Tenneille E. Ludwig, Ph.D. and
James A. Thomson, Ph.D
Wisconsin National Primate Research Center,
University of Wisconsin-Madison
The immortality and potentially unlimited developmental
capacity of human embryonic stem (ES)  cells ignite the
imagination. After months or years of growth in culture
dishes, these cells retain the ability to form cell types
ranging from heart muscle to nerve to blood-possibly any
cell in the body. Because of their unique developmental
potential, human ES cells have widespread implications for
human developmental biology, drug discovery, drug testing,
and transplantation medicine. Indeed, human ES cells promise
an essentially unlimited supply of specific cell types for
in vitro experimental studies and for transplantation
therapies for diseases such as heart disease, Parkinson's
disease, leukemia, and diabetes.
The derivation of a human ES cell line destroys a human
embryo. Thus, the derivation of human ES cells resurrected
a fierce controversy over human embryo research in the United
States, a controversy originally created by the development
of human in vitro fertilization decades ago, but
never completely resolved. In particular, the derivation
of human ES cells led to a re-examination of the role of
federal funding of human embryo research. In response to
the intense public interest, President George W. Bush reviewed
the potential of human ES cell research to improve the health
of Americans. In his national address on August 9, 2001,
he stated "Federal dollars help attract the best and brightest
scientists. They ensure new discoveries are widely shared
at the largest number of research facilities, and the research
is directed toward the greatest public good." On that
basis, he directed federal funding to "explore the
promise and potential of stem cell research," including,
for the first time, human ES cell research. However, the
President went on to restrict federal funding to research
that used only those human ES cell lines derived prior to
his address on August 9, 2001. This paper reviews progress
in human ES cell research in the wake of that decision.
Human ES Cell Publication Summary
Since their initial derivation, only a limited number of
independent (i.e., derived from different embryos) human
ES cell lines that meet President Bush's criteria have been
used in published research. Just nine human ES cell lines
meeting President Bush's criteria are currently listed by
the National Institutes of Health as readily available for
distribution to investigators. Despite limited availability
to date, research with human ES cells is proceeding. Forty
human ES cell primary research papers have been published
in peer-reviewed journals since the initial publication
of human ES cell isolation in 1998 (Table 1). Published
human ES cell research includes studies on the optimization
of the culture environment, characterization of human ES
cells, modification of the ES cell genome, and differentiation.
Table 1. Human ES Cell Research Publications
Area of interest
Publications to date
Feeder Layer Alternatives/Replacements
Differentiation into multiple lineages
Differentiation into specific lineages
Culture Optimization for Human ES Cells.
Improvement of culture conditions to enable large-scale
production and reduce safety concerns has been a major research
focus. The first two research groups that described the
derivation of human ES cell lines examined long-term proliferation,
karyotypic stability, developmental potential, and cell
surface marker expression by ES cells [1, 2] . Because
these first human ES cell lines remain the most extensively
characterized, most subsequent research has utilized them.
These human ES cell lines were derived on mouse fibroblast
feeder layers in the presence of fetal bovine serum. The
exposure to these and other sources of animal proteins has
raised concern that some yet unidentified pathogen(s) may
have been transferred to the ES cells by contact with cells
or proteins from other species, and that these pathogens
could be transferred to patients if these ES cells were
to be used for transplantation therapies. Thus, several
research groups have been actively working to reduce or
eliminate non-human cells or proteins from human ES cell
Significant progress has been made in eliminating serum,
and limited progress has been made in eliminating fibroblasts
from human ES cell culture. Serum is a complex, poorly
defined mixture of components, and there is significant
variation between lots  . Individual lots of serum,
therefore, must be carefully screened for their ability
to sustain undifferentiated ES cell growth. If basic fibroblast
growth factor is added to a proprietary serum substitute
(Gibco BRLÒ Knockoutä Serum Replacer), it supports
human ES cells and significantly reduces the batch variability
associated with serum  . However, this medium does
not eliminate all serum products from human ES cell culture
medium, as it still contains a bovine serum albumin component.
With this same medium, human ES cells can be cultured without
direct contact with feeder layers if the medium is first
conditioned by exposure to mouse embryonic fibroblasts 
. However, the medium still contains bovine serum products
and is exposed to fibroblasts, therefore cross-species contamination
with pathogens remains a concern.
Recent reports demonstrate that human ES cells can be maintained
on feeder layers of human origin. Feeder layers obtained
from human bone marrow  , fetal muscle or skin  ,
adult human fallopian tube epithelial cells  , or human
foreskin  support human ES cell proliferation and maintenance
of normal karyotype and developmental potential. These
results led to the growth of human ES cells in the complete
absence of non-human products  . New human ES cells
derived under these conditions would eliminate concerns
about cross-species contamination by pathogens, but such
cell lines could not, at present, be supported by federal
funding in the United States. Growth on human feeder layers
is a significant advance because of reduced safety concerns;
nonetheless, the preparation of these feeders remains laborious
and introduces a significant source of biological variability.
