Monitoring Stem Cell Research
The President's Council on Bioethics
(The following commissioned paper was prepared
at the request of the President’s Council on Bioethics;
the Council has not itself verified the accuracy of the information
contained therein, nor does it necessarily endorse any of the
author's conclusions or opinions. Additionally, the Council
has not edited this paper either for style or content.)
The Biology of Nuclear Cloning
and the Potential of Embryonic Stem Cells for Transplantation
Rudolf Jaenisch, M.D.
9 Cambridge Center,
An emerging consensus is that somatic cell nuclear transfer
(SCNT) for the purpose of creating a child (also called "reproductive
cloning") is not acceptable for both moral and scientific
reasons. In contrast, SCNT with the goal of generating an
embryonic stem cell line ("therapeutic cloning") remains a
controversial issue. Although therapeutic cloning holds the
promise of yielding new ways of treating a number of degenerative
diseases, it is not acceptable to many because the derivation
of an embryonic stem cell line from the cloned embryo (an
essential step in this process) necessarily involves the loss
of an embryo and hence the destruction of potential human
In this article, I will develop two main arguments that
are based on the available scientific evidence. 1) In contrast
to an embryo derived by in vitro fertilization (IVF),
a cloned embryo has little if any potential to ever develop
into a normal human being. This is because, by circumventing
the normal processes of gametogenesis and fertilization, nuclear
cloning prevents the proper reprogramming of the clone's
genome, which is a prerequisite for development of
an embryo to a normal individual. It is unlikely that these
biological barriers to normal development can be solved in
the foreseeable future. Therefore, from a biologist's
point of view, the cloned human embryo, used for the derivation
of an embryonic stem cell and the subsequent therapy of a
needy patient, has little if any potential to create
a normal human life. 2) Embryonic stem cells developed
from a cloned embryo are functionally indistinguishable from
those that have been generated from embryos derived by in
vitro fertilization (IVF). Both types of embryonic stem cells
have an identical potential to serve as a source for
therapeutically useful cells.
It is crucial that the ongoing debate on the possible therapeutic
application of SNCT is based on biological facts. The goal
of this article is to provide such a basis and to contribute
to a more rational discussion that is founded on scientific
evidence rather than on misconceptions or misrepresentations
of the available scientific data.
It is important to distinguish between "reproductive cloning"
and "nuclear transplantation therapy" (also referred to as
"SCNT" or "therapeutic cloning"). In reproductive cloning
a cloned embryo is generated by transfer of a somatic nucleus
into an enucleated egg with the goal to create a cloned individual.
In contrast, the purpose of nuclear transplantation therapy
is to generate an embryonic stem cell line (referred to as
"ntES cells") that is "tailored" to the needs of a patient
who served as the nuclear donor. The ntES cells could be used
as a source of functional cells that would be suitable for
treating an underlying disease by transplantation.
There is now experience from cloning of seven different
mammalian species that is relevant for three main questions
of public interest: 1) Would a cloned human embryo be "normal"?
2) Could the problems currently seen with cloning be solved
in the foreseeable future? 3) Would ES cells derived from
a cloned human embryo be "normal" and useful for cell therapy?
The arguments advanced in this article are strictly based
on molecular and biological evidence that has been obtained
largely in the mouse. I will not attempt to review the cloning
literature but only refer to selected papers on cloned mice.
The relevant literature on cloning of mammals can be found
in recent reviews (Byrne and Gurdon, 2002; Gurdon, 1999; Hochedlinger
and Jaenisch, 2002b; Oback and Wells, 2002; Rideout et al.,
2001; Wilmut, 2001; Young et al., 1998).
II. Most cloned animals die or are born with abnormalities
The majority of cloned mammals derived by nuclear transfer
(NT) die during gestation, and those that survive to birth
frequently display "Large Offspring Syndrome", a neonatal
phenotype characterized by respiratory and metabolic abnormalities
and enlarged and dysfunctional placentas (Rideout et al.,
2001; Young et al., 1998). In order for a donor nucleus to
support development into a clone, it must be reprogrammed
to a state compatible with embryonic development. The transferred
nucleus must properly activate genes important for early embryonic
development and also suppress differentiation-associated genes
that had been transcribed in the original donor cell. Inadequate
of the donor nucleus is most likely the principal reason for
developmental failure of clones. Since few clones survive
to birth, the question remains whether survivors are fully
normal or merely the least affected animals carrying through
to adulthood despite harboring subtle abnormalities that originate
in faulty reprogramming but that are not severe enough to
interfere with survival to birth or beyond.
III. Reprogramming of the genome during normal
development and after nuclear transfer
The fundamental difference between nuclear cloning and normal
fertilization is that the nucleus used in nuclear cloning
comes from a somatic (body) cell that has not undergone the
developmental events required to produce the egg and sperm.
Nuclear cloning involves the transplantation of a somatic
nucleus into the oocyte from which the nucleus has been removed.
