Blood Reviews (2005) 19, 321331

www.elsevierhealth.com/journals/blre

REVIEW

Therapeutic potential of embryonic stem cells

Paul H. Lerou, George Q. Daley *
Karp Family Research Building 7th Floor, Childrens Hospital, 300 Longwood Avenue,
Boston, MA 02115, USA

KEYWORDS

Summary Nearly 20 years after murine embryonic stem cells (mESC) were isolated, the first report of the derivation of human embryonic stem cells (hESC) in
1998 spawned the field of hESC research [Evans MJ, Kaufman MH, Establishment
in culture of pluripotential cells from mouse embryos. Nature 1981;292(5819):
1546; Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines
derived from human blastocysts. Science 1998;282(5391):11457]. Although this
field is only in its infancy, hESC represent a theoretically inexhaustible source of
precursor cells that could be differentiated into any cell type to treat degenerative,
malignant, or genetic diseases, or injury due to inflammation, infection, and
trauma. This pluripotent, endlessly dividing cell has been hailed as a possible means
for treating diabetes, Parkinsons disease, Alzheimers, spinal cord injury, heart
failure, and bone marrow failure. But the regenerative medicine applications of
embryonic stem cells are only one facet of hESC therapeutic potential. Human
ESC are an invaluable research tool to study development, both normal and abnormal, and can serve as a platform to develop and test new therapies. In addition to
discussing the therapeutic potential of hESC, this chapter will cover limitations to
using hESC for replacement cell therapy, strategies to overcome these limitations,
and alternative methods of deriving hESC.
c 2005 Elsevier Ltd. All rights reserved.

Human embryonic
stem cells;
Regenerative medicine;
Nuclear transfer

Introduction
Stem cells are defined by two basic properties: the
ability to self renew indefinitely and the ability to
differentiate into one or more specialized cell

*
Corresponding author. Tel.: +1 617 919 2013; fax: +1 617
730 0222.
E-mail addresses: paul.lerou@childrens.harvard.edu (P.H.
Lerou), george.daley@childrens.harvard.edu (G.Q. Daley).

types. Study of murine embryonic stem cells

(mESC) has paved the way for hESC research. Several tissues depend on an adult or somatic
stem cell pool for maintenance. The hematopoietic
system, skin, gut, and some parts of the central
nervous system fall in this category. Several diseases are characterized by a depletion of the stem
cell pool such as bone marrow failure due to malignancy of the hematopoietic stem cell (HSC) resulting in leukemia and lymphoma, or genetic defects
in the HSC itself (e.g., Fanconis Anemia). Other

0268-960X/$ - see front matter c 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.blre.2005.01.005

322
diseases involve destruction of tissues that may not
be robustly replenished from stem cells pools: Type
1 diabetes due to auto-immune destruction of the
pancreatic beta cells; liver failure due to cirrhosis
either from toxins or infectious agents. These diseases have been successfully treated by replacing
the stem cell pool as in bone marrow transplant
(BMT), or with direct organ transplant as in the
treatment of heart, liver, or pancreatic failure.
Several limitations render organ transplantation
less than ideal; the most glaring being the lack of
organ and tissue donors. Human ESC could potentially be directed to differentiate into adult stem
cells or tissues and be used clinically to reconstitute the depleted adult stem cell pool or degenerating organs. For organs and tissues not maintained
by an adult stem cell population, like the adult
pancreatic beta-cell,3 differentiation of hESC in vitro to derive the desired tissue would be required.
The other major limitation to organ transplantation
is the immune barrier, which necessitates immunosuppression to prevent graft rejection or, in the
case of BMT, graft versus host disease.
Human development is a complex choreography
of events, each taking place in a critical temporal
and spatial pattern. One of the principal challenges
of hESC research is unraveling the developmental
pathways that specify formation of specific
tissues within the embryo, so these pathways can
be recapitulated in vitro. The following cell types
have been derived from hES cells: neural tissue,36
insulin secreting cells,8 cardiomyocytes,811 hematopoietic cells,1315 endothelial cells,16 osteoblasts,17 and hepatocytes.18 The strategies used
to derive these specialized cell types are a
combination of culture conditions that favor differentiation towards the desired cell type, transgenic
approaches that exploit factors known to direct differentiation, and reporter systems to identify and
allow isolation of that cell type. The derivation of
cardiomyocytes from ESC (murine and human) is
well described, and illustrates how these different
strategies are used.

