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Mature cells can be reprogrammed to become pluripotent – John Gurdon and Shinya Yamanaka

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Innovation

Series E: 2; 7.1

In 1962, John B. Gurdon successfully cloned frogs. He took the nucleus of an adult frog cell – the part of the cell that holds the DNA – and put it into a frog egg cell. The egg was able to develop into a normal tadpole. These experiments showed that an adult, specialised cell still had the information needed to form a new tadpole. The same technique was later used to produce the famous cloned sheep, Dolly.

In 2006, Shinya Yamanaka’s work again took the scientific community by surprise and changed the way researchers think about how cells develop.Yamanaka showed that adult, fully specialised mouse cells could be reprogrammed to become cells that behave like embryonic stem cells – so-called induced pluripotent stem cells, which can develop into all types of cells in the body.

Gurdon and Yamanaka’s work is celebrated and explained in the award-winning documentary, Stem Cell Revolutions, by Clare Blackburn and Amy Hardie. The short clip above is taken from the film and links Gurdon and Yamanaka’s work (click the red button on the image above to watch the clip). Amy Hardie, who directed the film, commented: “So many scientists have said that Shinya Yamanaka has overturned our understanding of basic developmental biology. And he has – with the discovery of iPS cells. What Shinya Yamanaka himself points out and we were able to show in our film, Stem Cell Revolutions, is the lineage from John Gurdon who cloned frogs in Cambridge. Shinya’s groundbreaking discovery would not have been possible without Gurdon’s pioneering work.

Proc Natl Acad Sci U S A. 2013 Apr 9; 110(15): 5740–5741.

Published online 2013 Mar 28. doi:  10.1073/pnas.1221823110

Sir John Bertrand Gurdon, FRS, FMedSci (born 2 October 1933), is an English developmental biologist. He is best known for his pioneering research in nuclear transplantation^[2][3][4] and cloning.^[1][5][6][7] He was awarded the Lasker Award in 2009. In 2012, he and Shinya Yamanaka were awarded the Nobel Prize for Physiology or Medicine for the discovery that mature cells can be converted to stem cells.^[8]

The Nobel Prize in Physiology or Medicine 2012
Sir John B. Gurdon, Shinya Yamanaka

ohn Bertrand Gurdon (JBG), born 2 October 1933, was brought up in a comfortable home by his parents (fig.1) on the Surrey/Hampshire border in a village, Frensham in South England, endowed with a large amount of National Trust heathland and ponds. His mother, Marjorie Byass, was from an East Yorkshire farming family. Brought up on a farm, and educated in that region, she became a physical training teacher working for some time in an American private school. When her son and daughter (Caroline, who trained as a nurse) had been raised, she gave much time to the regional administration of the “Women’s Institute,” a voluntary organisation for educating women.

His father, William Gurdon, was from a longstanding Suffolk family whose ancestors go back to 1199 (fig. 2; Muskett, 1900; Cunnington, 2008); with the family motto “virtus viget in arduis” [virtue flourishes in adversity].

Figure 2. Paternal lineage of JBG.

Many of them had distinguished careers in government and as regional administrators, including Sir Adam Gurdon [Muskett, 1900]. JBG’s ancestors lived in a stately home, Assington Hall, in West Suffolk (fig. 3).

Figure 3. Assington Hall, Suffolk. Burnt down in 1957.

His grandfather had to leave the family home through lack of money to maintain it, due to repeal of the Corn Laws (1846) so that tenant farmers could no longer pay their rent, because of foreign imports. Assington Hall was requisitioned by the army during World War II, and was burnt down in a supposedly accidental fire in 1957. The remaining part of the house was partly restored and part of the original home, including its minarets, is still present in Assington. One of JBG’s ancestors married again after his first wife died and the outcome of a second marriage yielded a distinguished lawyer who accepted the hereditary title of Baron Cranworth. JBG’s father left school at the age of 16 and took a position in a rice broking firm in Burma. He was an early volunteer in the First World War and was decorated with the Distinguished Conduct Medal (DCM) before being commissioned to an officer rank. After that he led a career in banking in Assam and East India. He retired, in his forties, and in retirement, he gave much time to the transcribing of professional textbooks (especially legal) into Braille for the blind as voluntary work.

