Human Therapeutic Cloning (NTSC)

Stem Cell Reviews
Copyright © 2006 Humana Press Inc.
All rights of any nature whatsoever are reserved.
ISSN 1535–1084/06/2:265–276/$30.00
(Online) 1558–6804
Human Therapeutic Cloning (NTSC)
Applying Research From Mammalian Reproductive Cloning
Andrew J. French,*,1 Samuel H.Wood,1,2 and Alan O.Trounson3
1
Stemagen Corporation, La Jolla, California, USA 92037; 2The Reproductive Sciences Center, La Jolla California,
USA 92037; and 3Monash Immunology and Stem Cell Laboratories, Monash University,Victoria 3800 Australia
Abstract
Human therapeutic cloning or nuclear transfer stem cells (NTSC) to produce patientspecific stem cells, holds considerable promise in the field of regenerative medicine. The
recent withdrawal of the only scientific publications claiming the successful generation
of NTSC lines afford an opportunity to review the available research in mammalian reproductive somatic cell nuclear transfer (SCNT) with the goal of progressing human NTSC.
The process of SCNT is prone to epigenetic abnormalities that contribute to very low
success rates. Although there are high mortality rates in some species of cloned animals,
most surviving clones have been shown to have normal phenotypic and physiological
characteristics and to produce healthy offspring. This technology has been applied to an
increasing number of mammals for utility in research, agriculture, conservation, and biomedicine. In contrast, attempts at SCNT to produce human embryonic stem cells (hESCs)
have been disappointing. Only one group has published reliable evidence of success in
deriving a cloned human blastocyst, using an undifferentiated hESC donor cell, and it
failed to develop into a hESC line. When optimal conditions are present, it appears that
in vitro development of cloned and parthenogenetic embryos, both of which may be utilized to produce hESCs, may be similar to in vitro fertilized embryos. The derivation of
ESC lines from cloned embryos is substantially more efficient than the production of
viable offspring. This review summarizes developments in mammalian reproductive
cloning, cell-to-cell fusion alternatives, and strategies for oocyte procurement that may
provide important clues facilitating progress in human therapeutic cloning leading to
the successful application of cell-based therapies utilizing autologous hESC lines.
Index Entries: Cloning; embryonic stem cells; human; oocyte donation; somatic cell
nuclear transfer; therapeutic cloning.
Introduction
*Correspondence and reprint
requests to:
Andrew French,
Stemagen Corporation,
La Jolla, CA 92037.
E-mail: [email protected]
Human “therapeutic cloning” describes
the use of somatic cell nuclear transfer
(SCNT) to produce patient-specific stem
cells. If scientific advances allow therapeutic cloning to be performed in a consistent
and efficient manner, its potential in understanding and developing treatments for
degenerative diseases is potentially limitless. Given the recent voluntary withdrawal,
amid allegations of scientific impropriety,
of publications that claimed successful
human therapeutic cloning (1), it would
seem an opportune time to regroup and to
review the available data from mammalian
reproductive SCNT research that may ultimately suggest new strategies to progress
the goal of therapeutic cloning in humans.
As a starting point, it is useful to re-examine
the parallels and recent developments in
*According to the International Society for Stem Cell Research’s (ISSCR) Nomenclature
Statement (September 2, 2004).
265
266 _________________________________________________________________________________________________ French et al.
mammalian reproductive cloning that may ultimately provide
the conduit to the promise of safe and effective patient-specific
pluripotent cells for cell therapies, and tissues for regenerative medicine (2–8). Human nuclear transfer (NT) remains in
its infancy, with the one reliable report demonstrating the generation of a single cloned blastocyst from an undifferentiated
human embryonic stem cell (hESC) donor cell, and it failed to
reinitiate into a hESC line (9).
This review highlights recent strategies in mammalian biology that improve the efficiency of nuclear transfer and nuclear
reprogramming. These strategies include the use of pluripotent
cells as donor cells and the treatment of donor cells with cellular extracts to increase the access of reprogramming factors to
donor chromatin. Potential approaches to the challenging problem of procuring high-quality oocytes for use in the generation
of cloned human embryos is the critical first step toward the
accomplishment of SCNT for patient-specific ES cells.
Reproductive Cloning
The restoration of totipotency to a differentiated nucleus
following fusion with an enucleated oocyte, to produce healthy
cloned animals, has been progressively applied to an increasing number of animal species using a variety of donor cell
types (10–13). Although remarkable, the process is highly inefficient and subject to epigenetic errors that result in high rates
of morbidity and mortality throughout development.