Cardiomyocytes from hESC

When ESC are removed from culture conditions
that block differentiation, they aggregate and develop into cystic structures called embryoid bodies
that contain derivatives of all three embryonic
germ layers,19 including cardiomyocytes.2022
Methods for the differentiation of cardiomyocytes
from murine ESC are well defined and yield a variety of cardiomyocytes including atrial, ventricular,

P.H. Lerou, G.Q. Daley

and sinus-nodal like cells.9,20,21 Using a G418 resistance gene driven by a cardiomyocyte specific promoter, Klug and colleagues were able to select for
cardiomyocytes derived from murine ESC.23 When
transplanted into hearts of dystrophic mice, these
cardiomyocytes engrafted and functioned in situ.
Other investigators have demonstrated similar results in the setting of experimentally induced
infarction in mice.2426 Adenovirus vectors have
been used to introduce reporter genes into the
mESC whose expression is driven by the ventricular
specific myosin light chain-2 gene, thereby allowing isolation of a pure population of ventricular
cardiomyocytes.2629
Directed differentiation towards cardiomyocytes
from hESC is based on protocols used in
mESC.9,10,12,13,31 Cardiomyocyte differentiation
from hESC is enhanced by 5-aza-20 deoxycytodine,
but not retinoic acid, and the population can be
enriched via a Percoll gradient density centrifugation.31 However, these hESC derived cardiomyocytes are immature, and have structural and
functional properties consistent with fetal cardiomyocytes.10 This raises a concern that the in vitro
developmental pathways of ESC recapitulate
embryonic rather than adult cell development.32
This issue has been confronted in attempts to derive
blood stem cells, and the need to generate more
mature cell population may emerge as a challenge
in other cell systems.
In order for hESC to become a clinical source of
engraftable heart cells, or other tissues, methods
of generating a purified cell population will be required. In all the studies of ESC derived cardiomyocyte transplants mentioned earlier, a mixed
population of cardiomyocytes was transplanted.
In a clinical setting however, one would prefer to
engraft a specific type of cardiomyocyte. In the
setting of chronic heart failure or myocardial
infarction, for instance, the cell type needed is a
ventricular cardiomyocyte, not sinus-nodal type,
because the latter could be arrythymogenic and
might cause considerable morbidity.33
Ideally, the hESC derivatives that will be used
clinically would be free of transgenes that introduce unpredictable risks of mutagenesis, potentially negatively affecting the function of
transplanted cells in vivo. The transgenic approach
is helpful in defining development pathways, and
may allow scientists to develop culture conditions
that enrich for cardiomyocyte development without reliance on transgenes. However, if possible,
methods to purify the desired cell should rely on
its endogenous characteristics and cell surface
markers, without introducing reporter genes. In
the event that transgenes will be required, systems

Therapeutic potential of embryonic stem cells

that make this process safe will be of utmost
importance. It may be possible to introduce a suicide transgene for instance.34 In the event that
the transplanted hESC derived cells malfunction, a drug to which they are selectively sensitive can be administered to the patient to destroy
the transplanted cells.