World War II started in 1939 when JBG was aged six. It was a time of austerity. Limited rations of food were managed by his mother, and the garden was used to raise chickens. He did not see luxuries like a banana or an orange until well after the end of the war. At the age of eight he was sent to a local private school, Frensham Heights. In an intelligence test at that age, he was asked to draw an orange. He started drawing the stalk by which the orange would hang from a tree, reasoning that an orange would not exist in space. The teacher tore up the piece of paper and reported to his parents that he was mentally subnormal and would need special teaching. The teacher meant to say, draw a circle. He was moved to another private school in the village, namely Edgeborough, where he thrived. At that age he had an intense interest in plants and insects. In most of his spare time he collected butterflies and moths and raised their caterpillars.

At the age of 13, he started school at Eton as a boarder. He found life there intensely uncomfortable, because senior boys acted as despots, administering punishments for trivial misdemeanours. As a means of survival, he took up squash, and as a result of hard work rather than ability, he became eventually the school captain in this sport. While at school he continued his interest in Lepidoptera, raising large numbers of moths from their larval stage.

Gurdon attended Edgeborough and then Eton College, where he ranked last out of the 250 boys in his year group at biology, and was in the bottom set in every other science subject. A schoolmaster wrote a report stating “I believe he has ideas about becoming a scientist; on his present showing this is quite ridiculous.”^[9] Gurdon explains it is the only document he ever framed; Gurdon also told a reporter “When you have problems like an experiment doesn’t work, which often happens, it’s nice to remind yourself that perhaps after all you are not so good at this job and the schoolmaster may have been right.”^[10]

It was during his first term of being taught Science at the school, at the age of 15, that he received a totally damning report from the Biology master (fig. 4). This report resulted from JBG being placed in the bottom position of the lowest form in a group of 250 students of the same age. The report, sent to his housemaster, resulted in him being taken off any further study of Science of any kind at the school. For the rest of his school days, for the next three years, he was given no Science teaching and was placed in a class which studied Ancient Greek, Latin and a modern language, a course intended for those judged to be unsuited for studying any subject in depth.

Entrance to University was a problem: having sat the Entrance examination in Latin and Greek, the Admissions tutor at Christ Church Oxford University told JBG that he would be accepted for Entrance on condition that he did not plan to study the subject in which he took the Entrance (Classics). Later the Admissions tutor admitted that he had under-filled the college and had his mind on other things; he was Hugh Trevor-Roper, later Lord Dacre, and author of The Last Days of Hitler. In due course it emerged that JBG’s acceptance for Christ Church involved a complicated arrangement between JBG’s uncle, at that time a Fellow of Christ Church, JBG’s school housemaster and a friend of his uncle, Sir John Masterman, who was Master of Worcester College, Oxford and in charge of the wartime Enigma operation at Bletchley, agreeing to accept the housemaster’s son. Such a manoeuvre, and admission to Oxford on those terms, could never happen now. At that time, 1952, it was not very easy to fill a college with paying students. Before entering University, JBG had to take a year off to learn elementary Biology with a private tutor, generously funded by his parents who had already paid several years of Eton fees. He was told that he could formally enter the Department of Zoology course at Oxford if he passed the elementary exams in Physics, Chemistry and Biology in a preliminary year. He survived this and started the course in Zoology at Oxford in 1953. The course was extremely oldfashioned, by today’s standards. A major part of the teaching involved learning Palaeontology, and the names of skeletal parts of dinosaurs. JBG later became a personal friend of Sir Alister Hardy, the Head of that department, through his Oxford aunt (see later).

As the Zoology course came to an end, JBG enquired about the possibility of doing a PhD in Entomology, in accord with his continuing interest in insects. While still a student, he had got permission to go to Oxford University’s nature reserve, namely Wytham Woods, with his butterfly net. No butterflies were to be seen, but he caught the only moving thing, which was a kind of fly. He used the taxonomic reference works to try to identify this “fly.” Having realised that the fly was a Hymenopteron, he was still unable to identify it. He therefore went to the Natural History Museum in London for help. They pronounced that it was in fact a species of sawfly new to Britain. This must have been intensely irritating to the Professor of Entomology, whose main research project was to identify animals and plants in Wytham Woods. JBG was later rejected for PhD work in Entomology. This was a great blessing because the work he would have done in Entomology was not well regarded and had very little, if any, analytical component to it. By his immense good fortune, he was invited to do a PhD with the Oxford University lecturer who taught Developmental Biology, Dr Michael Fischberg.