However, the genes in the somatic nucleus are not in the same
state as those in the fertilized egg because nuclear transplantation
short-cuts the complex process of egg and sperm maturation
which involves extensive "reprogramming" of the genome, a
process that shuts some genes off and leaves others on. Reprogramming
during gametogenesis prepares the genome of the two mature
gametes with the ability to activate faithfully the genetic
program that ensures normal embryonic development when they
combine at fertilization (Fig 1a). This reprogramming of the
genome begins at gastrulation, when primordial germ cells
(PGCs) are formed, and continues during differentiation
into mature gametes resulting, in a radically different
chromatin configuration of sperm and oocyte (Rideout et al.,
Experiments have shown that uniparental embryos (embryos
whose genomes are derived solely from either the maternal
or paternal parent) do not develop normally. Uniparental embryos
first seem normal; they direct cleavage (early development
to the blastocyst stage) despite profound differences in their
epigenetic organization (Reik et al., 2001). However, uniparental
embryos fail soon after the implantation of the embryo into
the wall of the uterus, indicating that both parental genomes
are needed and functionally complement each other beginning
at this later step of embryogenesis. Presumably, the different
epigenetic organization of the two genomes is crucial for
achieving normal development. Moreover, it has been well established
that the imbalance of imprinted2
gene expression represents an important cause of embryonic
In order for cloned embryos to complete development, genes
normally expressed during embryogenesis but silent
in the somatic donor cell, must be reactivated. This complex
process of epigenetic3
remodeling (i.e., the reconfiguration of the genome by turning
on and turning off specific genes) that occurs during gametogenesis
in normal development ensures that the genome of the zygote
can faithfully activate early embryonic gene expression (Fig
1a). In a cloned embryo, reprogramming, which in normal gametogenesis
requires months to years to complete, must occur in a cellular
context radically different from gametogenesis and within
the short interval (probably within hours) between transfer
of the donor nucleus into the egg and the time when zygotic
transcription becomes necessary for further development. Given
these radically different conditions, one can envisage a spectrum
of different outcomes to the reprogramming process ranging
from (i) no reprogramming of the genome, resulting in immediate
death of the NT embryo; through (ii) partial reprogramming,
allowing initial survival of the clones, but resulting in
an abnormal phenotype and/or lethality at various stages of
development; to (iii) faithful reprogramming producing fully
normal animals (Fig 1b). The phenotypes observed over the
past five years in cloned embryos and newborns suggest that
complete reprogramming is the exception, if it occurs
IV. Development of clones depends on the differentiation-state
of the donor nucleus
The majority of cloned embryos fail at an early step of
embryonic development, soon after implantation in the wall
of the uterus, an early step of embryonic development (Hochedlinger
and Jaenisch, 2002b; Rideout et al., 2001). Those that live
to birth often display common abnormalities irrespective of
the donor cell type (Table 1). In addition to symptoms referred
to as "Large Offspring Syndrome", neonate clones often suffer
from respiratory distress and kidney, liver, heart or brain
defects (Cibelli et al., 2002). However, the abnormalities
characteristic of cloned animals are not inherited by their
offspring (Tamashiro et al., 2002), indicating that epigenetic
aberrations (i.e., failure of genome reprogramming) rather
than genetic aberrations (changes in the sequences within
the DNA) are the cause.
The efficiency of creating cloned animals is strongly influenced
by the differentiation-state of the donor nucleus (Table 1).
In the mouse, for example, only 1-3% of cloned blastocysts
derived from somatic donor nuclei, e.g., those prepared from
fibroblasts or cumulus cells, will develop to
adult cloned animals (Hochedlinger and Jaenisch, 2002b).
In certain cases, such as those using terminally differentiated
B or T cell donor nuclei, the efficiency of cloning is so
low as to preclude the direct derivation of cloned animals.
In stark contrast to these examples, cloning using donor nuclei
prepared from embryonic stem (ES) cells is significantly more
efficient (between 15 and 30 %, Table 1). This correlation
with differentiation-state suggests that embryonic nuclei
require less reprogramming of their genome, ostensibly because
the genes essential for embryonic development are already
active and need not be reprogrammed. In fact, the nucleus
of an embryonic cell such as an ES cell may well have the
same high efficiency to generate postnatal mice after nuclear
transfer as the nucleus prepared from a recently fertilized
egg (Table 1, compare Fig. 4). Nonetheless, most if not all
mice that have been cloned from ES cell donor nuclei, in contrast
to mice derived through natural fertilization from the zygote,
are abnormal, indicating that the processes of gametogenesis
(development of sperm and of egg) and fertilization endows
the zygote nucleus with the ability to direct normal
development. In summary, these data indicate that the potential
of a nucleus to generate a normal embryo is lost progressively
V. Adult cloned animals: how normal are they?
The observation that apparently healthy adult cloned animals
have been produced in seven mammalian species (albeit at low
efficiency) is being used by some as a justification for attempting
to clone humans. In fact, even those that survive to
adulthood, such as Dolly, may succumb relatively early
in adulthood because of numerous health problems. Insights
into the mechanisms responsible for clone failure before and
after birth have come from molecular and biological analyses
of mouse clones that have reached (i) the blastocyst stage,
(ii) the perinatal period and (iii) adulthood.
(i) Most clones fall short of activating key embryonic
genes and fail early.
As stated above in order for clones to develop, the genes
that are normally expressed during embryogenesis, but are
silent in the somatic donor cell, must be reactivated (Hochedlinger
and Jaenisch, 2002b; Rideout et al., 2001). It is the
failure to activate key "embryonic" genes that are required
for early development that leads to the demise of most clones
just after implantation. Recently, a set of about 70 key embryonic
genes termed "Oct-4 like" genes have been identified that
are active in early embryos but not in somatic donor cells.
Importantly, the failure to faithfully activate this set of
genes can be correlated with the frequent death of cloned
animals during the immediate post-implantation period (Bortvin
et al., 2003). These results define "faulty reprogramming"
as the cause of early demise of cloned embryos as the failure
to reactivate key embryonic genes that are silent in the donor
(ii) Newborn clones misexpress hundreds of genes.
Clones that survive to birth suffer from serious problems,
many of which appear to be due to an abnormal placenta. The
most common phenotypes observed in animals cloned from either
somatic or ES cell nuclei are fetal growth abnormalities such
as increased placental and birth weight. This has suggested
that surviving clones had accurately reprogrammed the "Oct-4
like" genes that are essential for the earliest stages of
development, i.e. those immediately following implantation
of the embryo into the uterus. The abnormal phenotype of those
clones that do survive through these early stages and develop
to birth indicates that other genes that are important for
later stages of development but are not essential for early
survival are not correctly reprogrammed. To assess the extent
of abnormal expression of various genes in the cells of clones,
global gene expression has been assessed by microarray analysis
of RNA prepared from the placentas and livers of neonatal
cloned mice, i.e., clones that survived development and were
viable at birth; these clones had been derived by nuclear
transfer (NT) of nuclei prepared either from cultured ES cells
or from freshly isolated cumulus cells (somatic cells that
surround the egg) (Humpherys et al., 2002). Direct comparison
of gene expression profiles of over 10,000 genes (of the 30,000
or so in the mammalian genome) showed that for both classes
of cloned neonatal mice, approximately 4% of the expressed
genes in their placentas differed dramatically in expression
levels from those in controls, and that the majority of abnormally
expressed genes were common to both types of clones. When
imprinted genes, a class of genes that express only one allele
(either from maternal or paternal origin), were analyzed,
between 30 and 50% were not correctly activated. These data
represent strong molecular evidence that cloned animals, even
those that survive to birth, suffer from serious gene expression
(iii) Cloned animals develop serious problems
The generation of adult and seemingly healthy adult cloned
animals has been taken as evidence that normal cloned animals
can be generated by nuclear transfer, albeit with low efficiency.