Although the potential applications of SCNT in research,
industry, and animal breeding are vast (10,14–19), they remain
hampered by the requirement to achieve competitive commercial outcomes. To date, SCNT, in combination with transgenic strategies, has been applied to laboratory and domestic
animals in the areas of:
1. Basic research to generate animal models of human disease to further our understanding of biological events
surrounding development and functional genomics;
2. Animal breeding programs to increase biological efficiency at a reduced cost;
3. Preclinical testing of new therapeutic interventions for
human medicine; and
4. Genetic rescue of endangered mammals by cross-species
SCNT.
To achieve full-term development, the cloned embryo has
to overcome a significant number of molecular and cellular
challenges, including the regulatory environment of the recipient oocyte, the plasticity of reprogramming in the donor cell,
recipient and donor cell cycle coordination, technical competence of micromanipulation, chromatin remodeling and reprogramming of gene expression, parthenogenetic activation of
the reconstructed cell, and exposure to culture conditions used
to reinitiate embryonic development.
The rate of generation of cloned offspring by nuclear transfer remains low in all species, irrespective of donor cell type
(20–23). In many species, and within strains of species, it remains
technically challenging to produce cloned offspring.
Conversely, nuclear transfer in the mouse and bovine has been
used successfully to generate ESC lines from somatic cells with
relative ease using a variety of genotypes and cell types from
both genders (24,25). These ESCs have been used to rescue
immune-deficient phenotypes (26); however, it remains to be
determined if ESC lines have the same functional characteristics as those derived from fertilized embryos. Recent evidence
in the mouse suggests a high degree of equivalency between
nuclear transfer stem cell (NTSC)-derived ESCs and those from
fertilized mouse blastocysts (27). Collectively, the data indicates that the generation of ESCs from NTSC (therapeutic
cloning) is far more efficient (up to 10 times higher in the mouse)
and hence less exacting, than the demands for complete development to term of SCNT reconstructed embryos (28–32).
Mammalian SCNT
Although significant variations exist, the basic features of
SCNT in both laboratory and domestic animals involve the transplantation of a diploid nucleus into a mature oocyte cytoplast.
The procedure was first successfully accomplished in mammals
in 1996 with the birth of a cloned lamb (33) that established that
cell differentiation and commitment to end-stage tissue type
can be reversed to enable complete resetting of the embryonic
developmental program. To date, reproductive cloning has been
successfully used to derive offspring in 12 species using approx
12 different donor cell types from among the approx 210 adult
differentiated cell types that exist in mammals (22). Some cell
types, including fetal fibroblasts (11,34), ESCs (35–37), and
oviduct epithelial cells (38), appear more suitable as donor nuclei
for generating offspring than others, although this hypothesis
has not been conclusively established (39,40).
Around 6% of all embryos transferred to the synchronized
reproductive tracts of surrogate mothers result in healthy
long-term surviving clones (41). The majority of cloned
embryos fail to thrive as a result of developmental anomalies
in both the fetus and placenta (see Fig. 1). These developmental abnormalities are likely to be as a result of aberrations in
gene regulation mechanisms, including errors of epigenetic
reprogramming of the donor genome following nuclear transfer (35,36,42–44) and are later manifested as precocious or
absent patterns of gene expression during development.
Abnormalities have been detected in DNAmethylation (44–46),
chromatin modification (47), X-chromosome inactivation
(48,49), and expression of imprinted and nonimprinted genes
(50–56). Even apparently healthy cloned animals show epigenetic defects affecting the expression of genes activated later
in development or adulthood (57).
A likely source of variability may be species-specific,
including but not limited to speed of development, response
to parthenogenetic activation stimuli, and differing metabolic requirements during in vitro culture, or they may
derive from variations in the methodologies used. The progeny from clones appear to be free of both developmental
and phenotypic anomalies (41,54). The physiology of surviving postpubertal cloned bulls and the quality of their
collected semen apparently share equivalent reproductive
potential to their original cell donor with no evidence of any
deleterious effects in their progeny (58–60). Although confirmation is required at the molecular level, it appears epigenetic errors prevalent during in vivo development of
cloned animals are subsequently erased during gametogenesis, preventing transmission of aberrant cloning phenotypes to the offspring of clones.
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Fig. 1. Developmental competence of in vivo, IVM/IVF, and IVM/SCNT bovine embryo production systems.
The reasons for the apparent markedly reduced developmental competence of SCNT embryos when compared with in
vivo embryos and those from other assisted reproductive technologies such as in vitro fertilization (IVF), has been the subject of considerable scientific debate. Perhaps unexpectedly
then, a direct comparison of the in vitro development rates of
cloned vs IVF embryos may serve to clarify this matter (25,61).
