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Colloquium
Nuclear reprogramming and stem cell creation
J. B. Gurdon*, J. A. Byrne, and S. Simonsson
Wellcome Trust/Cancer Research UK Institute, Tennis Court Road, Cambridge CB2 1QR, United Kingdom; and Department of Zoology, University of
Cambridge, Cambridge CB2 3EJ, United Kingdom
The transplantation of a somatic cell nucleus to an enucleated egg
results in a major reprogramming of gene expression and switch in
cell fate. We review the efficiency of nuclear reprogramming by
nuclear transfer. The serial transplantation of nuclei from defective
first-transfer embryos and the grafting of cells from such embryos
to normal host embryos greatly increases the proportion of nuclei
that can be seen to have been reprogrammed. We discuss possible
reasons for the early failure of most nuclear transfers from differ-
entiated cells and describe the potential value of growing oocytes,
rather than unfertilized eggs, as a source of nuclear reprogram-
ming molecules and for the eventual identification of these mol-
ecules. Nuclear transfer provides a possible route for the creation
of stem cells from adult somatic cells.
Nuclear reprogramming is a term used to describe changes in
gene activity that are induced experimentally by introducing
nuclei into a new cytoplasmic environment. When nuclei from
partially or fully differentiated cells are transplanted to enucle-
ated eggs of Amphibia or mammals in second meiotic meta-
phase, blastula or blastocyst embryos can be obtained, and these
can form a wide range of tissues and cell types. The multipotency
of these nuclear transplant embryos means that they share some
characteristics of early stem cells. Indeed those nuclear trans-
plant embryos that undergo growth (after feeding in Amphibia,
and after implantation in mammals) to become adults must
contain stem cells for renewing tissues. To this extent nuclear
transplantation can achieve the creation of stem cells or stem-
like cells from somatic cells of very restricted developmental
potential.
In contrast, the identification and isolation of natural stem
cells from normal tissues is a difficult process and has not yet
been successful for most vertebrate tissues (1~. Furthermore the
differentiated state of cells is very stable, and it is hard to induce
cells that have embarked on one pathway of differentiation to
switch to another. Therefore nuclear transplantation is at
present the most reliable way of deriving multipotential cells
from a tissue of any kind. For this reason, nuclear reprogram-
ming is of interest as a means of creating a range of replacement
cells of the same genetic type as the donor source, thereby
avoiding the need for immunosuppression as is required with
most genetically nonhomologous grafts or implants. The aim of
this article is to summarize the efficiency of nuclear reprogram-
ming by nuclear transfer and hence to comment on its potential
as a source of stem cells.
Nuclear Transplant Embryo Development
How efficiently and effectively can somatic cell nuclei be repro-
grammed to an embryonic state? In the case of Amphibia, these
questions have been addressed by the early nuclear transfer
experiments carried out with Rana and Xenopus. For reasons
that are still not clear, nuclear transfer success declines rapidly
with increasing donor age in Rana (2), and nuclear transplant
development is much more successful in Xenopus. In the
endoderm lineage, which has been analyzed in greatest detail (3,
4), it is now known that cells express the endoderm-lineage
www.pnas.org/cgi/doi/10.1 073/pnas.1 834207100
marker endodermin (5) from the late neurula stage onward.
Well before this stage, endoderm cells are specified [i.e., they
form only endoderm derivatives as explants (6, 7~] and are
determined ti.e., they form only endoderm derivatives if trans-
planted to ectopic sites (844. Yet nuclei transplanted from more
advanced-stage endoderm cells into unfertilized eggs form func-
tional muscle and nerve cells in ~20% of all cases (Table 1~.