Indeed, a routine physical and clinical laboratory examination
of 24 cloned cows of 1 to 4 years of age failed to reveal
major abnormalities (Lanza et al., 2001). Cloned mice of a
corresponding age as that of the cloned cows (2 - 6 months
in mice vs. 1 - 4 years in cows) also appear "normal" by superficial
inspection. However, when cloned mice were aged, serious problems,
not apparent at younger ages, became manifest. One study found
that the great majority of cloned mice died significantly
earlier than normal mice, succumbing with immune deficiency
and serious pathological alterations in multiple organs (Ogonuki
et al., 2002). Another study found that aged cloned mice became
overweight with major metabolic disturbances (Tamashiro et
al., 2002). Thus, serious abnormalities in cloned animals
may often become manifest only when the animals age.
Firm evidence about aging and "normalcy" of cloned farm
animals is incomplete or anecdotal because cloned animals
of these species are still comparatively young (relative to
their respective normal life span). For example, the premature
death of Dolly (Giles and Knight, 2003) is entirely consistent
with serious abnormalities in cloned sheep that become manifest
only at later ages. Also, two of the analyzed cloned cows
developed disease soon after the study on "healthy and normal
cattle" (Lanza et al., 2001) had appeared: one animal developed
an ovarian tumor and another one suffered brain seizures (J.
Cibelli, pers. comm.). While it cannot be ruled out that these
are "spontaneous" maladies unconnected with the cloning procedure,
a more likely alternative is that these problems were direct
consequences of the nuclear transfer procedure.
(iv) Are there any "normal" clones?
It is a key question in the public debate whether it is
ever possible to produce a normal individual by nuclear cloning,
even if only with low efficiency. The available evidence suggests
that it may be difficult if not impossible to produce normal
clones for the following reasons: 1) As summarized above,
all analyzed clones at birth showed dysregulation of hundreds
of genes. The development of clones to birth and beyond despite
widespread epigenetic abnormalities suggests that mammalian
development can tolerate dysregulation of many genes. 2) Some
clones survive to adulthood by compensating for gene dysregulation.
Though this "compensation" assures survival, it may
not prevent maladies to become manifest at later ages. Therefore,
most if not all clones are expected to have at least subtle
abnormalities that may not be so severe as to result in an
obvious phenotype at birth but will cause serious problems
later as seen in aged mice. Clones may just differ
in the extent of abnormal gene expression: if the key "Oct-4
like" genes are not activated, clones die immediately after
implantation. If those genes are activated, the clone may
survive to birth and beyond.
As schematically shown in Fig. 2, the two stages when the
majority of clones fail are immediately after implantation
and at birth. These are two critical stages of development
that may be particularly vulnerable to faulty gene expression.
Once cloned newborns have progressed through the critical
perinatal period, various compensatory mechanisms may counterbalance
abnormal expression of other genes that are not essential
for the subsequent postnatal survival. However, the stochastic
occurrence of disease and other defects at later age in many
or most adult clones implies that such compensatory mechanisms
do not guarantee "normalcy" of cloned animals. Rather, the
phenotypes of surviving cloned animals may be distributed
over a wide spectrum from abnormalities causing sudden demise
at later postnatal age or more subtle abnormalities allowing
survival to advanced age (Fig. 2). These considerations illustrate
the complexity of defining subtle gene expression defects
and emphasize the need for more sophisticated test criteria
such as environmental stress or behavior tests. However, the
available evidence suggests that truly normal clones may be
It should be emphasized that "abnormality" or "normalcy"
is defined here by molecular and biological criteria that
distinguish cloned embryos or animals from control animals
produced by sexual reproduction. The most informative data
for the arguments presented above come from the mouse. There
is, however, every reason to believe that these difficulties
associated with producing mice and a variety of other mammalian
embryos by nuclear transplantation will also afflict the process
of human reproductive cloning (Jaenisch and Wilmut, 2001).
(v) Is it possible to overcome the problems
inherent in reproductive cloning?
It is often argued that the "technical" problems in producing
normal cloned mammals will be solved by scientific progress
that will be made in the foreseeable future. The following
considerations argue that this may not be so.
A principal biological barrier that prevents clones from
being normal is the "epigenetic" difference (such as distinct
patterns of DNA methylation4)
between the chromosomes inherited from mother and from father,
i.e. the difference between the "maternal" and the "paternal"
genome of an individual. Such methylation of specific DNA
sequences is known to be responsible for shutting down the
expression of nearby genes. Parent-specific methylation marks
are responsible for the expression of imprinted genes and
cause only one copy of an imprinted gene, derived either from
sperm or egg, to be active while the other allele is inactive
(Ferguson-Smith and Surani, 2001). When sperm and oocyte genomes
are combined at fertilization, the parent-specific marks established
during oogenesis and spermatogenesis persist in the genome
of the zygote (Fig 3A). Of interest for this discussion is
that within hours after fertilization, most of the
global methylation marks (with the exception of those on imprinted
genes) are stripped from the sperm genome whereas the genome
of the oocyte is resistant to this active demethylation process
(Mayer et al., 2000). This is because the oocyte genome is
in a different "oocyte-appropriate" epigenetic state than
the sperm genome. The oocyte genome becomes only partially
demethylated within the next few days by a passive demethylation
process. The result of these post-fertilization changes is
that the two parental genomes are epigenetically different
(as defined by the patterns of DNA methylation) in the later
stage embryo and remain so in the adult in imprinted as well
as non-imprinted sequences.
In cloning, the epigenetic differences that are established
during gametogenesis may be erased because both parental genomes
of the somatic donor cell are introduced into the egg from
the outside and are thus exposed equally to the demethylation
activity present in the egg cytoplasm (Fig 3B). This predicts
that imprinted genes should be particularly vulnerable to
inappropriate methylation and associated dysregulation in
cloned animals. The results summarized earlier are consistent
with this prediction. For cloning to be made safe, the two
parental genomes of a somatic donor cell would need to be
physically separated and separately treated in an "oocyte-appropriate"
and a "sperm-appropriate" way, respectively. At present, it
seems that this is the only rational approach to guarantee
the creation of the epigenetic differences that are normally
established during gametogenesis. Such an approach is beyond
our present abilities. These considerations imply that serious
biological barriers exist that interfere with faithful
reprogramming after nuclear transfer. It is a safe conclusion
that these biological barriers represent a major stumbling
block to efforts aimed at making nuclear cloning a safe reproductive
procedure for the foreseeable future.