Although variation between laboratories and subtle differences
between species impact the overall success of reproductive
cloning, it appears that once optimal conditions have been established in any given laboratory, the in vitro development of both
cloned and parthenogenetic embryos approaches that of in vitro
fertilized embryos (see Fig. 2; 25,61) and both may be used to
derive ESCs. Thus, it may be that attaining the “Gold Standard”
for in vitro fertilized embryos will emerge as the key indicator
of proficiency in nuclear transfer programs.
SCNT Methodologies
SCNT is a technically demanding, multistep procedure with
the potential for cumulative errors that can impact developmental potential and each stage of development. Although
numerous variations exists, the basic features of the SCNT procedure, used in both laboratory and domestic animals, have
remained the same since nuclear transfer in mammals was first
performed in the early 1980s and can be summarized as follows:
1. Enucleation of the oocyte to form a cytoplast;
2. Merger of the donor cell or nucleus with the cytoplast to
form a reconstructed one-cell embryo;
3. Parthenogenetic activation either before of after reconstruction of the reconstructed one-cell embryo; and
4. Preimplantation embryo in vitro culture.
Maternal chromosomes are most commonly removed (enucleated) by aspirating or extruding a membrane-bound portion of cytoplasm containing the metaphase plate, or by
bisection containing the metaphase plate. Other methods of
enucleation include centrifugation, inactivation, or destruction by ultraviolet or laser irradiation, or noninvasively
expelled by chemically induced extrusion (11). Successful enucleation is confirmed by incubating the oocyte in Hoechst
33342, a nontoxic DNA interchalating agent, which fluoresces
in the presence of ultraviolet light (11). Reconstructed onecell embryos are formed when the donor cell or karyoplast is
deposited into the perivitelline space and adjacent membranes
are fused by exposure to a series of electrical pulses, inactivated Sendai virus, or treatment with chemicals such as polyethylene glycol. Alternatively, donor nuclei have been directly
injected into the cytoplasm of the cytoplast in manner akin to
intracytoplasmic sperm injection (ICSI).
Oocyte Source and Availability
The rapid progress seen in optimizing the reproductive
cloning of domestic animals was in no small part the result
of the availability of an alternative and almost unlimited supply of developmentally competent oocytes; those harvested
from ovaries opportunistically scavenged from abattoir processing. There is little doubt that the quality of the oocyte,
i.e., its intrinsic developmental potential is a key factor determining the proportion of in vitro fertilized or nuclear transfer embryos that grow to the blastocyst stage (62), and it also
influences the overall success of any assisted reproductive
technology. An eloquent demonstration of this effect was
shown when the superovulation protocol in the bovine was
manipulated to produce an almost maximal number of competent
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Fig. 2. SCNT proficiency in the bovine—a retrospective profile of in vitro production systems to the blastocyst stage. A yearly profile of in vitro
produced bovine blastocysts showing percents of in vitro matured (presence of polar body [PB], metaphase II), in vitro fertilized (IVF blastocyst
rate), parthenogenetically activated (PA blastocyst rate), and cloned (SCNT blastocyst rate). Linear trend lines shown for each of the four groups
(ivm – solid; parthenogenetic – solid; IVF – dotted; SCNT – dashed lines). Modified from ref. 61.
cumulus–oocyte complexes (COCs) as determined by blastocyst formation and significantly improved in vitro embryo
production systems (63).
SCNT Improvements
Cell Synchronization
Coordination of the cell cycle between the donor cell nucleus
(karyoplast) and the recipient cell cytoplasm (cytoplast) is
essential to minimize DNA damage and to maintain euploidy
in the reconstructed cloned embryo (64). Cloned offspring have
been obtained using cytoplasts at telophase (second meiotic
division) and interphase of the first cell cycle and at metaphase
II (second meiotic division) (64,65). The readily available
metaphase II cytoplasts have typically become the recipient of
choice for SCNT experiments.
Cell cycle inhibitors have been used to increase cell cycle
synchronization. Recent work in the bovine has shown
that donor cells treated with a new metaphase arrestor
(2-methoxyestradiol) permitted increased recovery of mitotic
cells following shaking. When these cells, arrested at early G1,
were subsequently used for nuclear transfer, the resulting SCNT
embryos were shown to have a higher developmental competence (blastocysts and live birth rates) (66).
