John B. Gurdon and Shinya Yamanaka fortheir pioneering researchon stem
cells.
-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 totransfer 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 embryonicundifferentiated 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 nucleusof 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.
Gurdon's 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
thelining of tadpole intestines (Figure 1). ThusGurdon succeeded
whereBriggs 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
ledhim 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 ofBriggs and
King. "Indeed," Gurdon said in the post-prize announcement
interviewwith 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 frogswere 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 it's entirely reasonable for the skeptics to say,
'well, these well-established people have already done the experiment
andhere is a graduate student from Europe whois disagreeing with them…
why should we pay attention to that?'"
SOURCE: NOBEL FOUNDATION
Figure 1: John gurdon used ultraviolet light to destroy the cell
nucleus in a frog egg (1). He then replaced the cell nucleus with a
differentiated intestinal epithelial cell from a tadpole (2). Many
manipulated eggs did not develop, but in some cases normal swimming
tadpoles were generated(3). This showed that the genetic information
required to generate the differentiated cells in a tadpole remained
intact in the donor cell nucleus. Later studies have shownthat mammals
can also becloned by this technique (4).
Gurdon's 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 SCNThad 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
quiescence—non-dividing state—which 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, afterGurdon's 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 itpossible 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.
Yamanaka's research
Then came Shinya Yamanaka, who believed otherwise. He approached the
problem of reprogramming adult somatic cells systematically.
Interestingly, Yamanaka had started out his careeras an orthopaedic
surgeon but found that "he was not so good at surgery". He felt that,
as asurgeon, he was not able to help many patients. Hewas also
concerned about finding cure for intractable diseases such as the
motor neuron disease amytrophic lateral sclerosis (ALS). Andso he
decided to take up basic medical sciences. Hedid his PhD in
pharmacology. During hispostdoctoral 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 theNara 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 pluripotencyin 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
(iPScells), appeared at a very low frequency.
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