It has been argued that the problems in mammalian cloning
are similar to those encountered with IVF 30 years ago: Thus,
following this argument, the methods of culture and embryo
manipulations just would need to be improved to develop reproductive
cloning into a safe reproductive technology that is as acceptable
as IVF. This argument appears to be fundamentally flawed.
It is certainly correct that merely "technical" problems needed
to be solved to make IVF efficient and safe. It is important
to distinguish between the perfection of technical skills
to imitate a biological event and the development of wholly
new science to overcome the blocks to events that have severe
biological restrictions. Nuclear cloning faces serious biological
barriers that cannot be addressed by mere adjustments in experimental
technique. Indeed, since the birth of Dolly no progress
has been made in solving any of the underlying biological
issues of faulty gene reprogramming and resulting defective
VI. Therapeutic applications of SCNT
(i) Reproductive cloning vs. therapeutic cloning
In spite of the biological and ethical barriers associated
with reproductive cloning, nuclear transfer technology has
significant therapeutic potential that is within our grasp.
There is an enormous distinction between the goals and the
end product of these two technologies. The purpose of reproductive
cloning is to generate a cloned embryo that is then implanted
in the uterus of a female to give rise to a cloned individual.
In contrast, the purpose of nuclear transplantation therapy
is to generate an embryonic stem cell line that is derived
from a patient (referred to as "ntES cells") and can be used
subsequently for tissue replacement.
Many scientists recognize the potential of NtES cells for
organ transplantation (for recent review see (Hochedlinger
and Jaenisch, 2003). This procedure is currently complicated
by immune rejection due to immunological incompatibility.
Thus, virtually all organ transplants undertaken at present
involve the use of donor organs that are recognized as foreign
by the immune systems of the recipient and thus are targeted
for destruction by these immune systems. To treat this "host
versus graft" disease, immunosuppressive drugs are routinely
given to transplant recipients in order to suppress this organ
rejection. Such immunosuppressive treatment has serious side
effects including increased risks of infections and malignancies.
In principle, ES cells can be created from a patient's
nuclei using nuclear transfer. Because ntES cells will be
genetically identical to the patient's cells, the risks of
immune rejection and the requirement for immunosuppression
are eliminated. Moreover, ES cells provide a renewable source
of replacement tissue allowing for repeated therapy whenever
needed. Finally, if ES cells are derived from a patient carrying
a known genetic defect, the mutation in question can be corrected
in the ntES cells using standard gene targeting methods before
introducing these ES cells (or derived tissue-specific stem
cells) back into the patient's body.
(ii) Combining nuclear cloning with gene and
In a "proof of principle" experiment, nuclear cloning in
combination with gene and cell therapy has been used to treat
a mouse genetic disorder that has a human counterpart (Figure
4). To do so, the well-characterized Rag2 mutant mouse
was used as "patient" (Rideout et al., 2002). This mutation
causes severe combined immune deficiency (SCID), because
the enzyme that catalyzes immune receptor rearrangements in
lymphocytes is non-functional. Consequently, these mice are
devoid of mature B and T cells, a disease resembling human
Omenn syndrome (Rideout et al., 2002).
In a first step, somatic (fibroblast) donor cells were isolated
from the tails of Rag2-deficient mice and their nuclei
were injected into enucleated eggs. The resultant embryos
were cultured to the blastocyst stage and isogenic ES cells
were isolated. Subsequently, one of the mutant Rag2
alleles was targeted by homologous recombination in ES cells
to restore normal Rag2 gene structure and function.
In order to obtain somatic cells for treatment, these genetically
repaired ES cells were differentiated into embryoid bodies
and further into hematopoietic precursors by expressing HoxB4,
a transcription factor that is responsible for programming
the behavior of the hematopoietic stem cells, i.e., those
cells that are able to generate the full range of red and
white cells in the blood. Resulting hematopoietic precursors
were transplanted into irradiated Rag2-deficient animals
in order to treat the disease caused by their Rag2
mutation. Initial attempts to engraft these cells were, however,
unsuccessful because of an increased level of natural killer
(NK) cells in the Rag mutant host. ES cell derived
hematopoietic cells express low levels of the MHC antigens
and thus are a preferred target for NK mediated destruction.
Elimination of NK cells by antibody depletion or genetic ablation
allowed the ntES cells to efficiently populate the myeloid
and to a lesser degree the lymphoid lineages of these mice.
Functional B and T cells that had undergone proper rearrangements
of their immunoglobulin and T cell receptor alleles as well
as serum immunoglobulins were detected in the transplanted
mutants. Hence, important cellular components of the immune
system were restored in mice that previously were unable to
produce these cells.
This experiment demonstrated that embryonic stem (ES) cells
derived by NT from somatic cells of a genetically afflicted
individual can be combined with gene therapy to treat the
underlying genetic disorder. Because Rag2 deficiency causes
an increase in NK activity and necessitated the elimination
of NK cells prior to transplantation in the above-described
experiments, some have concluded that "The experiment failed
to show success with therapeutic cloning" (Coalition and Ethics,
2003) and that "This indicates that the only successful therapy
using cloned embryos would be through 'reproductive'
cloning, to produce born clones who can serve as tissue donors
for patients" (Prentice, 2002). This is a troubling misinterpretation
of the data. (i) It has been shown that ES cell-derived hematopoietic
cells can successfully engraft and rescue lethally irradiated
mice indicating that increased NK activity is a peculiarity
of Rag2-deficiency (Kyba et al., 2002). Therefore, it would
seem that for most diseases, no anti-NK treatment would be
required to assure engraftment of ES cell-derived somatic
cells. (ii) It is correct that treatment of a human patient
with Omenn syndrome, which is equivalent to Rag2 deficiency,
by SCNT may also require anti-NK treatment to transiently
reduce NK activity. This would allow the transplanted cells
to engraft as in the mouse experiment. Once these cells are
successfully engrafted, there is every reason to believe that
such anti-NK treatment would no longer be necessary.