Fischberg was born in St Petersburg, Russia, in 1919. He was educated in Switzerland and was a PhD student of E. Hadorn. Hadorn in turn was a student of F. Baltzer, who was a student of H. Spemann, himself a student of T. Boveri. This German-Swiss lineage of eminent Developmental Biologists turns out to be the background of a great many of the successful Developmental Biologists of the mid-1950s. Most of those that did not have this background can trace their own training back to R. G. Harrison (1870–1959) of the USA, who pioneered cell culture. Having finished his PhD with Hadorn, Fischberg took a position in the Institute of Animal Genetics under Waddington in Edinburgh, from where he accepted his appointment in the Oxford Zoology department, headed by Professor Sir Alister Hardy, an eminent marine biologist [Royal Society memoirs].

Starting his PhD work in 1956, Fischberg suggested to JBG that he should try to carry out somatic cell nuclear transfer in Xenopus, a procedure for this having been recently published by Briggs and King (1952). The advisability and technical problems that arose at this point are described in the accompanying papers (Gurdon 2013 a,b). Once these technical obstacles had been overcome, largely as a result of good luck, JBG’s work proceeded extraordinarily fast; strongly motivated by early success, he became an intensely hard worker. By the end of his PhD he had succeeded in obtaining normal development of intestinal epithelium cell nuclei transplanted to enucleated eggs of Xenopus. When these tadpoles had eventually reached sexual maturity, he was able to publish a paper entitled “Fertile intestine nuclei.”This was the first decisive evidence that all cells of the body contain the same complete set of genes. This answered a long-standing and important question in the field of Developmental Biology. However it also showed very clearly, as was commented on in JBG’s papers at the time, the remarkable ability of eggs to reprogram somatic cell nuclei back to an embryonic state. Eventually this phenomenon attracted increasingly large interest, and led to the idea of cell replacement using accessible adult cells, such as skin. A key future discovery was that of Martin Evans (Nobel Prize, 2006) that a permanently proliferating embryonic stem cell line could be established from mouse embryos. Under appropriate conditions these cells could be caused to differentiate into all different cell types. The combination of somatic cell nuclear transfer and the derivation of embryonic stem cells in mammals made it realistic to think of cell replacement for human diseases. A huge boost for this idea was later provided by Takahashi and Yamanaka (2006), with their discovery that the overexpression of certain transcription factors can also yield embryonic stem cells from adult somatic tissue. The accompanying Nobel lecture provides more detail of the later scientific part of JBG’s career.

A visit by the Nobel Laureate George Beadle to the Fischberg Group in the Oxford Zoology department in 1960 led to an offer from the California Institute of Technology (CalTech) (previous chairman George Beadle) for JBG to do postdoctoral work there. Fischberg very wisely advised JBG to accept the CalTech offer of postdoctoral work rather than offers from other nuclear transplant labs. Stimulated by his mother’s adventurous spirit, JBG decided to buy a secondhand Chevrolet in New York and drive across the USA to California, using the famous Route 66 (now replaced). He gave lectures as he travelled across the USA and stopped at laboratories of Briggs and King, Alexander Brink (paramutation) etc. He had hoped to become a post-doctoral student of R. Dulbecco at CalTech (Nobel Prize), but the chairman of that department advised against this because JBG had no training in virology. Therefore JBG did his postdoctoral work with Robert Edgar on Bacteriophage Genetics. JBG found he had no aptitude at all for Phage Genetics and decided to return to Britain after one year at CalTech. Nevertheless, that year at CalTech was extremely formative because it provided some acquaintance with Molecular Biology, which had so far entirely escaped his training. During that year he met Sturtevant, a student of Morgan, who pioneered the whole field of Drosophila Genetics. He also got to know Ed Lewis (future Nobel Laureate). Thanks to James Ebert (director of the Department of Embryology, Carnegie Institute of Washington, in Baltimore) JBG visited various labs in the USA at the end of his post-doctoral period and met Donald Brown in Baltimore on that visit. Meantime, the success of the nuclear transfer work in Oxford had led to Michael Fischberg being offered a head of department professorship in Geneva, Switzerland. JBG was offered the teaching position in Oxford vacated by M. Fischberg. JBG returned from California to England via Japan and many other countries over a two-month period. One month of that time he spent in Japan and met Tokindo Okada and made other friends in Japan, including M. Furusawa and subsequently Koichiro Shiokawa.

While doing graduate and postdoctoral work in Oxford, JBG made other contacts and friendships. His mother’s sister lived in Oxford, and he spent much time at her house and visiting famous gardens, fostering a lifelong interest in plants. Through that connection he met Miriam Rothschild, and became a lifelong friend of hers (Van Emden and Gurdon, 2006). This friendship contained, through Miriam Rothschild’s generosity, ski mountaineering holidays based in her house in Wengen. JBG had achieved the British ski club’s Gold standard ski medal, again through relentless practice rather than any natural ability. Also, in accord with his interest in the open air and dogged determination, he became a reasonably accomplished ice figure skater.