Chromatin Transfer
The general consensus of laboratories performing reproductive cloning in animals is that future improvements are
most likely to come from a greater understanding of the molecular mechanisms of reprogramming. In the bovine, a novel
system allowing remodeling of mammalian somatic nuclei
in vitro before SCNT has been developed (67). Donor cells
are permeabilized to allow a mitotic extract access to the chromatin and to facilitate removal of nuclear factors solubilized
during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes before activation. When compared with nuclear transfer embryos, these
chromatin transfer embryos appear to exhibit a pattern of
markers that more closely resembles those of normal IVF
embryos.
Initial results indicate that calves born after chromatin transfer may have a greater survival (see Fig. 3; A French unpublished observations). More importantly, this technique provides
a method for directly manipulating the somatic donor chromatin before transplantation, and thus it may be a useful tool
to investigate mechanisms of nuclear reprogramming and to
enable improvements to the efficiency of both reproductive
and therapeutic cloning.
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Fig. 3.The efficiency of a generation of bovine SCNT calves using traditional nuclear transfer, chromatin transfer, and hand-made cloning methodologies.The developmental competence of bovine SCNT embryos from a number of somatic fibroblast cell lines using traditional nuclear transfer, handmade cloning,and chromatin transfer methodologies against IVF embryos from the same laboratory (2003–2005; A French unpublished observations).
Therapeutic Cloning
There is a clear distinction between reproductive cloning
and the derivation of embryonic cells for their potential use in
regenerative medicine. Human reproductive cloning is not
only unethical, it is illegal throughout much of the world.
Therapeutic cloning, on the other hand, is becoming increasingly accepted with substantial governmental funding
available in some localities. These promising initiatives have
been guided by principles (68,69) that address the associated
wide-ranging ethical and societal concerns (70–73), that may
ultimately permit the accrual of benefits to society though
improvements in regenerative medicine. Undoubtedly,
continued research designed to improve the efficiency of
reproductive cloning in laboratory and domestic animals,
and to develop alternate somatic cell reprogramming
techniques, will prove useful in parallel studies of methodologies to increase the efficiency of patient-specific stem cell
strategies.
Therapeutic Cloning in Animal Models
In 2000, the utility of therapeutic cloning was first demonstrated in the mouse, when autologous ESC were derived
from a cloned mouse blastocyst (74,75); this observation
was subsequently reaffirmed in a number of similar publications (26,28,76). However, although autologous cell
transplantations using isogenic ESCs have not been
reported, synergic transplantations have elicited a partial
rescue of a deficient phenotype (26). Homologous recombination in the Rag 2 –/– allele was used to correct the genetic
defect in a nuclear transfer embryonic-stem cell line derived
from this immuno-deficient mouse. Partial rescue of the
phenotype was achieved following transplantation of
hematopoietic precursors differentiated from the corrected
nuclear transfer ESC line.
Therapeutic Cloning in Human
and Nonhuman Primates
At present, only one reliable published report describing
the production of a human NTSC blastocyst exists. This
blastocyst was derived following fusion of an undifferentiated ESC (9) with an oocyte obtained from a follicular-reduction procedure. Unfortunately, this blastocyst failed to
develop into an ESC line. Importantly, this report suggested
that the oocyte source and age may influence NTSC
outcomes. Other preliminary reports have generated only
early cleavage stage nuclear transfer embryos (77b) and
using cytoplasts obtained from aged human IVF oocytes
that had failed to fertilize (77a). Lu et al. (2003) have also
reported to have cloned human embryos using fetal fibroblasts and granulosa cells, although the reliability of the
finding is uncertain since no DNA fingerprinting or other
genetic confirmation demonstrating that they were indeed
clones was provided (77c).
The generation of SCNT blastocysts in nonhuman primates
appears to be far more inefficient as compared with other
species, and attempts at therapeutic cloning have been entirely
unsuccessful. The failures to date have been attributed to misaligned chromosomes and defects in microtubule kinetics
resulting from removal/interruption of microtubule proteins
(nuclear mitotic apparatus protein [NuMA] and kinesinrelated protein [HSET]) at the time of enucleation (78,79).
However, primate embryonic NT has been used successfully
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Fig. 4. ESCs for cell-based therapies.
to derive offspring (80). The establishment of therapeutic
cloning in nonhuman primates could be important for investigating the effects of undifferentiated and differentiated ESCs
in transplantation studies and may allow extrapolation to
human therapeutic intervention. Potential strategies for the
derivation of human ESC lines for cell-based therapies are
shown in Fig. 4.