In conclusion, the mouse experiment indicates that, unlike
the situation with reproductive cloning, no biological
barriers exist that in principle prevent the use of SCNT to
treat human diseases. The technical issues in using
SCNT and human stem cells for therapeutic purposes need, however,
to be solved, but there are no indications at present that
these represent formidable problems that will resist relatively
VII. Faulty reprogramming after nuclear transfer:
does it interfere with the therapeutic potential of ES cells
derived by SCNT?
As summarized above, most if not all cloned animals are
abnormal because of faulty reprogramming after nuclear transfer.
Does this epigenetic dysregulation affect the potential of
ntES cells to generate functional somatic cells that can be
used for cell therapy? To address this question, I will first
compare the in vivo development of embryos with the
in vitro process of ES cell derivation from explanted
embryos. This will be followed by discussing the epigenetic
state of the ES cell genome. Finally, I will contrast the
phenotype of cloned mice derived from ES cell donor nuclei
with that of chimeric mice generated by injection of ES cells
(i) The phenotype of an embryo is determined
by its donor nucleus
As mentioned repeatedly above, embryos can be derived from
the fertilized egg or from a somatic nucleus by SCNT. The
potential of the resulting blastocyst, when implanted into
the womb, to develop into a fetus and a postnatal animal depends
strictly on the nature of the donor nucleus (Fig 5): (i) When
derived from the zygote, most embryos develop to birth and
generate a normal animal; (ii) Similarly, most blastocysts
cloned from an embryonic stem cell donor nucleus develop to
birth but, in contrast to the normally fertilized embryo,
the great majority of the cloned animals will be abnormal
("Large offspring syndrome") (Eggan et al., 2001; Humpherys
et al., 2001); (iii) The great majority of cloned blastocysts
derived from somatic donor nuclei such as fibroblasts or cumulus
cells will die soon after implantation and only a few clones
will survive to birth and these too will be abnormal, suffering
once again from the Large offspring syndrome (Wakayama and
Yanagimachi, 2001); (iv) Finally, the likelihood of cloned
blastocysts derived from another type of somatic donor nuclei
- those present in terminally differentiated lymphoid cells
- to generate a cloned animal is extremely low and has not
been achieved except by using a two step procedure involving
the intermediate generation of embryonic stem cells (Hochedlinger
and Jaenisch, 2002a). These observations suggest that a blastocyst
retains an "epigenetic memory" of its donor nucleus. This
memory determines its potential for fetal development: while
a fertilized embryo develops normally, any embryo derived
by SCNT will be abnormal though the efficiency of a given
clone to develop to birth is strongly influenced by the differentiation
state of the donor cell (see Table 1). In other words, the
cloned embryo after implantation into the womb will be abnormal
because the cloned blastocyst retained an epigenetic memory
of its donor nucleus and this causes faulty fetal development.
This epigenetic memory is erased when a blastocyst, either
derived by nuclear cloning or from the fertilized egg, is
explanted into tissue culture and grown into an embryonic
stem cell. Erasure of the epigenetic memory has major consequences
for the "normalcy" of embryonic stem cells.
(ii) The derivation of embryonic stem cells is a highly
selective process that erases the "epigenetic memory" of the
Embryonic stem cells, regardless of whether they have been
generated from a fertilized egg or by SCNT, are derived from
the cells of a blastocyst that have been explanted and propagated
in tissue culture. Of the blastocyst cells that are explanted
in this way, those that derive from the portion of the blastocyst
termed the inner cell mass (ICM) initially express "key" embryonic
genes such as Oct-4. However, soon after explantation, most
ICM cells extinguish Oct-4 expression and cease proliferating
(Buehr et al., 2003). Only one or a few of the ICM-derived
cells will eventually re-express Oct-4 and these few Oct-4-positive
cells are those that resume rapid proliferation, yielding
the cell populations that we designate as "embryonic stem"
cells. These cells represent a cell population that has no
equivalent in the normal embryo and may be considered a tissue
culture artifact, though a useful one (Fig. 6).
The important point for this discussion is that the propagation
of blastocyst cells in vitro results in a rare population
of surviving cells that have erased the "epigenetic memory"
of the donor nucleus. This process results ultimately in ES
cells that have, regardless of donor nuclear origin, an identical
developmental potential. In other words, ES cells derived
from embryos produced by normal fertilization and those produced
from cloned embryos are functionally indistinguishable (Hochedlinger
and Jaenisch, 2002b; Rideout et al., 2002; Wakayama et al.,
2001). Because the ES cells that derive from normally fertilized
embryos are able to participate in the generation of all normal
embryonic tissues, we can conclude that the ES cells derived
from cloned embryos have a similar potential to generate the
full range of normal tissues.
(iii) ES cells, epigenetic instability and
Epigenetic instability appears to be a consistent characteristic
of ES cells. This was shown when individual ES cells were
analyzed for expression of imprinted genes: even cells in
a recently subcloned ES cell line differed strongly in the
expression of genes such as H19 or Igf2. The variable expression
was correlated with the DNA methylation status of the genes,
which switched from an unmethylated to a methylated state
between sister cells (Humpherys et al., 2001). This was a
surprising result in view of the known potential of ES cells
to generate terminally differentiated cells that function
normally after transplantation into an animal. Possible explanations
include (i) that epigenetic instability in ES cells is a consequence
of propagation of cells in tissue culture or (ii) that epigenetic
instability is a prerequisite for cells to be pluripotent,
i.e., this instability may be a manifestation of a plasticity
in the gene expression program that is required to enable
the ES cells to generate a wide variety of differentiated
Whatever the explanation for the observed epigenetic instability
of ES cells may be, it supports the view that the process
of generating ES cells erases all epigenetic memory of the
donor nucleus and, as a consequence of the selection process,
generates epigenetic instability in the selected cells. In
other words, epigenetic instability appears to be an intrinsic
characteristic of ES cells regardless of whether derived by
SCNT or from a fertilized egg. This is consistent with
the conclusion that both types of ES cells have an equivalent
potency to generate functional cells in culture and, in the
longer term, fully normal differentiated tissues upon implantation
of these cells in vivo.
(iv) ES cells form normal chimeras but abnormal
As outlined above, faulty reprogramming leads to abnormal
phenotypes of cloned mice derived from ES cell donor nuclei.
Why is faulty reprogramming and epigenetic instability a problem
for reproductive cloning but not for therapeutic applications?
