptics to say, well, these well-established people have already done the experiment and here is a graduate student from Europe who is disagreeing with them why should we pay attention to that?

Gurdons discovery introduced a new research field based on somatic cell nuclear transfer (SCNT) as a method to understand how cells change as they become specialised and also how this process could be reversed. This formed the basis for the first cloned mammal, the sheep Dolly, by Ian Wilmut and Keith Campbell, which the SCNT had created by turning an adult mammary gland epithelial cell into an enucleated sheep egg. One significant modification that Wilmut and Campbell did was to induce the mammary epithelial cells into quiescencenon-dividing statewhich was found to be better to synchronise with the embryo in the early development phase. Since Dolly, many mammalian species have been cloned using SCNT, including mouse, cow, pig, wolf and African wildcat.

A fundamental question remained, however, after Gurdons path-breaking work. What Gurdon had shown was that a differentiated cell nucleus had the capacity to revert to an undifferentiated pluripotent state. But is it possible to induce this reversal in an intact differentiated cell without any nuclear transfer? This was considered to be impossible or at the very least requiring very complex reorganisation in the cell to unlock the differentiated state.

Yamanakas research

Then came Shinya Yamanaka, who believed otherwise. He approached the problem of reprogramming adult somatic cells systematically. Interestingly, Yamanaka had started out his career as an orthopaedic surgeon but found that he was not so good at surgery. He felt that, as a surgeon, he was not able to help many patients. He was also concerned about finding cure for intractable diseases such as the motor neuron disease amytrophic lateral sclerosis (ALS). And so he decided to take up basic medical sciences. He did his PhD in pharmacology. During his postdoctoral work at the Gladstone Institute in the mid-1990s involving knock-out (KO) mice, he came across embryonic stem (ES) cells. He had identified a new gene that seemed to have significance for cancer. To study that gene, he made a KO mouse and discovered that the gene was very important for pluripotency in mouse ES cells.

Back in Japan, he set up his own laboratory at the Nara Institute of Science and Technology to study this problem of reprogramming. His laboratory focussed on transcription factors expressed in ES cells that were important for maintaining pluripotency in ES cells. From his work and that of the others, it seemed that a large number of factors were responsible. It was also known then that ES cells could induce pluripotency in somatic cell nuclei after induced cell fusions between ES cells and somatic cells. Armed with this knowledge, Yamanaka identified a set of 24 factors as candidates to reinstate pluripotency.

In one of his first experiments, he introduced all 24 genes, encoding these transcription factors into skin fibroblasts (connective tissue cells), and a few of them actually generated cell colonies that resembled ES cells. He whittled down the number of genes one by one to identify finally a combination of only four transcription factors (Myc, Oct3/4, Sox2 and Klf4) that were sufficient to convert mouse embryonic fibroblasts to pluripotent stem cells (Figure 2). These pluripotent stem cells, which he called induced pluripotent stem cells (iPS cells), appeared at a very low frequency.

iPS cells thus derived were found to display the important pluripotency characteristics of ES cells, including the formation of teratomas-containing cells from all three germ layers (endoderm, mesoderm and ectoderm), stem cell markers and being able to differentiate into different tissues when injected into mouse embryos at very early stages of development, including the formation of chimeric mice (mice exhibiting different genetic makeup in patches derived from two or more embryos by experimental intervention). However, in the first study, iPS cell germ line transfer was not achieved. But, a year later, Yamanakas group, in parallel with two other groups working independently, refined the iPS cell selection process and the resulting cells showed germ line transmission. In the landmark paper of 2006, Yamanaka had used a combination of four factors: Myc, Oct4, Sox2 and Klf4.

Yamanakas finding of iPS cells marks a truly fundamental discovery, as it was the first-ever reprogramming of an intact differentiated somatic cell into ES-like pluripotent state. It was based on the principle of gene conservation in differentiation that Gurdons experiment had established. This discovery has opened new vistas of research, and the amazingly simple technology to generate iPS cells is now being used in many laboratories of the world. Stem cell research, prior to Yamanakas discovery, had been troubled by the ethically problematic issue of the use of embryos. Now, use of iPS cells in research, and eventually in therapy, bypasses the ethical issue by obviating the need for embryos and ES cells derived from them.

In his interview, Gurdon, who spends considerable time in his laboratory even at his advanced age, spoke about further possibilities in this direction. He said: From the early point that almost all cells of the body have the same genes, I think it was reasonably clear that given time it should be possible to achieve complete reversal. Once the principle is there, that cells have the same genes, my own personal belief is that we will, in the end, understand everything about how cells actually work.

The technology to generate iPS cells has now improved in many ways. An important development is that the transcription factors can now be delivered without the use of retroviral vectors. This original technique had an inherent problemthe vectors DNA could get integrated randomly in the host genome and cause degradation of nearby endogenous genes and lead to tumour formation. However, recent research using non-integrating adenoviruses or transient plasmids and episomal vectors has shown that genomic integration itself is not necessary for reprogramming to occur. Similarly one of the transfected factors is a potentially cancer-causing gene. We are closer to overcoming these major hurdles, Yamanaka said in an interview he had given earlier this year to Frontline (May 18 and June 1).

Stem cells, including iPS cells, can be potentially used to replace diseased or lost cells in degenerative disorders such as Parkinsons disease and Type 1 diabetes. The potential therapeutic use of iPS cells in cell replacement therapy will allow autologous cell grafting that would be devoid of the problem of immune rejection. iPS cells can be derived from a patients own cells. This is called regenerative medicine.

However, as Yamanaka pointed out in his Frontline interview, there is the possibility of mutations getting introduced in the iPS cells, which would render them unsuitable for cell therapy. Still a lot of work needs to be done to realise the prospect of using pluripotent iPS cells in cell therapy. From this perspective, an important immediate application of iPS cells is, however, disease modelling . iPS cells derived from patients with genetic and other disorders can be used to study their differentiation in vitro so that new insights can be gained into the disease process and appropriate therapeutic intervention developed (Figure 3). Also iPS cell-base platforms could be designed for toxicology testing and drug development for such disorders. Thus iPS cell-based in vitro differentiated cells are also increasingly being used as screening platforms for the development and validation of therapeutic compounds. These techniques are being used for a large number diseases such as ALS, Rett syndrome, spinal muscular atrophy (SMA), spinal cord injury, Alzheimers disease and other neurogenic disorders.

When asked after the prize announcement as to what was his greatest hope for stem cell technologies at the moment, Yamanaka said, I will bring this technology to clinics. I really want to help as many patients as possible.I still feel I am a doctor, I am a physician, so I really want to help patients. So my goal, all my life, is to bring this stem cell technology to the bedside, to patients, to clinics.