Limitations to Human Therapeutic Cloning
SOURCE OF HUMAN OOCYTES
Undoubtedly, a significant impediment to progress in the area
of human therapeutic cloning is the very limited availability of
high-quality human oocytes for this purpose. In the human,
the emergence of IVF as a highly successful treatment option
for infertility has given rise to 25 yr of experience in ovarian
stimulation (81). The ability in the human to induce multiple
dominant follicles by gonadotrophin stimulation and to grow
and produce mature, developmentally competent oocytes has
improved the chance of conception both in vivo (empirical
ovarian stimulation with or without intrauterine insemination) and with IVF (82). Ovarian stimulation permits the
retrieval of numerous COCs and thus compensates for
inefficiencies in oocyte maturation, fertilization, in vitro culture, and subsequent in vivo development that might result
from having these events occur in vitro rather than in vivo (81).
The success of SCNT will very likely be influenced by the
age and fertility status of the woman donating her oocytes, with
younger women without a history of infertility presumably
being the best donor candidates. Although ethical and legal
considerations strongly impact the availability of donor oocytes,
several prospects exist for consenting patients to provide
oocytes for compelling research proposals. To date, sources of
donated oocytes for research have ranged from altruistic oocyte
donation in which no cost or benefit accrues to the donor (9) to
a more traditional oocyte-donation model in which donors
receive compensation, either monetary or in the form of reduced
treatment fees. Although compensation in excess of expenses
is controversial and prohibited by law in some localities, a level
of compensation that is not viewed as an undue inducement
might prove to be practically necessary to obtain the quantity
of oocytes necessary for research in this area. As has been prescribed for reproductive oocyte donation, this compensation,
if allowed, would acknowledge the very real fact that ovarian
stimulation and oocyte retrieval are not without risk or discomfort (83,84).
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Source of donated of oocytes for the purposes of SCNT may
include the following categories:
1. Oocytes that fail to fertilize following either IVF or ICSI.
However, no data exists at present to support evidence
that aged oocytes, or those that have failed to fertilize
in IVF or ICSI, provide a suitable ooplasm for reprogramming donor nuclei (9,77), their utility may be
restricted to proficiency testing.
2. Oocytes recovered following follicle reduction, a procedure performed to minimize the risk of multifetal pregnancy following unintended excessive gonadotrophic
stimulation during infertility treatment.
3. Immature oocytes obtained from ovarian tissue following oophorectomy, hysterectomy, and caesarian section.
4. Oocytes obtained after ovarian stimulation and oocyte
retrieval that are excess to reproductive requirements (9).
There is a need in some of these categories for effective in
vitro maturation protocols to be optimized for human oocytes,
so that immature oocytes can complete their normal maturation at a practical rate, thus allowing fertilization and early
preimplantation embryonic development to occur. Immature
oocytes recovered opportunistically at surgery and those recovered as a proportion of COCs following ovarian stimulation for
IVF, also require in vitro culture for cytoplasmic maturation to
be completed. Immature human oocytes obtained from patients
undergoing gynecological surgery or ovulation induction can
be matured and fertilized in vitro (82). Following transfer of
these embryos to the patient’s uterus, pregnancies and live births
have been achieved (85). However, the developmental capacity
of in vitro matured oocytes is reduced compared with those
retrieved after maturation in vivo (86,87). An alternate source,
one that involves the nurturing of immature oocytes from fetal
ovarian tissue (88), is unlikely to have ethical support.
As the technology of human SCNT matures and the application of vitrification techniques for oocyte cryopreservation
is increasingly adopted (89), it is foreseeable that cryobanks of
human ova available for donation could be established to provide a readily available source of cytoplasts for NTSC and therapeutic purposes (90). An alternative supply of human oocytes
for NTSC could come from the differentiation of ESCs into germ
stem cells and putative ova in vitro. Preliminary studies in the
mouse have shown that oocyte-like cells can be produced following differentiation of mouse ESCs (91,92). It does appear
that functioning oogonia express markers restricted to this cell
type (91,92), although confirmation of their ability to fertilize
and produce viable young will be required to fully confirm this
potential as a source of reprogramming cytoplasts (93).
Reprogramming and Remodeling
Critical to successful SCNT is the resetting or reprogramming of the epigenetic modifications that maintain the
differentiated state of the somatic genome, a state conserved
by DNA methylation and by the covalent modification of
nucleosome histones. For embryonic development to proceed, the oocyte cytoplasm must interact with the transferred
donor nuclei to silence expression of its somatic cell-specific
genes and upregulate embryonic-specific genes in the correct spatio-temporal manner. Although relatively little is
known of the cellular factors responsible for remodeling or
reprogramming, studies in both Xenopus (94) and mammals
(95) have revealed a role for nucleoplasmin in the removal
of histones and for the imitation switch in the removal of
TATA-binding proteins (TBP) from DNA (96). The mechanisms governing nuclear reprogramming appear to be widely
conserved with oocyte components from different species
having the ability to reprogram gene expression in a variety
of somatic cells from other species (97). The remodeling of
nuclear structures by oocyte components results in the reactivation of genes associated with pluripotency in early
embryos and pluripotential stem cells (98). The failure or
incomplete silencing of gene products that relate specifically
to the donor cell types can generate profoundly altered characteristics that lead to severe consequences for the development of cloned embryos (99).