Hematopoietic stem cells from hESC

The hematopoietic stem cell is the best understood
somatic stem cell. It has been used therapeutically
in bone marrow transplants dating back to the
1950s,35 and has been extensively characterized
in both mice and humans (Reviewed in 36). Blood
formation from mouse ESC is readily obtained,
and was reported nearly 2 decades ago when murine ESC were first differentiated into embryoid
bodies.19 However, achieving stable blood engraftment of irradiated mice with ESC-derived HSCs has
proved challenging.3739 In most reports ESCderived hematopoietic cells represent primitive
embryonic progenitors that do not support longterm engraftment. But, more recently, using the
homeobox gene HoxB4 to stimulate HSC expansion,
normal long-term multi-lineage hematopoiesis derived entirely from mESC has been achieved in lethally irradiated mice.40 The cdx4 gene, which
induces hox genes like hoxB4, has also been shown
to play an important in role in deriving HSC and in
promoting high grade blood chimerism in engrafted
animals.41,42 One hypothesis suggests that the
Cdx4-Hox pathway can induce primitive embryonic
progenitors to acquire properties characteristic of
the adult hematopoietic stem cell. Another
hypothesis argues that Cdx4-HoxB4 expands a rare
definitive stem cell that arises unpredictably during
ES cell differentiation. Additional research is
needed to define the specific conditions for differentiating ESC into adult definitive HSC so that this
process can be made efficient and reproducible.
As the morphogens of hematopoietic differentiation are defined and their specific mode and period
of action are elucidated, scientist should achieve
conditions for production of HSC from hESC.
The niche in which stem cells reside is critical
for providing the appropriate milieu of signals for
stem cell self-renewal, survival, and differentiation. When bone marrow failure is due to an intrinsic defect of the hematopoietic stem cell, as in the
case of leukemia or Fanconis anemia, a HSC transplant can be curative. However, bone marrow
transplant for myelofibrosis is rarely curative because the disease process entails destruction of

323
the HSC niche. Other disease processes where the
stem cell niche has been destroyed may also not
be good candidates for monocellular therapy:
burns, hepatic cirrhosis, and certain types of neural
injury. These conditions may still benefit from
stem cell replacement strategies, but these will
need to accompany therapies aimed at the pathophysiologic process extrinsic to the cell that is
being replaced. The capacity of the HSC to home
to its niche in the bone marrow, take up residence
there, and resume function, is one reason for the
long history of success with bone marrow transplants. The niche of others tissues may not be so
readily accessible. Though there is great hope that
hESC based therapies will prove useful for the
treatment of central nervous system disease, delivering cells to the proper niche represents a major
obstacle, as outlined below.

Central nervous sytem repair from hESC

Formation of the nervous system is an early event
in human development, and it is therefore not surprising that neuronal tissue is found in embryoid
bodies formed from hESC.13,43 Mouse embryonic
stem cells allowed to differentiate in monolayer
culture have also yielded neuronal tissue.6,44,45
Neuronal tissue is a vast collection of highly specialized cell types, each with unique roles. Directed differentiation and isolation of specific
neuronal subtypes will need to be achieved. By
using morphogens and transcription factors known
to promote neuronal development, several
researchers have accomplished such directed differentiation from mESC. Jessell and colleagues
used retinoic acid to commit mESC to ectodermal
fates. Subsequent exposure to sonic hedgehog led
to formation of spinal motor neurons which successfully engrafted in the embryonic spinal cord
of chicks, extended axons, and formed synapses
with target muscles.46 Murine ESC derived neuronal
tissue has also been used to generate motor neurons that engraft and restore function in a rat model
of spinal cord injury47 and hemiplegia.48 McKay and
colleagues used sonic hedgehog and FGF8 to instruct mESC differentiation to ventral midbrain
fates, resulting in dopaminergic neurons. In several
instances, mESC-derived dopaminergic neurons
have been shown to repair motor dysfunction in a
rodent model of Parkinsons.4951 Several groups
have demonstrated directed development of neurons from hESC.36
Even if directed differentiation is able to yield
specific neuronal subtypes, other barriers to their

324
use remain. Unlike cells of the hematopoietic system, there is no evidence that when delivered via
the circulation, neurons can home to their appropriate location in the nervous system; thus, they
must be delivered to the correct site surgically.
In order to properly function, neurons require synaptic input from neighboring neurons. Although
synaptic connections are highly plastic, the principle anatomic relationships among neurons and the
major signaling tracts are all established during
embryonic and fetal development. Regenerated
neurons will need to integrate properly with existing, fully developed neurons to re-establish functional neural networks. Perhaps this can be
accomplished by delivering the cells in a precursor
state, which when targeted to the proper niche will
develop further in vivo to achieve proper integration and function. The propensity for cell types derived from ESC to express embryonic phenotypes
may predispose to a certain degree of intrinsic
plasticity that would allow functional integration.
In addition to not restoring function, failure to
integrate properly could result in epileptiform
activity. Moreover, transplanting cells that retain
the ability to differentiate and expand increases
the risk of inappropriate development in situ, leading to disabling deformation or even teratoma
formation.2,43,52
Another safety concern for the clinical use of
hESC-derived products takes note of the fact that
most existing hESC lines have been cultured in
the presence of non-human animal products (e.g.,
cells, serum factors), raising the specter of transmission of animal pathogens. Undifferentiated
growth has been shown on laminin- or Matrigelcoated plastic surfaces supplemented with mouse
embryonic fibroblast conditioned media,53 and isolation of self renewal factors should allow for derivation of new lines under pristine conditions. More
recently, undifferentiated hESC growth has been
shown in the presence of human fibroblasts, adult
epithelial cells, and foreskin cells.5456