The complete elimination of feeder layers and serum from
human ES cell culture medium and their replacement by defined,
cloned products remains an important goal for the future
and is an active area of research for several groups.
Genetic Modification of Human ES Cells
Although the human genome project is essentially completed,
we are ignorant about the function of most human genes.
Human ES cells provide a powerful new model for identifying
the function of any human gene, and this requires efficient
methods for genetic modification of human ES cells. Genetic
manipulation of human ES cells is essential to elucidate
gene function; direct the differentiation of ES cells to
specific lineages; purify desired differentiated cell types
from mixed populations of ES cell derivatives; use the differentiated
derivatives of ES cells as a vehicle for gene therapy; and
modulate the immune response to transplanted ES cell derivatives.
Transfection methods routinely used for mouse ES cells
generally fail to transfect human ES cells efficiently,
but there have now been several approaches developed for
human ES cells. Transient  and stable  integration
of plasmids into human ES cells can be accomplished through
specific transfection reagents, the best reagents yielding
stable (drug-selectable) transfection rates of about 10-5.
Recently more labor-intensive, HIV-based, lentivirus vectors
have been shown to transduce human ES cells at an efficiency
rate of over 90% [20, 21] . This should allow complex mixtures
of genes to be screened for specific phenotypic effects
by a process termed "expression cloning"  .
Homologous recombination allows the defined modifications
of specific genes in living cells [42, 43] and has been
used extensively with mouse ES cells. However, the differences
between mouse and human ES cells delayed the development
of homologous recombination in human ES cells. Except for
viral approaches, high stable transfection efficiencies
in human ES cells have been difficult to achieve, and in
particular, electroporation protocols established for mouse
ES cells do not work in human ES cells  . Also, in
contrast to mouse ES cells, human ES cells proliferate inefficiently
from single cells, making screening procedures to identify
rare homologous recombination events difficult  . We
have recently developed modified electroporation protocols
to overcome these problems and have successfully targeted
a ubiquitously expressed gene (HPRT1), an ES cell-specific
gene (POU5F1), and a tissue-specific gene (Tyrosine
hydroxylase: TH) in human ES cells [22, 44] . The overall
targeting frequencies for the three genes suggest that homologous
recombination is a broadly applicable technique in human
Homologous recombination in human ES cells will be important
for studying gene function in vitro and for lineage
selection. For therapeutic applications in transplantation
medicine, controlled modification of specific genes should
be useful for purifying specific ES cell-derived, differentiated
cell types from a mixed population  ; altering the antigenicity
of ES cell derivatives; and giving cells new properties
(such as viral resistance) to combat specific diseases.
Homologous recombination in human ES cells might also be
used for approaches combining therapeutic cloning with gene
therapy  . In vitro studies using homologous
recombination in human ES cells will be particularly useful
for learning more about the pathogenesis of diseases where
mouse models have proven inadequate. For example, HPRT-deficient
mice fail to demonstrate an abnormal phenotype, yet defects
in this gene cause Lesch-Nyhan disease in children 
. In vitro neural differentiation of HPRT-deficient
human ES cells or transplantation of ES cell-derived neural
tissue to an animal model could help to understand the pathogenesis
of Lesch-Nyhan syndrome.
Human ES Cells as a Model of Early Human Development
The excitement surrounding the prospective role of human
embryonic stem (ES) cells in transplantation therapy has
often overshadowed a potentially more important role as
a basic research tool for understanding the development
and function of human tissues. The use of human ES cells
is particularly valuable to derive tissue for study that
is difficult to obtain otherwise, and for which animal models
Human ES cells offer a new and unique window into the
early events of human development, a period critical for
understanding infertility, birth defects, and miscarriage.
Because manipulation of the early post-implantation human
embryo could jeopardize the health of the resulting child,
it has never been possible to examine this important period
of human development experimentally. Nearly all of what
is known about early human development, especially in the
early post-implantation period, is based on very rare histological
sections of human embryos, or on an imperfect analogy to
experimental studies in the mouse. The mouse has been the
mainstay of mammalian experimental embryology because of
its historical use, well-defined genetics, and favorable
reproductive characteristics. However, early mouse development
and early human development differ significantly. For example,
human and mouse embryos differ in the expression of embryonic
antigens; timing of embryonic genome expression; formation,
structure, and function of the fetal membranes and placenta;
and formation of an embryonic disc instead of an egg cylinder.
Thus, if one is interested in the development of a human
tissue known to differ significantly from the corresponding
mouse tissue, such as the yolk sac or the placenta, studying
a human model is desirable.