Even at the heartbeat stage, when the endoderm has begun
regional differentiation (9, 10), 13% of nuclear transfers from
the endoderm can form functional muscle and nerve cells (Table
1~. Likewise, the region of the mesoderm destined to form
muscle expresses the myogenic genes MyfS and MyoD by the late
gastrula stage (11, 12), and cells from this region continue to
express muscle markers even when these cells are transplanted
singly to the endoderm (134. Yet the nuclei of myogenic cells
generate a functional nervous system in >5% of nuclear trans-
plants. These results cannot be attributed to escaped germ cells
or other rare cell types residing in the endoderm or muscle,
because the success rate is too high.
Using nuclei from differentiated or adult cells, the success rate
of nuclear transfers is much lower than from larval or embryo
cells (Table 14. In the case of adult Xenopus tissues, the cells that
grow out from explants are of fibroblastic morphology and often
do not express differentiation markers. However, for the even-
tual purposes of cell replacement, the accessibility of adult tissue,
as in the case of skin or blood, is much more important than the
definition of cell type. In Xenopus experiments, cells from adult
skin have been obtained by outgrowth in culture and retain
expression of an epidermal keratin marker. Nuclei from these
cells give nuclear transfer results with the same efficiency as cells
from other adult organs (14~. About 1% of eggs receiving
transplanted nuclei from cells of adult skin reach the muscular
response stage and therefore have functional muscle and nerve
cells (Table 1~. Work with mammals has given comparable
results (15), although relatively few experiments have been done
in which nuclei of defined cell types have been transplanted to
enucleated eggs.
The overall conclusion from these direct nuclear transfer
experiments is that a substantial proportion of nuclei from
specified or determined embryonic cells expressing differentia-
tion markers undergo major reprogramming when transplanted
to enucleated eggs.
Serial Nuclear Transfers and Grafts
The question arises as to whether the low percentages of nuclear
transfer success shown in Table 1 for nonendoderm nuclei mean
that only a minority of cells in a tissue have the capacity to be
reprogrammed or that this capacity exists but has not been
demonstrated for technical or other reasons. In Amphibia, a
This paper results from the Arthur M. Sackier Coiloquium of the Nationai Acaclemy of
Sciences, "Regenerative Medicine," held October 18-22, 2002, at the Arnoicl ancl Mabei
Beckman Center of the National Acaclemies of Science and Engineering in irvine, CA.
*To whom correspondence shouicl be aciciressecl. E-maii: jbg1000@hermes.cam.ac.uk.
@) 2003 by The Nationai Acaclemy of Sciences of the USA
PNAS | September 30, 2003 I vol. 100 I suppl. ~ | 11819-~822
, .
i,
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Table 1. First nuclear transfers using determined and differentiated cells
% of total
nuclear transfers
reaching muscular
Species Donor tissue Donor stage response stage Ref.
Xenopus la evis Endoderm Muscular response stages 23-26 17-24 3
X. Iaevis Endoderm Heart beatstage 36 9-13 3
X. Iaevis Muscle Muscular response stage 26 2 42
X. Iaevis Intestinal epithelium Earlyfeeding tadpole stage47 1-3 4
X. Iaevis Skin outgrowth Adult 1.3 14
Cow Innercell mass cells Embryonic 0.6 43
Primate 4-32 cell embryos Embryonic 1.4 44
Sheep Mammary Adult 0.4 45
Mouse Cumulus Adult 2.8 46
substantial proportion of nuclear transplant embryos cleave
abnormally and die as partial blastulae within 24 h of nuclear
transfer. In the case of nuclei from differentiated or adult cells,
partial cleavage results from one-quarter to one-third of all
nuclear transfers and is far more frequent than complete cleav-
age (4~. However, it has been found that the normal-appearing
cells of partial blastulae can be used as donors for a second, serial
set of nuclear transfers to more enucleated eggs, an experimental
design first used by King and Briggs in 1956 (164. When this is
done with partial blastulae derived from intestinal epithelium
cells of Xenopus, it is found that many of the serial nuclear
transplant embryos develop remarkably well, sometimes reach-
ing the normal tadpole stage (49. These serially derived tadpoles
reflect the developmental potential of the originally trans-
planted intestinal epithelium cell nucleus, even though this
potential was not revealed by the first nuclear transfers.