Confronting the immune barrier

Transplantation of tissues or organs between
genetically unrelated individuals typically provokes an immune response that leads to rejection
of the graft. The targets of the immune response
are alloantigens on the graft in the form of major
and minor histocompatibility complex (MHC) antigens and ABO blood group antigens. In the undifferentiated state, hESC express low levels of
MHC-I, and differentiation of the cells in vitro

P.H. Lerou, G.Q. Daley

causes a 24-fold increase in expression. Though
relatively low compared to most somatic cells,57
this expression level would be sufficient to cause
hESC and derivatives which are not an HLA match
with the recipient to be destroyed in a T cell
dependent manner. Differentiated hESC might also
be eliminated by delayed-type hypersensitivity.58
Absence of MHC-I molecule expression targets a
cell for destruction by NK cells, but hESC and their
derivatives do not appear subject to elimination
by NK cells in spite of their low level class I
expression.57 Cytotrophoblasts at the materno-fetal interface express very low levels of MHC-I antigens, but are protected from NK cell destruction
due to expression of the non-classical MHC-I molecule, HLA-G.59 HLA-G is not expressed by hESC
and their derivatives, so it has been postulated
that the low expression of MHC-I is sufficient to
protect hESC from NK-cell targeted destruction.57
It remains likely that hESC and their derivatives
will be subject to the same immune barriers that
apply to tissue and organ transplants. Strategies
to minimize rejection of hESC derivatives have
been expertly reviewed by Drukker and
Benvenisty.58
In some clinical contexts, as in transplantation
of non-HLA-matched fetal-derived dopaminergic
neurons in Parkinsons patients, the transplants
survived for years, even in the absence of immunosuppression.60 The durability of such transplants
may follow from the immune-privileged status of
the brain. To overcome the immune barrier in
other therapeutic contexts, it has been proposed
to create hESC banks. Unfortunately, the allele
combination of the three MHC-I genes alone generates 11 million haplotypes, and a billion diploid
combinations.61 Perhaps genetic engineering would
allow for the creation of immunologically matched
hESC lines, but this would be an arduous task.
Yet another strategy to minimize rejection is to
induce immunologic tolerance to donor cells. It has
been shown that mixed hematopoietic chimerism
of the host and donor results in specific tolerance
to tissue grafts from the same donor (reviewed in
62). For example, transplantation of purified HSCs
in mice can produce mixed T cell chimerism and induce tolerance to donor-matched neonatal heart
grafts.63 Fandrich and colleagues injected rat
embryonic stem cell-like cells of WKY origin into
the portal vein of fully MHC-mismatched DA rats
and achieved permanent engraftment and mixed
chimerism without supplementary host conditioning. The mixed chimeras accepted subsequent
WKY donor origin allograft heart transplants.64
Co-transplantation of ES-derived HSCs and any
other differentiated tissue might enable a broad