Mitochondrial Heteroplasmy
The unfertilized mammalian oocyte contains approx 100,000
mitochondria. Following fertilization and during early cleavage development, the small number of paternally derived mitochondria, transferred with sperm, are eliminated by an as yet
unknown mechanism. The activity of ubiquitin, and the lysosomal apparatus of the egg appear to be involved in the proteolytic destruction of bovine-sperm mitochondria inside egg
cytoplasm, although the mechanism of ubiquitination of sperm
mitochondria leading to their destruction appears speciesspecific (100). By the time the blastocyst stage of development
is reached, cells contain approx 100 mitochondria (indicating
there is no replication) of entirely maternal contribution
(101,102). Rarely, however, mitochondrial heteroplasmia does
occur in humans (103) and in other mammals (102,104).
Mitochondrial heteroplasmy has been observed following
SCNT in other mammalian species (105–107), although not in
all cloned offspring (108,109). The modified somatic cell
nuclear-transfer technique, termed “hand-made cloning,”
involves the construction of several fused cytoplasts so that
the SCNT blastocyst, although genomically identical, could
have mitochondrial DNAcontributions from two to six oocytes
(110). In an experiment conducted in the bovine, there was no
evidence of any harmful effect of mitochondrial DNA heteroplasmy or differences in reproductive efficiency when compared with the standard nuclear-transfer procedure using a
single oocyte (110). There is a possibility that species-specific
or methodological differences could exist that could lead to
variations in the level of mitochondrial heteroplasmy that may
have implications for human therapeutic cloning and this warrants further investigation (111,112). Arecently described ubiquitination method may represent a mechanism for the
elimination of paternal mitochondria during fertilization in
some species (100).
Parthenogenetic Activation
Critical to the reprogramming of the donor nucleus and the
development of NTSC embryos is the resumption of meiosis
and restoration of cell cycle progression (113). During normal
fertilization, sperm penetration activates the phosphoinositide
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pathway (114,115), thereby triggering Ca(2+) release and egg
activation through a prolonged series of intracellular free calcium ion oscillations that continue for several hours. Although
the precise signaling pathway initiated by the sperm remains
unknown, recent evidence favors the delivery of a soluble
protein factor by the sperm that increases the production of
inositol 1,4,5-triphosphate, which acts to open Ca(2+) channels in the endoplasmic reticulum thereby releasing Ca(2+)
into the cytosol.
A variety of electrical and chemical activation methods that
influence maturation promoting factor activity and mitogenactivated protein (MAP) kinase activity have been devised to
duplicate or closely mimic the changes in the oocyte cytoplasm
that normally are triggered by the sperm during fertilization
(116). However, most treatments cause only a single transient
Ca(2+) spike, which serves to activate only a fraction of target
oocytes, one that increases with the time after ovulation.
Attempts to prolong the Ca(2+) oscillations have been successful
with less mature oocytes but have been unwieldy (117,118).
A popular and highly effective oocyte activation method
involves inducing an elevation in intracellular Ca(2+) with a
calcium ionophore while maintaining low levels of maturation promoting factor using a protein synthesis inhibitor, for
several hours after the initial Ca(2+) elevation. Regardless of
the activation procedure utilized, however, both pregnancy
and survival rates after birth of cloned animals remain low.
Parthenogenetic activation is therefore likely to be a cocontributor to the inefficiency of cloning. New activation protocols that more closely mimic fertilization and the physiological
actions of sperm may yield improvements in viability.
Human oocytes, like those of other mammals, can be artificially activated by stimuli that elevate cytosolic Ca(2+) levels,
with both fresh and aged oocytes subsequently undergoing
pronuclear formation and early cleavage division (119).
However, as other studies have found that calcium-ionophore
treatment alone does not activate human oocytes in a reliable
fashion (120,121), the commonly used activation protocols in
human studies also combine a calcium ionophore along with
protein synthesis or protein kinase inhibitors (122–124).
Although these combination treatments have induced pronuclei formation in activated human oocytes, the success rates
in terms of preimplantation development to the blastocyst
stage are still very poor when compared with embryo development after in vivo or IVF.
