This commissioned working paper was prepared for the July 2003 meeting and is solely to aid discussion
and does not represent the official views of the Council or of the
United States Government.
The biology of nuclear cloning and the potential of embryonic
stem cells for transplantation therapy
9 Cambridge Center
Cambridge, MA 02142
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
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
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
"reprogramming" of the donor nucleus is most likely the principal
reason for developmental failure of clones (for definition of "reprogramming",
see footnote 1). 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
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., 2001).
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 imprinted gene expression represents
an important cause of embryonic failure (for definition of "imprinting",
see footnote 2).
In order for cloned embryos to complete development, genes normally
expressed during embryogenesisbut silent in the somatic donor
cell,must be reactivated. This complex process of epigenetic
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; definition
of "epigenetics", see footnote 3). 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 at all.
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
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, areabnormal, 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 with development.
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 cell.
(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 abnormalities.
(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 the exception.
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
(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 methylation, for definition of "methylation", see footnote
4) 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
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 certainlycorrect
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
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 cell
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 rapid solution.
VII. Faulty reprogramming after nuclear transfer: does
it interfere with the therapeutic potential of ES cells derived
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 into blastocysts.
(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 donor
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 therapeutic
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 withthe 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 cell lineages.
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
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
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
Fig 1: Reprogramming in normal development and nuclear
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 at all.
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 cloned animals
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
B: 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 equalized.
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
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, 2002a).
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
Fig. 7: Normal and cloned embryos have three possible
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 abnormalbabies
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 are
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).
1) 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 footnote 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.
2) 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 footnote 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.
3) 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.
4) 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 active.
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