Therapeutic potential of embryonic stem cells

array of tissue therapies from a limited set of human ESC lines.65,66
Theoretically, the most attractive strategy to
not only minimize, but eliminate immune barriers
is to create isogenic hESC lines by one of several
methods: somatic cell nuclear transfer, somatic
cell fusion with ESC, and parthenogenesis. Somatic
cell nuclear transfer (SCNT) to an enucleated oocyte results in the formation of a blastocyst from
which ESC can be derived. Generating ESC from somatic nuclear cell transfer embryos in mice is well
established.6669 In 2004, a group of scientists from
Korea reported deriving the first hESC line from a
human blastocyst created using somatic cell nuclear transfer.71 It remains to be seen whether mitochondrial DNA which would be oocyte derived,
will pose a significant immune barrier though this
is unlikely.72
Short of nuclear transfer, fusion of somatic cells
with mESC can reprogram somatic nucleus to give
rise to hybrid cell lines that retain pluropotency,
but contain an abnormal number of chromosomes.73 Dr. Paul J. Verma (Monash University,
Australia) has developed fusion protocols to generate stable heterokaryons between diploid (2N) cells
and polyploid (4N or 6N) ESC. Upon differential
centrifugation, the polyploid ESC nucleus is selectively expelled from the heterokaryon, leaving behind the diploid nucleus. Fusion of mouse
embryonic fibroblasts (MEF) with ESC generates
reprogrammed hybrids that express gene markers
of pluripotency (e.g., Oct 4) from the MEF nucleus.74 These results suggest that ESC can reprogram embryonic genes, but additional studies are
needed to show whether the fusion-based ESC-like
cells are indeed pluripotent.
A third means of generating genetically matched
ESC is parthenogenesis, the process by which an
unfertilized oocyte is activated and divides. ESC
can be isolated from a parthenogenetic blastocyst,
as recently demonstrated in a non-human primate.75 Parthenogenetic cells are diploid, but are
homozygous for half the maternal chromosomal
complement, including the HLA locus. Assuming
recombination due to crossing over did not occur,
parthenotes are 50% match with the oocyte donor,
as well as her offspring. It is suggested that tissue
derivatives from these cells might suffer less immune rejection by virtue of haploidentity at the
HLA locus,75 but indeed may be rejected by NK
cells in a phenomenon resembling hybrid resistance.76 Crossing over in meiosis I might generate
parthenote ESC that maintain heterozygosity at the
HLA locus and would therefore be matched to the
oocyte donor.77 Based on genetic distance considerations (the MHC locus maps 20 centimorgans

325
from the centromere) roughly one in five parthenote ESC lines made from the oocytes of a F1 hybrid mouse will be a recombinant that will be
perfectly matched at histocompatibility loci. Another means to generate fully isogenic ESC would
be a technique called parthenogenetic cloning.
Parthenogenetic cloning entails inhibiting the
extrusion of the first polar body by blocking
the first meiotic cleavage with cytochalasin D.78
The resulting oocytes can be activated and will
form embryos that are isogenic to the oocyte donor
mouse.

Sources of hESC
On August 9, 2001, during a prime time television
address, President Bush announced that United
States federal funding would only be provided
for human embryonic stem cell lines derived prior
to that date.79 Although, in a sense endorsing the
importance of studying human embryonic stem
cells, this announcement has created limitations.
Currently, only 22 hES cell lines are available for
study using US federal funding. The lines vary in
their extent of characterization and conditions of
derivation. Even though these cells replicate
indefinitely, due to prolonged growth in culture
changes to their genome occur that render them
less useful for research purposes. For all these
reasons researchers have begun to derive new
hESC lines. Doug Melton from Harvard University
recently reported in the New England Journal of
Medicine on the derivation of 17 such lines.80
The federally approved hESC, as well as those recently created in the Melton lab, were derived
from donated frozen embryos created in the
course of fertility treatments. Human ESC derived
in this manner are invaluable from a research perspective. Their clinical usefulness is limited by the
immunological barriers discussed above. Isogenic
hESC lines using nuclear transfer techniques at
present hold the greatest promise for a clinical
source of hESC.

The debate over reproductive versus

research cloning
Much of the spirited public debate on hESC technology centers on the concern that it will facilitate
reproductive cloning. The concept of nuclear
transfer was first proposed in 1938 as a fantastical
experiment by an embryologist, Hans Speman.81