The main reason for this seeming paradox is that, in contrast
to reproductive cloning, the therapeutic application of NT
does not require the formation of a fetus. Therapeutic applications
involve the ability of cloned ES cells to form a single tissue
or organ, not to recapitulate all of fetal development. For
example, normal fetal development requires faithful expression
of the imprinted genes. As outlined above, nuclear cloning
causes between 30% and 50% of imprinted genes to be dysregulated
consistent with the notion that disturbed imprinting is a
major contributing factor to clone failure. As most imprinted
genes have no known function in the postnatal animal, the
dysregulation of imprinting would not be expected to impede
functionality of in vitro differentiated ES cells because
this process does not require the formation of a fetus. Therefore,
the functionality of mature cells derived in culture from
ES cells would not depend on the faithful reprogramming of
the imprinted genes. Dysregulation of some imprinted genes
such as Igf2 are known, however, to cause disease in
the adult. Thus, it will be important to test whether dysregulation
of such genes has adverse effects on the function of somatic
cells derived from ES cells.
When injected into a blastocyst, ES cells form normal chimeras.
It appears that the presence of surrounding "normal" cells,
i.e. cells that are derived from a fertilized embryo, prevents
an abnormal phenotype of the chimera such as the "Large Offspring
Syndrome" that is typical for cloned animals. Any therapeutic
application creates, of course, a chimeric tissue where cells
derived from ntES cells are introduced into a diseased adult
individual and interact with surrounding "normal" host cells.
Therefore, no phenotypic abnormalities, such as those seen
in cloned animals, would be expected in patients transplanted
with cells derived from ntES cells.
VIII. SCNT for cell therapy: destruction of potential human
A key concern raised against the application of the nuclear
transplantation technology for tissue therapy in humans is
the argument that the procedure involves the destruction of
potential human life. From a biological point of view, life
begins with fertilization when the two gametes are combined
to generate a new embryo that has a unique combination of
genes and has a high potential to develop into a normal baby
when implanted into the womb. A critical question for the
public debate on SCNT is this one: is the cloned embryo equivalent
to the fertilized embryo?
In cloning, the genetic contribution is derived from one
individual and not from two. Obviously, the cloned embryo
is the product of laboratory-assisted technology, not the
product of a natural event. From a biological point of view,
nuclear cloning does not constitute the creation of new life,
rather the propagation of existing life because no meiosis,
genetic exchange and conception are involved. Perhaps more
important is, however, the overwhelming evidence obtained
from the cloning of seven different mammalian species. As
summarized above, the small fraction of cloned animals that
survive beyond birth, even if they appear "normal" upon superficial
inspection, are likely not so. The important conclusion is
that a cloned human embryo would have little if any potential
to develop into a normal human being. With other words, the
cloned human embryo lacks essential attributes that characterize
the beginning of normal human life.
Taking into account the potency of fertilized and cloned
embryos, the following scenarios regarding their possible
fates can be envisaged (Fig. 7). Fertilized embryos that are
"left over" from IVF have three potential fates: disposal,
generation of normal embryonic stem cells or generation of
a normal baby when implanted into the womb. Similarly, the
cloned embryo has three potential fates: it can be destroyed
or could be used to generate a normal ntES cell line that
has the same potential for therapy as an ES cell derived from
a fertilized embryo. In contrast to the fertilized embryo,
the cloned embryo has little if any potential to ever generate
a normal baby.An embryonic stem cell line derived by nuclear
may, however, help sustain existing life when used as a source
for cell therapy that is "tailored" to the need of the patient
who served as its nuclear donor.
If SCNT were accepted as a valid therapeutic option, a major
concern of its implementation as medical procedure would be
the problem of how to obtain sufficient numbers of human eggs
that could be used as recipients. Commercial interests may
pressure women into an unwanted role as egg donors. The recent
demonstration that embryonic stem cells can be coaxed into
a differentiation pathway that yields oocyte-like cells (Hubner
et al., 2003) may offer a solution to this dilemma. If indeed
functional oocytes could be generated from a generic human
ES cell line, sufficient eggs could be generated in culture
and serve as recipients for nuclear transfer without the need
of a human egg donor. It seems that technical issues, not
fundamental biological barriers, need to be overcome so that
transplantation therapy can be carried out without the use
of human oocytes.
Fig 1:Reprogramming in normal development and
a. The genome of primordial germ cells (PGCs) is
hypomethylated ("reset", white boxes). Reprogramming and establishment
of parent specific epigenetic marks occurs over the course
of gametogenesis so that the genome of sperm and egg are competent
to express the genes that need to be activated in early embryonic
(box with wavy lines) and later (hatched box) development.
During cleavage and early postimplantation development "embryonic"
genes, such as Oct 3/4, become activated (black box) and are
repressed at later stages (stippled boxes) when tissue specific
genes (hatched boxes) are activated in adult tissues (A, B,
C). Epigenetic reprogramming of imprinted and non-imprinted
genes occurs during gametogenesis in contrast to X inactivation
and the readjustment of telomere length which take place postzygotically.
b. Reprogramming of a somatic nucleus following nuclear
transfer may result in (i) no activation of "embryonic" genes
and early lethality, (ii) faulty activation of embryonic genes
and an abnormal phenotype, or (iii) in faithful activation
of "embryonic" and "adult" genes and normal development of
the clone. The latter outcome is the exception if it occurs
Fig. 2: The phenotypes are distributed
over a wide range of abnormalities. Most clones fail at
two defined developmental stages, implantation and birth.
More subtle gene expression abnormalities result in disease
and death at later ages.
Fig 3: Parental epigenetic differences in normal and
A: The genomes of oocyte
and sperm are differentially methylated during gametogenesis
and are different in the zygote when combined at fertilization.
Immediately after fertilization the paternal genome (derived
from the sperm) is actively demethylated whereas the maternal
genome is only partially demethylated during the next few
days of cleavage. This is because the oocyte genome is in
a different chromatin configuration and is resistant to the
active demethylation process imposed on the sperm genome by
the egg cytoplasm. Thus, the methylation of two parental genomes
is different at the end of cleavage and in the adult. Methylated
sequences are depicted as filled lollipops and unmethylated
sequences as empty lollipops.
3B: In cloning a somatic nucleus is transferred
into the enucleated egg and both parental genomes are
exposed to the active demethylating activity of the egg cytoplasm.
Therefore, the parent specific epigenetic differences are
Fig. 4:Scheme for therapeutic cloning combined
with gene and cell therapy.