The best explanation for this apparent improvement in nuclear
transfer success is the following. It is believed that nuclei from
slow-dividing somatic cells cannot complete their chromosome
replication in time for the first cleavage of a recipient egg, which
always takes place according to the time schedule of the egg, for
example at 1.5 h for the first cleavage in Xenopus. Somatic cells
take some 6 h to complete chromosome replication. When their
transplanted nuclei are forced into early mitosis with incom-
pletely replicated chromosomes, they are likely to suffer chro-
mosomal damage (17, 18) and generate chromosomally defective
embryos, which cannot survive. However, it sometimes happens
that a transplanted nucleus fails to undergo chromosome seg-
regation when the recipient egg divides into two cells and the
whole replicating transplanted nucleus moves into one of the first
two blastomeres. It then has a second chance to complete the
replication of its chromosomes before undergoing mitosis when
the egg goes from a two- to four-cell stage. As a result, partial
blastulae are obtained because one of the first two blastomeres
undergoes cleavage with the transplanted nucleus, while the
other blastomere, having no nucleus, dies. These partial blastu-
lae are more likely to contain nuclei with completely replicated
chromosome sets than are those nuclear transplant embryos that
undergo chromosome segregation at the first mitosis.
The result of carrying out serial nuclear transplantation shows
that a substantial proportion of the partially cleaved blastulae
contain nuclei with wide developmental potential. When this is
taken into account, the proportion of original intestinal epithe-
lium cells whose nuclei can promote muscle and nerve differ-
entiation rises to 20% (Table 2 and ref. 4~.
Using a similar line of thinking, it was found that the normal-
appearing cells of partial blastulae can be grafted to host
embryos and then reveal a wide range of developmental poten-
tial. Using GFP-marked donor nuclei, it has been calculated that
at least 16% of differentiated larval intestinal epithelium cells
contain nuclei capable of completely different pathways of
differentiation (193. In conclusion, the use of serial nuclear
transfer and grafts shows that a much higher proportion of
differentiated cells contain nuclei that undergo a major repro-
gramming by egg cytoplasm than the 1-3% apparent when
considering only first nuclear transfers.
Possible Reasons for Failures
After the transfer of nuclei from differentiated cells to enucle-
ated eggs, whether in Amphibia or mammals, only a few of the
nuclear transplants develop into adult animals. There are several
possibilities that could account for this low success rate, and the
primary reason for developmental failure may differ between
Amphibia and mammals. In Amphibia, the majority of eggs
receiving transplanted nuclei from differentiated or adult cells
undergo only a few irregular cleavages or fail to divide at all.
Thus, the primary loss in amphibian somatic cell nuclear transfer
is during the very earliest cleavages. In mammals, the develop-
mental loss tends to be greater after the initial cleavages. In
primates, development to the early eight-cell stage has been
shown to be similar in somatic and embryonic cell nuclear
transfers and after intra-cytoplasmic sperm injection (ICSI)
(between 80~o alla 90% in every case). However, the develop-
ment of cloned embryos to the later blastocyst stage was
markedly different; only 1% of somatic cell nuclear transfers
reach the blastocyst stage, whereas 34% of embryonic nuclear
transfers and 46% of ICSI controls reach this stage (204.
Table 2. The combination of first and serial nuclear transfer results
% of total
nuclear transfers
reaching muscular
Species Donor tissue Donor stage response stage Ref.
X. Iaevis Intestinal epithelium Early feeding tadpole stage47 20 4
X. Iaevis Skin outgrowth Adult 11-12 14
X. Iaevis Kidney, lung, heart Adult 13 47
11820 1 www.pnas.org/cgi/doi/10.1073/pnas.1834207100
., it,
. .
Gurdon et al.