The first differentiation event in mammalian embryos is
the formation of the trophectoderm, the outer epithelial
layer of the blastocyst. The trophectoderm is crucial for
implantation of the embryo and gives rise to specialized
populations of trophoblast cells in the placenta [48, 49]
. Mouse and human placentas differ in structure and function,
and these differences are clinically significant. For example,
the placental hormone chorionic gonadotropin, which has
an essential role in establishing and maintaining human
pregnancy, is not even produced by the mouse placenta.
When formed into chimeras with intact preimplantation embryos,
mouse ES cells rarely contribute to the trophoblast, and
the manipulation of external culture conditions has, to
date, failed to direct mouse ES cells to form trophoblast
Spontaneously differentiated rhesus monkey  or human
ES cells  do secrete modest amounts of chorionic gonadotropin,
indicating the differentiation of trophoblast cells 
. Recently it was discovered that a single growth factor
(BMP4) would induce human ES cells to differentiate to a
pure population of early trophoblast  . These early
human trophoblast cells have never before been available
for detailed study, and already this new experimental model
has provided information about the specific genes that control
the early development of the human placenta  . The derivation
of other early lineages from human ES cells in vitro
to provide a more complete understanding of early human
development is an active area of research.
Cardiovascular disease is the leading cause of death in
the United States, taking the lives of more people each
year than the next five leading causes of death combined
 . Cardiovascular disease and its related disorders
affect more than 68 million Americans, at a cost of more
than 350 billion dollars annually. Heart disease alone
accounts for 229 billion dollars in health care costs each
year. Adult heart tissue cannot be expanded in culture,
and thus, there are no human heart cell lines available
for research. The limited amount of physiological research
done directly on human heart cells has generally relied
on biopsy samples, which are small, erratically available,
and usually obtained from diseased hearts. In contrast,
human ES cells are already providing a reliable in vitro
supply of human heart cells for experimental study [30-34]
Animal models, such as the mouse, have historically been
used for the study of the heart. However, there are clinically
significant physiological differences between animal and
human cardiomyocytes that limit the usefulness of these
models. For example, the mechanisms regulating the QT polarization
interval-the time required for repolarization of the heart
muscle between beats-differ significantly between species.
A prolonged QT polarization interval in humans is related
to ventricular arrhythmias and cardiac arrest and has been
a significant side effect of a wide range of drugs in early
human clinical trials. Drugs exhibiting this serious side
effect must be withdrawn from clinical trials, and such
drugs have been responsible for patients' deaths. Because
the mechanisms that regulate repolarization of the heart
muscle cells differ appreciably between human and mouse
models, screening drugs on mouse hearts does not reliably
detect this side effect. Yet, because they do not divide
in culture, human heart cells have not been previously available
Human ES cells differentiate spontaneously to heart muscle
cells, and several research groups have reported the characterization
of these cells [23, 31, 54] . Human ES cells allowed to
differentiate in unattached clumps (termed "embryoid bodies")
form synchronized contracting areas that express appropriate
cardiac markers [23, 31, 32] . Co-culture of human ES cells
with visceral, endoderm-like cells also causes differentiation
to cardiomyocytes  , and 5-aza-2'-deoxycytidine or density
gradient separation allows some enrichment of cardiomyocyte
populations  . Human ES cell-derived cardiomyocytes
display many of the functional properties of native cardiomyocytes,
including the generation of synchronized action potentials
and response to cardioactive drugs [30-33] . Heart cells
exhibiting action potentials characteristic of nodal, atrial,
and ventricular cardiomyocytes are all present, and the
human-specific mechanisms regulating QT interval are functional
 . Thus, human ES cell-derived heart cells are already
useful for drug screening, and their use should make the
drug development process quicker, cheaper, and safer.
There is also a great interest in using human ES cell-derived
heart cells for transplantation, but this will likely be
challenging. Studies in animal models demonstrate that
cell transplantation is effective in increasing the myocyte
population in damaged or diseased cardiac tissue  .
However, when heart cells die in a heart attack, it is not
because the heart cells themselves are defective, but because
the blood supply is cut off. Thus, to be successful, transplanted
heart cells would have to integrate functionally with the
surrounding heart cells, obtain a new blood supply, and
avoid immune rejection. Each of these problems has potential
solutions, but will require significant time and effort
to solve. Precursors of vascular tissue can also be derived
from human ES cells  , and such cells may be useful
in supporting co-transplanted heart cells.
Because of the country's aging population, neural degenerative
disorders such as Parkinson's disease and Alzheimer's disease
are becoming increasingly prevalent in the United States.
Historically, one of the difficulties in studying the pathogenesis
of neural disease has been the very limited access to the
specific neural cells involved in these diseases. Neural
precursor (or stem) cells cultured from fetal and adult
brains have been extensively studied, but appear to have
limited developmental potential. For example, the sustainable
differentiation of neural stem cells to dopaminergic neurons,
the cell defective in Parkinson's disease, has not yet been
achieved. Mouse ES cells, for example, differentiate efficiently
to dopaminergic neurons, and several groups are beginning
to apply approaches used with mouse ES cells to human ES
cells. Human ES cells should offer an improved supply of
neural tissue, for both in vitro experimental studies
and transplantation therapies.