A piece of tail from a mouse homozygous for the recombination
activating gene 2 (Rag2) mutation was removed and cultured.
After fibroblast-like cells grew out, they were used as donors
for nuclear transfer by direct injection into enucleated MII
oocytes using a Piezoelectric driven micromanipulator. Embryonic
stem (ES) cells isolated from the NT-derived blastocysts were
genetically repaired by homologous recombination. After repair,
the ntES cells were differentiated in vitro into embryoid
bodies (EBs), infected with the HoxB4iGFP retrovirus, expanded,
and injected into the tail vein of irradiated, Rag2-deficient
mice (after (Rideout et al., 2002)).
Fig. 5: Blastocysts
retain epigenetic memory of donor nucleus
Blastocysts can be derived from the fertilized egg or by
nuclear transfer. After implantation development of the embryo
strictly depends on the donor nucleus: Blastocysts derived
from a fertilized egg will develop with high efficiency to
normal animals; blastocysts derived by NT from an ES
cell donor will develop with high efficiency to abnormal
animals; blastocysts derived by NT from a fibroblast or cumulus
cell donor will develop with low efficiency to abnormal
animals; blastocysts derived by NT from B or T donor cells
will not develop to newborns by direct transfer into the womb
(only by a 2 step procedure, compare (Hochedlinger and Jaenisch,
Fig. 6: The establishment of ES
cells from blastocysts erases epigenetic memory of donor nucleus
Most cells of the inner cell mass turn off Oct-4 like genes
and die after explantation of blastocysts into tissue culture.
Only one or a few cells turn on the Oct-4 like genes and proliferate.
The surviving cells will give rise to ES cells. During this
highly selective outgrowth of the surviving cells all epigenetic
memory of the donor nucleus is erased. Therefore, regardless
of donor nucleus (fertilized egg or somatic nucleus in cloned
embryos), all ES cells have an equivalent potency to generate
functional differentiated cells.
Fig. 7: Normal and cloned embryos
have three possible fates
Embryos derived by IVF ("left over embryos") have three
fates: they can be disposed, create normal babies if
implanted or can generate ES cells if explanted into tissue
culture. Cloned embryos have also three fates: they can be
disposed, can generate abnormal babies if any
when implanted or can generate ES cells when explanted. The
ES cells derived from an IVF embryo or a cloned embryo are
indistinguishable (same potency, see figure 6)
(% of blastocysts)
30 - 50 %
Nuclear transfer from
15 - 30 %
Most if not all clones
Cumulus cell, fibroblast
1 - 3 %
B, T cell
Development of normal embryos and embryos cloned from ES
cell and somatic donor nuclei. Note that normal and ES cell
derived blastocysts have a similar potency to develop to term
if calculated from the fraction of transplanted blastocysts.
1: (Eggan et al., 2001; Eggan et al., 2002; Rideout et al.,
2000); 2 (Wakayama et al., 1998; Wakayama and Yanagimachi,
1999); 3 (Hochedlinger and Jaenisch, 2002a).
I thank my colleagues Bob Weinberg, Gerry Fink, George Daley
and Andy Chess for critical and constructive comments on this
Reprogramming: The genome of
a somatic cell is in an epigenetic state that is appropriate
for the respective tissue and assures the expression of
the tissue specific genes (in mammary gland cells, for
example, those genes important for mammary gland function
such as milk production). In cloning, the somatic nucleus
must activate those genes that are needed for embryonic
development but which are silent in the donor cell in
order for the cloned embryo to survive. The egg cytoplasm
contains "reprogramming factors" that can convert the
epigenetic state (see endnote 3) characteristic of the
somatic donor nucleus to one that is appropriate for an
embryonic cell. This process is very inefficient leading
to inappropriate expression of many genes and causes most
clones to fail early.
Imprinted genes: For most genes, both copies, the
one inherited from father and the one inherited from mother,
are expressed. In contrast, only one of the two copies
of an imprinted gene, either the maternal one or the paternal
one, is active. The two copies are distinguished by methylation
marks (see endnote 4) that are imposed on imprinted genes
either during oogenesis (maternally imprinted genes) or
during spermatogenesis (paternally imprinted genes). Thus,
the two copies of imprinted genes are epigenetically different
in the zygote and remain so in all somatic cells. These
epigenetic marks distinguish the two copies and cause
only one copy to be expressed whereas the other copy remains
silent. It is estimated that between 100 and 200 genes
(of the total of 30,000 genes) are imprinted. Disturbances
of normal imprinted gene expression lead to growth abnormalities
during fetal life and can be the cause of major diseases
such as Beckwith-Wiedeman or Prader-Willi syndrome.
Epigenetic changes: Cells of a multicellular organism
are genetically identical but express, depending on the
particular cell type, different sets of genes ("tissue
specific genes"). These differences in gene expression
arise during development and must be retained through
subsequent cell divisions. Stable alterations of this
kind are said to be "epigenetic", as they are heritable
in the short term (during cell divisions) but do not involve
mutations of the DNA itself.
DNA methylation: Reversible modification of DNA
(methylation of the base cytosine) that affects the "readability"
of genes: usually, methylated genes are silent and unmethylated
genes are expressed. DNA methylation represents an important
determinant of the "epigenetic state" of genes and affects
the state of the chromatin: methylated regions of the
genome are in a "silent" state and unmethylated regions
are in an "open" configuration that causes genes to be
Bortvin, A., Eggan, K., Skaletsky, H.,
Akutsu, H., Berry, D. L., Yanagimachi, R., Page, D. C.,
and Jaenisch, R. (2003). Incomplete reactivation of Oct4-related
genes in mouse embryos cloned from somatic nuclei. Development
Buehr, M., Nichols, J., Stenhouse, F.,
Mountford, P., Greenhalgh, C. J., Kantachuvesiri, S., Brooker,
G., Mullins, J., and Smith, A. G. (2003). Rapid loss of
oct-4 and pluripotency in cultured rodent blastocysts and
derivative cell lines. Biol Reprod 68, 222-229.
Byrne, J. A., and Gurdon, J. B. (2002).
Commentary on human cloning. Differentiation 69,
Cibelli, J. B., Campbell, K. H., Seidel,
G. E., West, M. D., and Lanza, R. P. (2002). The health
profile of cloned animals. Nat Biotechnol 20, 13-14.
Coalition, and Ethics, A. f. R. (2003).