Nobel Lecture by Sir John B. Gurdon (42 minutes)

Sir John B. Gurdon delivered his Nobel Lecture on 7 December 2012 at Karolinska Institutet in Stockholm. He was introduced by Professor Urban Lendahl, Chairman of the Nobel Committee for Physiology or Medicine.
Credits: Sveriges Television AB (production)

Copyright © Nobel Media AB 2012

The Nobel Prize in Physiology or Medicine 2012    Lecture (pdf)

Nuclear transfer

In 1958, Gurdon, then at the University of Oxford, successfully cloned a frog using intact nuclei from the somatic cells of a Xenopus tadpole.[14][15] This work was an important extension of work of Briggs and King in 1952 on transplanting nuclei from embryonic blastula cells[16] and the successful induction of polyploidy in fish Stickleback, Gasterosteus aculatus, in 1956 by Har Swarup reported in Nature.[17] However, he could not yet conclusively show that the transplanted nuclei derived from a fully differentiated cell. This was finally shown in 1975 by a group working at the Basel Institute for Immunology in Switzerland.[18] They transplanted a nucleus from an antibody-producing lymphocyte (proof that it was fully differentiated) into an enucleated egg and obtained living tadpoles.

Gurdon’s experiments captured the attention of the scientific community and the tools and techniques he developed for nuclear transfer are still used today. The term clone[19] (from the ancient Greek word κλών (klōn, “twig”)) had already been in use since the beginning of the 20th century in reference to plants. In 1963 the British biologist J. B. S. Haldane, in describing Gurdon’s results, became one of the first to use the word “clone” in reference to animals.

Messenger RNA expression

Gurdon and colleagues also pioneered the use of Xenopus (genus of highly aquatic frog) eggs and oocytes to translate microinjected messenger RNA molecules,[20] a technique which has been widely used to identify the proteins encoded and to study their function.

Recent research

Gurdon’s recent research has focused on analysing intercellular signalling factors involved in cell differentiation, and on elucidating the mechanisms involved in reprogramming the nucleus in transplantation experiments, including the role of histone variants,[21][22] and demethylation of the transplanted DNA.[23]

Reprogramming of Mature Cells

Our lives begin when a fertilized egg divides and forms new cells that, in turn, also divide. These cells are identical in the beginning, but become increasingly varied over time. As a result of this process, our cells become specialized for their location in the body – perhaps in a nerve, a muscle, or a kidney. It was long thought that a mature or specialized cell could not return to an immature state, but this has been proven incorrect.

In 1962, John Gurdon removed the nucleus of a fertilized egg cell from a frog and replaced it with the nucleus of a mature cell taken from a tadpole’s intestine. This modified egg cell grew into a new frog, proving that the mature cell still contained the genetic information needed to form all types of cells. In 2006, Shinya Yamanaka succeeded in identifying a small number of genes within the genome of mice that proved decisive in this process. When activated, skin cells from mice could be reprogrammed to immature stem cells, which, in turn, can grow into all types of cells within the body. In the long-term, these discoveries may lead to new medical treatments.

Shinya Yamanaka

A winding road to pluripotency

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/yamanaka-lecture.pdf

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/ypdfamanaka-lecture_slides.

Nobel Lecture

A scandal involving Japanese stem-cell research took a surprising turn Monday when the nation’s most revered researcher in the field, Nobel Prize laureate Shinya Yamanaka, apologized for what he described as poor record-keeping.

The apology came after months of soul-searching in Japan over research ethics. A researcher at the prestigious Riken institute, Haruko Obokata, apologized earlier this month after admitting errors in a paper in the journal Nature that described a possible new method of creating stem cells.

Last week, the head of the Riken panel investigating Dr. Obokata had to resign from the panel after admitting that a paper he co-authored used some of the same improper methods of cutting and pasting images that he had criticized in Dr. Obokata’s work.

On Monday evening, Dr. Yamanaka, a professor at Kyoto University, spoke at a news conference after questions arose about an image in a 2000 paper on which he was the lead author. In the paper, Dr. Yamanaka, then at Nara University, described a protein that played a role in turning embryo cells into cells specific to a part of the body.

The university said it conducted an investigation after Dr. Yamanaka informed administrators about allegations he discovered online that an image in the paper was doctored.