326
Building on the foundations laid by Briggs and King
in the 1950s and John Gurdon in the 1960s, Ian Wilmut and colleagues introduced the world to Dolly
the sheep the first mammal to be cloned by nuclear transfer using an adult donor cell.82 Since
then several other mammalian species have been
cloned: mouse,83 cow,84,85 goat,86 pig,87 cat,88 rabbit,89 and rat.90
Procreative cloning using somatic (adult) donor
cells is an inefficient and error-prone process. All
reports of successful cloning were achieved after
many failed attempts. Even in experienced hands
the success rate viable offspring is probably
no greater than 3%, with the majority of cloned
organisms dying during gestation or in the immediate perinatal period (reviewed in 91). For development to occur after nuclear transfer, the
specialized gene expression profile of the donor
cell needs to be erased and reset to activate the
correct sequence of embryonic genes. When one
considers the complexity of embryonic development, it is remarkable that somatic cell nuclear
transfer works at all. Even in the rare instances
when reprogramming enables embryonic and fetal
development to produce live clones, most are not
clinically normal. Cloned animals suffer from overgrowth syndromes, respiratory distress, defects of
the kidney, liver, and heart.9193 In clones that survive into later life other abnormalities become
apparent unexplained obesity, premature death,
and propensity for tumors.94,95 Examination of the
genome of those animals demonstrates errors in
reprogramming.9698 Apart from any philosophical
considerations, the error-prone nature of nuclear
transfer makes any attempt at reproductive cloning in humans unacceptable because of safety concerns alone.

Nuclear transplantation to enable

combined gene and cell therapy
The ability to introduce transgenes in ESC has been
an indispensable tool for scientists studying ESC
biology. In many models of directed differentiation
in ESC, transgenes have been used to instruct cells
to follow a specified developmental pathway, or to
allow selection for them after differentiation. As
mentioned above, it is preferable to avoid gene
modification altogether when considering clinical
application of cells. Much of the early excitement
of gene therapy has been muted by several highly
publicized adverse events. In 1999, the death of
an 18-year old with ornithine transcarbamylase
deficiency from an acute inflammatory response

P.H. Lerou, G.Q. Daley

to an adenoviral vector raised serious concern
about the safety of this therapeutic modality.99
The first successful gene therapy for treatment of
X-linked severe combined immunodeficiency was
reported in 2002.100 However, a year later the
investigator reported that 2 of the 10 patients treated developed T-cell leukemia101 due to retroviral
insertion near the LMO2 oncogene.102 These high
profile negative outcomes highlight the risk associated with viral vectors, as random insertion of the
therapeutic gene compromises the safety of this
approach.
In contrast to viral vector-based gene delivery,
homologous recombination in embryonic stem cells
allows for site-specific gene repair.103,104 Because
gene repair is performed ex vivo, in a readily
expandable cell population, tests to ensure safety
and proper function can be done prior to transplanting the genetically modified cells. An experiment performed in 2002 demonstrated that
somatic cell nuclear transfer can be coupled with
gene therapy to treat disease in this case an
immunodeficient mouse.70 Nuclear transfer cloning
from tail-tip cells of an immunodeficient Rag2/
mouse was performed to generate a Rag2/ nuclear transfer ESC (ntESC) line. One allele was then
repaired by homologous recombination. The
repaired Rag2+/ ntESC line was differentiated into
blood progenitors in vitro, transduced with HoxB4,
and engrafted into immunodeficient mice. The
recipient mice showed partial reconstitution of B
and T cell populations, Ig and TcR gene rearrangement, and IgM, IgG, and IgA in peripheral blood.
This proof of principle experiment established
the technical feasibility of somatic cell nuclear
transfer with gene therapy.
Nuclear transfer is theoretically appealing a
means to generate customized, patient-specific
ESC. But this is a cumbersome, labor-intensive,
inefficient, and expensive technique. A long road
must still be traveled for technology to move from
to the clinical stage. The main obstacle is the low
efficiency of the process. Mice are the species in
which scientist have the most comprehensive experience deriving ntESC lines. It is estimated that
approximately 151000 oocytes (depending on
the somatic cell) are required to derive a murine
ntESC line.105 Experience in the human comes from
a single study in which 242 donated human oocytes
were reconstructed using autologous cumulus cells
to derive a single hESC line.71 ESC derived cloned
blastocysts are known to harbor multiple defects
owing to improper or incomplete epigenetic reprogramming.9698 In addition to inherent epigenetic
errors resulting from the nuclear transfer process,
ESC cloned by SCNT demonstrate epigenetic