OCR for page 5
These variations in the stage of developmental loss probably
reflect different problems encountered after somatic cell nuclear
transfer in Amphibia and mammals. In Amphibia the primary
problem may be the previously mentioned difficulty that somatic
cell nuclei have in completing chromosome replication within
the very limited time (only 90 min in Xenopus) before eggs
undergo cleavage (21~. In mammals there are 20 h for the mouse,
or longer for humans, between the time of fertilization and the
first cleavage division. This makes it unlikely that incomplete
chromosome replication is a problem in mammals. However,
mammals have imprinted genes (22) that Amphibia do not (23),
and it has been suggested that the developmental loss observed
after mammalian somatic cell nuclear transfer could be caused
by the incomplete reprogramming of various imprinted genes (as
well as Oct4) (24~. Another recent suggestion for mammalian
developmental failure is that enucleation removes maternal
spindle proteins required to maintain ploidy through the initial
cleavages (25~.
Although amphibian cloned embryos tend to suffer from
chromosomal damage caused by incomplete chromosome rep-
lication and mammalian cloned embryos tend not to express
imprinted genes correctly, there is also a range of other factors
that may affect both amphibian and mammalian nuclear trans-
plants. Quantitatively incomplete/incorrect reprogramming of
gene expression has been found in both amphibian (19) and
mammalian (26) cloned embryos, and it has been suggested that
this may affect development (27~. Also, it has been suggested
that the stage of the donor cell cycle may be critical to avoid
aneuploidy through re-replication of the donor genome (28~.
Another idea concerns the centriole; this is normally introduced
with the sperm at fertilization and the eggs of most animals do
not contain their own centriole. Perhaps most differentiated cells
that are no longer required to divide do not contain a fully
functional centriole. It is possible that technical factors may also
be important. Failure to rupture a donor cell would certainly
account for the total lack of cleavage. On the other hand, the
position in an egg at which a transplanted nucleus is deposited
is presumed not to matter on the grounds that sperm, entering
from the surface of the egg, always find their way to the correct
central position of the egg.
In conclusion, there is no definitive explanation for the high
frequency with which nuclei transplanted from differentiated or
adult cells fail to elicit any cleavage or development of recipient
eggs. It is probably a combination of aneuploidy, genetic damage,
and incomplete epigenetic reprogramming. For the purposes of
cell replacement, this is a serious problem only if the supply of
recipient eggs is strictly limited, as might be the case for humans.
The Cell Differentiation Potential of an Imperfect Genome
A fundamental idea behind the original vertebrate nuclear
transfer experiments was that a complete genome is required for
an egg to develop to a normal adult. This is very likely to be true;
in fact, the completeness of a genome might be defined in this
way. But this does not at all mean that each individual pathway
of cell differentiation also depends on ~ complete genome.
Nuclei that lack essential genes for one developmental pathway
may nevertheless be able to proceed along other differentiation
routes. Nuclear transfer experiments in both Amphibia and
mammals have given support to this idea. In Xenopus it has been
found that many of the partially cleaved nuclear transplant
embryos (that are developmentally defective) have quantita-
tively aberrant expression of early zygotic genes (19~. Despite
this, healthy cells from such embryos, genetically marked by
GFP, were able, after grafting to host embryos, to participate in
the differentiation and growth of normal muscle, notochord,
epidermal, and other cells (19~. In mice, it was discovered that
whereas only 2% of nuclear transplant embryos could develop
into adult animals, 9% of nuclear transplant embryos could
Gurdon et a/.
Oocyte growth: 9 months
Blastula
Development: 7 hours
7 hours at 1 8°C
DNA replication
Cell division
Unfertilized egg No transcription Blastula
1 cell 10,000 cells
4daysatl8°C
No DNA replication
No cell division
Oocyte
1 cell Intense transcription
Oocyte
1 cell
4 days at 37°C ~
-
Unfertilized egg
1 cell
DNA replication.
Slow cell division.