Cytosolic extracts from sperm can cause Ca(2+) oscillations
in a range of different mammalian oocytes, including humans
(114,125,126). In a further refinement of this approach, Saunders
et al. (127) isolated a novel protein from sperm that is a specific
isoform of phospholipase C (PLC), referred to as PLCζ (zeta) (128),
which is responsible for generating Ca(2+) release and inducing InsP3 production. Homologs for this protein exist in humans
and primates (129). Microinjection of cRNA encoding human
PLCζ induced a prolonged series of calcium oscillations in aged
human oocytes that closely mimicked the repetitive nature of
the Ca(2+) stimulus provided by the sperm during human fertilization. At low concentrations, injection of PLCζ; induced
parthenogenesis and development to the blastocyst stage (130).
The use of sperm extracts and PLCζ to activate human oocytes
for the purposes of generating both cloned and parthenogenetic
stem cell lines certainly warrants further investigation.
Parthenogenetically activated oocytes themselves represent
a possible source of pluripotential stem cell lines that are not
embryonic. A nonhuman ESC line was established by Cibelli
et al. (131,132) from parthenogenetic activation of monkey
oocytes that showed all the characteristics of traditional human
ESCs. Recently, Brevini et al. (133) have reported the production of human ESCs derived from parthenogenetically activated human oocytes. Oocytes were activated by 5 µM
ionomycin (5 min) and 2 mM 6-dimethylaminopurine
(6-DMAP) (3 h). Two cell lines extensively proliferated as
diploid, undifferentiated cells for more than 25 passages and
maintained the expression of pluripotential markers. They
showed differentiation as embryoid bodies in the three primary germ layers (endoderm, mesoderm, and ectoderm) in
vitro. This data suggests that nonembryonic ESCs can be established from unfertilized human oocytes and this should be further explored as a source of new pluripotent stem cells.
Human Embryo Culture
Culture environment during the preimplantation period of
development profoundly influences the physiology and viability of mammalian embryos. Susceptibility to environmental stimuli decreases as development proceeds. The degree to
which abnormal gene expression and altered imprinting patterns can be reduced or averted by using more physiological
and environmental conditions is presently unknown.
Improvements in the success of the assisted reproductive
technologies for the treatment of infertility have coincided with
the development of highly complex, biphasic culture media
and culture conditions, which offer improved in vitro development, pregnancy rates, and live births (134). A multitude of
commercially sourced media, manufactured under stringent
manufacturing conditions, are available for this purpose.
However, culture media specifically optimized for the development of SCNT is not yet available. Previous work indicates
that NTSC embryos may require different culture requirements
as a result of altered physiology and gene metabolism expression pathways (135). Thus, optimizing such culture conditions
may be important for understanding the kinetics and mechanisms of nuclear reprogramming and to improving the overall process.
Alternative Strategies for Therapeutic Cloning
Cell Fusion Based
The first demonstration that a pluripotent cell could
reprogram a somatic cell following cell fusion to form a stable heterokaryon was in the mouse (136), and this phenomenon has been used to study nuclear reprogramming and gene
expression (137). In an analogous human study, ESCs have
recently been shown to efficiently reprogram human adult skin
cells following cell to cell fusion (138). The current limitation
remains the removal of the original ES cell nuclei to form a
diploid isogenic ESC line, although recent work indicates that
the use of centrifugation both before and after fusion can accomplish this task (139,140). Spontaneous heterokaryon formation
is known to occur in vivo, as fused cell types have been observed
following transplantation of bone-marrow-derived cells into
adult mice brain and liver (141–143). Although this technology has the potential to replace the use of human oocytes in
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the reprogramming process, the low rates of spontaneous
fusion, the inefficiency of producing large numbers of reprogrammed diploid cells, and a variety of safety concerns may
ultimately limit its use (144).
Interspecies Therapeutic Cloning
The scarcity of human oocytes available for this type of
research has led some researchers to generate human ESCs by
reprogramming somatic cells with oocytes from another species
(145). ESCs generated by this process appear to retain the characteristics of those derived from human embryos (145). The
apparent universally conserved ability of oocyte components
to reprogram gene expression in somatic cells is generating
considerable interest leading to its exploration in several species
(146–149). The utility of this approach requires further investigation. To overcome the concerns of mitochondrial heteroplasmy, it has been suggested that the ooplasm, mitochondria,
as well as other ultrastructural components be replaced with
human equivalents in a process similar to cytoplasmic transfer to allow a generation of blastocyst made up entirely of
human material (8).
Extract-Mediated Transdifferentiation
The elucidation of the mechanisms associated with nuclear
reprogramming could eventually lead to a direct reprogramming of human somatic cell nuclei without the use of eggs.