Therapeutic potential of embryonic stem cells

instability in culture similar to natural ESC.97 Fortunately, in chimeric mice ESC reconstitute normal
tissue development, and it has been argued that
cloned tissue is likely to function normally and be
useful therapeutically.106 However, it is unlikely
that each ntESC line will have the same developmental potential and therefore, for clinical practice one may need several ntES lines per patient.
Mombaerts explains how the inefficiency of nuclear
transfer technology will prohibit its widespread use
in the following manner:105 assuming an optimistic
efficiency of 30 oocytes per ntESC line derive (a 10fold improvement over the Korean group), a
requirement of three ntESC lines per patient, at a
cost of $1000$2000 (US) per oocyte, the cost for
oocytes alone would be $100,000$200,000.
Including cost of labor, additional material, and
safety testing would escalate the cost to preclude
practical application of this technology. Furthermore, it is unlikely that practical sources of human
oocytes most likely donated specifically for this
purpose would satisfy demand, even if profound
improvements in efficiency took place.
In the course of science, however, it is difficult
to anticipate how technical advances might accelerate discovery. Derivation of ntESC from human
somatic cells transplanted into enucleated rabbit
oocytes has been reported.107 While this process
might address the challenge of procuring adequate
numbers of oocytes the resulting ntESC might carry
rabbit mitochondria, which may limit clinical utility. Exciting work has shown that mESC have the
potential to give rise to oocytes, in vitro.108 Thus,
one might envision creating specific hESC lines to
produce oocytes in vitro that could serve to reprogram somatic cells. The goal of much scientific effort is to solve the mystery of how the ooplasm
reprograms a somatic nucleus. Improved understanding could allow scientists to reprogram adult
somatic cells directly, without the need for nuclear
transfer and the generation of cloned embryos.
Although highly speculative, the tremendous clinical potential of reprogrammed somatic cells should
serve as incentive for scientists to turn such fantasy into reality.

327
cardiomyocytes derived from stem cells in vitro
could be used to screen new chemical entities for
hepatotoxicity and cardiotoxicity, two leading
causes of failure in preclinical development of
new therapeutic drugs.109 Human liver cells have
proven useful for investigating drug metabolism
and toxicology110,111 and the molecular genetics
of drug metabolism pathways.112,113 Human ESC
could offer a limitless supply of hepatocytes for
such studies.18 Kamp and colleagues have shown
that hESC differentiate into a number of cardiomyocyte classes, including embryonic atrial, ventricular, and nodal subtypes, each faithfully
recapitulating their respective electrophysiologic
properties and pharmacologic responses.9 Schultz
and colleagues recently performed a chemical
screen to identify agents that induce neurogenesis
in ESC, thereby establishing the proof-of-principle
for using stem cell differentiation in assays for drug
discovery.114 Embryonic stem cells can serve
to screen chemical compounds for teratogenic
effects.114117 One could even envision how isogenic hESC could be used to perform an in vitro
screen for an individual patients response to various therapeutic agents or how a bank of hESC could
be used on a larger population scale to further
investigate pharmacogenomics. Applications in
toxicology and pharmacology are expertly reviewed by Davila et al.109

Conclusion
The therapeutic potential of hESC is responsible for
tremendous excitement both in the scientific community as well as the general public. In order to
realize this potential considerable hurdles remain,
compelling scientists to press on to overcome the
daunting, though not insurmountable obstacles
outlined above. Fundamental questions about the
basic biology of these cells, human development,
pathophysiology, genetics, and epigenetics remain
unanswered. In answering these questions we are
bound to advance bold new strategies for treating
disease.

hESC: applications in toxicology and

pharmacology

Acknowledgements

Stem cells have many potential applications in the

fields of toxicology and pharmacology. Stem cellbased systems can allow for the screening of compounds, making them extremely useful in several
phases of drug development. Hepatocytes and

Work in the senior authors laboratory was supported by grants from the Bekenstein Family, the
National Institutes of Health, and the NIH Directors Pioneer Award of the NIH Roadmap for Medical Research. G.Q.D. is a recipient of the Burroughs

328
Wellcome Fund Clinical Scientist Award in Translational Research, and the Birnbaum Scholar of the
Leukemia and Lymphoma Society of America. P.L.
is supported by NIH training grant, Pathobiology
of Newborn and Developmental Diseases (T32:
HD07466).

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