Transcription starts Blastocyst
~100 cells
Fig. 1. Diagrams to show the time scale of nuclear reprogramming in
Xenopus and the mouse. (A) Oogenesis and early development of Xenopus.
(B) In Xenopus egg nuclear transfers, reprogrammed gene expression is seen
at the late blastula stage after at least 12 cell division cycles. (C) In Xenopus
oocyte nuclear transfers, reprogrammed gene expression is seen in the com-
plete absence of DNA replication and cell division. (D) In mouse nuclear
transfer to eggs, reprogrammed gene expression has been seen at the blas-
tocyst stage.
produce embryonic stem cell lines (29~. This finding suggests that
defective cloned mammalian embryos, which are incapable of
developing into an entire mouse, can still produce useful stem
cell lines. An interesting future direction of research will be to
investigate the differentiation capacity of transplanted nuclei
carrying known chromosomal or gene deficiencies. Perhaps
genetically deficient cells may be entirely suitable for somatic cell
replacement.
Reprogramming Without Replication
The great majority of nuclear transfer experiments in both
mammals and Amphibia have been carried out with eggs in
second meiotic metaphase as recipient cells. In all of these cases,
the first response of an enucleated egg to a transplanted nucleus
is the induction of DNA synthesis and cell division (Fig. 1~. In
Amphibia, new transcription, and hence evidence of nuclear
reprogramming, commences after 12 cell cycles at the midblas-
tula transition, 5 h after nuclear injection. In mice, new tran-
scription starts at the early two-cell stage, ~24 h after nuclear
injection, and new transcription starts later in other mammalian
species. This situation raises the possibility that nuclear repro-
gramming requires DNA replication and/or cell division to
reset an epigenetic program, a suggestion made by Tada and
Tada (30~.
PNAS | September 30, 2003 I vol. 300 | suppl. ~ | 11821
OCR for page 6
To test this possibility, we have transplanted somatic cell
nuclei into nondividing amphibian oocytes in the prophase of
first meiosis. These cells cannot be fertilized, are inactive in DNA
synthesis, but are intensely active in transcription (Fig. 1~. Their
active genes are maximally packed with RNA polymerase, as
seen in the spectacular transcription complexes of Miller (31~.
We transplanted between 10 and 100 somatic cell nuclei to a
single oocyte to obtain a detectable response, and the results
were assessed by 2D protein analysis. The injected nuclei un-
derwent a large increase in volume and dispersion of their
chromatin over the several days for which injected oocytes can
be cultured. Protein analysis showed that new proteins were
synthesized by oocytes injected with mammalian nuclei from
cultured HeLa cells, although the identity of the proteins was not
known (32, 33~. In the case of nuclei from one species of
amphibian (Pleurodeles) transplanted to oocytes of Xenopus,
some new proteins were synthesized with the size and charge
properties of oocyte expressed genes (34~. In other experiments,
oocyte-specific 5S genes were activated when Xenopus somatic
cell nuclei with inactive oocyte-type SS genes were injected into
oocytes (35) and liver-specific enzymes were inhibited in exper-
iments with two species of Ambystoma (364.
Very recently we have extended our analysis of nuclear
transfer in Xenopus oocytes. Quite surprisingly, we find that the
nuclei of differentiated adult cells of mice (thymocytes) and
humans (white blood cells) can be to some extent reprogrammed
by Xenopus oocytes (37~. In particular, the diagnostic pluripo-
tency stem cell marker gene oct4 is induced in these mammalian
nuclei after injection into the germinal vesicle (enlarged oocyte
nucleus'. The oct4 transcripts have the human or mouse se-
quence when oocytes are injected with human or mouse somatic
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cell nuclei, respectively. The ability to activate oct4 expression in
the nuclei of adult somatic cells may increase the probability of
deriving embryonic stem cells from nuclear transplant embryos
(38~. Evidently amphibian oocytes contain molecules and con-
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Gurdon et a/.
. .
,
,~ .
. ,
Representative terms from entire chapter:
nuclear transfers