Such an approach would ameliorate many of the difficulties
associated with current nuclear transfer procedures and hasten the generation of replacement cells for therapeutic purposes. The demonstration that adult stem cells have broader
differentiation potential than anticipated and that they can
contribute to tissues other than those in which they reside,
has led to the development of a novel process that directly
converts one somatic cell into another (a process known as
transdifferentiation) (150,151). Functional reprogramming of
a somatic fibroblast cell using a nuclear and cytoplasmic extract
derived from another somatic cell type (T-cells) was demonstrated by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodeling complex,
changes in chromatin composition, and activation of lymphoid
cell-specific genes. The reprogrammed cells expressed surface
molecules specific to T-cells and exhibited apparently normal
regulatory functions. In vitro cell reprogramming may also
allow an examination of the mechanisms of nuclear reprogramming. Its applicability remains limited by the large
amount of cell extract needed to reprogram a single cell; it is
estimated that the extract from 300 cells is required to initiate
transdifferentiation in a single cell. This approach may be useful in the identification and characterization of new molecules
involved in the remodeling/reprogramming process.
Conclusions
Although therapeutic cloning for the purpose of creating
patient-specific stem cells has considerable promise, its realization depends on the development of robust SCNT technologies utilizing human oocytes. Since the birth of the first
mammalian clone almost 10 yr ago, the technique has been
successfully accomplished in a wide range of mammalian
species. Although the rapid rate of progress in this area is certainly impressive, both cloned embryos and offspring exhibit
variations in regulation of gene expression as a consequence
of the epigenetic interplay between the original somatic cell
and its new embryonic environment. As a result, only a small
percent of cloned embryos result in viable offspring; however,
surviving clones appear to have equivalent physiological and
reproductive capacities when compared with their noncloned
counterparts. As a result of these studies, a considerable body
of work has been accumulated on the technical, biological, and
molecular aspect of nuclear transfer in mammals. Key factors
in the success of SCNT appear consistent across mammals and
include cell type, degree of cell-cycle synchrony, effectiveness
of parthenogenetic activation, and in vitro culture conditions.
Recent developments have focused on events surrounding
reprogramming and the initiation of embryonic development
in an effort to more closely replicate the well-orchestrated
events observed in more developmentally competent IVF and
in vivo embryos. It appears that when fully optimized, in vitro
development rates of cloned and parthenogenetic embryos
may approach those of in vitro fertilized embryos, suggesting
an important gauge of SCNT proficiency in any given species.
Of increasing relevance to human therapeutic cloning, is that
the efficiency of deriving ESC lines from cloned embryos is
substantially more efficient than the production of viable offspring. Moreover, ES lines derived from cloned blastocysts
appear to share identical characteristics to those derived from
IVF embryos.
However, it is possible that a greater proportion of ESC lines
might have a high rate of epigenetic errors because statistically many of the original embryos would not have resulted
in viable animals. Nonetheless, recent reports (152) showing
transcriptional profiles consistent with normal development
of ES cell lines derived from cloned and fertilized mouse blastocysts, were functionally indistinguishable and had identical
therapeutic potential. The derivation of ESC from cloned
embryos appears to rigorously select for those immortal cells
that have erased the “epigenetic memory” of the donor nucleus,
in contrast to aberrant cloning phenotypes observed in the
embryonic and fetal development of cloned animals (152).
Progress in SCNT in humans has been disappointing and
is likely to be constrained by the ethical considerations
involved in the procurement of high-quality oocytes. However,
the enormous potential of patient-specific and disease-specific
ESC lines has reinvigorated the research effort in human therapeutic cloning. With research burgeoning in this area, it seems
it is only a matter of time before it will be successfully accomplished. The full realization of the therapeutic utility of NTSC
is likely to be dependent on the ability to establish human ESC
lines in defined conditions that allow the rapid multiplication
of a purified population of an undifferentiated homogenous
cell type, whereas avoiding contamination by animal products and pathogens.
Advances in human SCNT for the purpose of ESC line
derivation are likely to result from a careful examination of
ongoing research undertaken for mammalian reproductive
cloning and other synergistic technologies associated with
reprogramming. Hopefully, the use of this information will
allow a higher level of efficiency for human therapeutic
cloning and assist in developing guiding principles that will
facilitate the accomplishment of autologous ESC lines for
cell-based therapies (153–156). In the meantime, it is apparent
Stem Cell Reviews ♦ Volume 2, 2006
274 _________________________________________________________________________________________________ French et al.
that parthenogenetically activated oocytes are capable of
producing nonembryonic pluripotential stem cells and this
source of stem cells for therapeutic application should be
explored in detail.
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