Do no harm - Reality check: proof of "therapeutic cloning"?
Eggan, K., Akutsu, H., Loring, J., Jackson-Grusby,
L., Klemm, M., Rideout, W. M., 3rd, Yanagimachi, R., and
Jaenisch, R. (2001). Hybrid vigor, fetal overgrowth, and
viability of mice derived by nuclear cloning and tetraploid
embryo complementation. Proc Natl Acad Sci U S A 98,
Eggan, K., Rode, A., Jentsch, I., Samuel,
C., Hennek, T., Tintrup, H., Zevnik, B., Erwin, J., Loring,
J., Jackson-Grusby, L., et al. (2002). Male and female
mice derived from the same embryonic stem cell clone by
tetraploid embryo complementation. Nat Biotechnol 20,
Ferguson-Smith, A. C., and Surani, M. A.
(2001). Imprinting and the epigenetic asymmetry between
parental genomes. Science 293, 1086-1089.
Giles, J., and Knight, J. (2003). Dolly's
death leaves researchers woolly on clone ageing issue. Nature
Gurdon, J. B. (1999). Genetic reprogramming
following nuclear transplantation in Amphibia. Semin Cell
Dev Biol 10, p239-243.
Hochedlinger, K., and Jaenisch, R. (2002a).
Monoclonal mice generated by nuclear transfer from mature
B and T donor cells. Nature 415, 1035-1038.
Hochedlinger, K., and Jaenisch, R. (2002b).
Nuclear transplantation: Lessons from frogs and mice. Curr
Opin Cell Biol 14, 741-748.
Hochedlinger, K., and Jaenisch, R. (2003).
Nuclear transplantation, embryonic stem cells, and the potential
for cell therapy. New England Journal of Medicine 349,
in press (July 17, 2003).
Hubner, K., Fuhrmann, G., Christenson,
L. K., Kehler, J., Reinbold, R., De La Fuente, R., Wood,
J., Strauss, I. J., Boiani, M., and Scholer, H. R. (2003).
Derivation of Oocytes from Mouse Embryonic Stem Cells. Science.
Humpherys, D., Eggan, K., Akutsu, H., Friedman,
A., Hochedlinger, K., Yanagimachi, R., Lander, E., Golub,
T. R., and Jaenisch, R. (2002). Abnormal gene expression
in cloned mice derived from ES cell and cumulus cell nuclei.
Proc Natl Acad Sci U S A 99, 12889-12894.
Humpherys, D., Eggan, K., Akutsu, H., Hochedlinger,
K., Rideout, W., Biniszkiewicz, D., Yanagimachi, R., and
Jaenisch, R. (2001). Epigenetic instability in ES cells
and cloned mice. Science 293, 95-97.
Jaenisch, R., and Wilmut, I. (2001). Developmental
biology. Don't clone humans! Science 291, 2552.
Kyba, M., Perlingeiro, R. C., and Daley,
G. Q. (2002). HoxB4 confers definitive lymphoid-myeloid
engraftment potential on embryonic stem cell and yolk sac
hematopoietic progenitors. Cell 109, 29-37.
Lanza, R. P., Cibelli, J. B., Faber, D.,
Sweeney, R. W., Henderson, B., Nevala, W., West, M. D.,
and Wettstein, P. J. (2001). Cloned cattle can be healthy
and normal. Science294, 1893-1894.
Mayer, W., Niveleau, A., Walter, J., Fundele,
R., and Haaf, T. (2000). Demethylation of the zygotic paternal
genome. Nature 403, 501-502.
Oback, B., and Wells, D. (2002). Donor
cells for cloning-many are called but few are chosen. Cloning
Stem Cells 4, 147-168.
Ogonuki, N., Inoue, K., Yamamoto, Y., Noguchi,
Y., Tanemura, K., Suzuki, O., Nakayama, H., Doi, K., Ohtomo,
Y., Satoh, M., et al. (2002). Early death of mice
cloned from somatic cells. Nat Genet 30, 253-254.
Prentice, D. (2002). Why the "Successful"
Mouse "Therapeutic" Cloning Really Didn't work. wwwcloninginformationorg/info/unsuccessful_mouse_therapyhtm.
Reik, W., Dean, W., and Walter, J. (2001).
Epigenetic reprogramming in mammalian development. Science
Rideout, W. M., 3rd, Hochedlinger, K.,
Kyba, M., Daley, G. Q., and Jaenisch, R. (2002). Correction
of a genetic defect by nuclear transplantation and combined
cell and gene therapy. Cell 109, 17-27.
Rideout, W. M., Eggan, K., and Jaenisch,
R. (2001). Nuclear cloning and epigenetic reprogramming
of the genome. Science 293, 1093-1098.
Rideout, W. M., Wakayama, T., Wutz, A.,
Eggan, K., Jackson-Grusby, L., Dausman, J., Yanagimachi,
R., and Jaenisch, R. (2000). Generation of mice from wild-type
and targeted ES cells by nuclear cloning. Nat Genet 24,
Tamashiro, K. L., Wakayama, T., Akutsu,
H., Yamazaki, Y., Lachey, J. L., Wortman, M. D., Seeley,
R. J., D'Alessio, D. A., Woods, S. C., Yanagimachi,
R., and Sakai, R. R. (2002). Cloned mice have an obese phenotype
not transmitted to their offspring. Nat Med 8, 262-267.
Wakayama, T., Tabar, V., Rodriguez, I.,
Perry, A. C., Studer, L., and Mombaerts, P. (2001). Differentiation
of embryonic stem cell lines generated from adult somatic
cells by nuclear transfer. Science 292, 740-743.
Wakayama, T., Whittingham, D. G., and Yanagimachi,
R. (1998). Production of normal offspring from mouse oocytes
injected with spermatozoa cryopreserved with or without
cryoprotection. J Reprod Fertil 112, 11-17.
Wakayama, T., and Yanagimachi, R. (1999).
Cloning of male mice from adult tail-tip cells. Nat Genet
Wakayama, T., and Yanagimachi, R. (2001).
Mouse cloning with nucleus donor cells of different age
and type. Mol Reprod Dev 58, 376-383.
Wilmut, I. (2001). How safe is cloning?
Cloning 3, 39-40.
Young, L. E., Sinclair, K. D., and Wilmut,
I. (1998). Large offspring syndrome in cattle and sheep.
Rev Reprod 3, p155-163.