Human ES cell-derived embryoid bodies produce both neural
precursor cells and cells expressing markers of mature neurons
and glia [26-29] . The percentage of neural precursors
can be enriched by alteration of culture conditions [26-28]
or by purification using cell surface markers  . Human
ES cell-derived neural cells are able to synthesize and
respond to neurotransmitters, form synapses and voltage-dependent
ion channels capable of generating action potentials, and
generate electrical activity  . Some human ES cell-derived
neurons express tyrosine hydroxylase, the rate-limiting
enzyme involved in dopamine synthesis and a marker of dopaminergic
neurons [26, 27] .
Human ES cell-derived neural precursors transplanted into
the mouse brain differentiate into all three types of central
nervous system cells (neurons, glia cells, and oliogodendrocytes)
[26, 27] . These differentiated cells migrate, following
host developmental cues, into various areas of the brain
(including cortex, hippocampus, striatum olfactory bulb,
septum, thalamus, hypothalamus, and midbrain) [26, 27] .
One of the concerns about using human ES cell-derived neural
cells in transplantation therapy is the fear that undifferentiated
ES cells may be transplanted with the differentiated cells
and form teratomas in the host. To date, transplantation
of isolated, human ES cell-derived neural precursor cells
into mice has not produced teratomas [26, 27] , suggesting
that appropriate selection procedures can eliminate undifferentiated
ES cell contamination. However, longer-term testing is
still needed to address the teratoma formation issue more
Human ES cells are already providing a sustainable source
of hematopoietic cells for in vitro studies [36,
37] . Hematopoietic stem cells are by far the most studied
adult stem cells, and bone marrow transplants are the most
common and effective form of stem cell-based therapy. However,
despite several decades of research by hundreds of laboratories,
hematopoietic stem cells have not yet been successfully
expanded in clinically useful amounts, and these cells must
instead be transferred directly from the donor. When cultured
in vitro, hematopoietic stem cells do not self-renew,
but instead differentiate to specific blood cells, and thus
quickly disappear. This makes the in vitro study
of human hematopoiesis difficult, as researchers must continually
return to patients to obtain hematopoietic stem cells from
bone marrow, peripheral blood, or placental cord blood.
Human ES cells can differentiate into hematopoietic precursor
cells through co-culture with murine bone marrow or yolk
sac cells  . Enrichment of ES cell-derived hematopoietic
precursors is accomplished by treatment with cytokines or
BMP-4  . Cell sorting using hematopoietic-specific
cell surface markers yields myeloid, erythroid, and megakaryocyte
precursors  .
There are three major areas where human ES cell hematopoiesis
should impact human medicine. First, because human ES cells
can be expanded without limit, human hematopoiesis can be
studied without the need to continually return to patients
for tissue donations. The knowledge of these in vitro
studies is likely to improve therapies based on adult hematopoietic
stem cells. Second, human ES cell-derived blood cells could
be used either in bone marrow transplants, or as a source
of blood products such as red blood cells and platelets.
And third, ES cell-derived hematopoietic stem cells could
aid in ES cell-based transplantation therapies for other
(non-hematopoietic) tissues. Transplantation of ES cell-derived
hematopoietic stem cells could be used to reduce or eliminate
immune rejection by creating hematopoietic chimerism in
patients receiving co-transplantation of other human ES
cell-derived tissues [45, 56-58] .
Type 1 diabetes offers one of the most promising applications
of human ES cell-based transplantation therapy. The destruction
of pancreatic islet $-cells results in type 1 diabetes.
$-cells produce insulin, and as their numbers dwindle, the
ability to appropriately control blood glucose levels is
lost. Even with current insulin therapies, type 1 diabetes
reduces a patient's life expectancy by 10 to 15 years, and
these patients often develop serious complications such
as blindness and kidney failure  . Recently, the transplantation
of $-cells from cadavers has proven to be an effective treatment
for some forms of uncontrollable diabetes, but the source
of tissue for transplantation is severely limiting and will
never come close to meeting the demands of over one million
people with type 1 diabetes in the United States. Spontaneous
in vitro differentiation of human ES cells reveals
a percentage of cells that produce insulin and express other
$-cells specific markers, offering hope of a scalable source
of $-cells for transplantation 
The challenges for using human ES cell-derived $-cells
for transplantation are significant and parallel those that
face the entire field of ES cell-based transplantation therapies.
First, pancreatic development is incompletely understood,
and it is not yet possible to direct ES cells to $-cells
efficiently. However, given the pace of advances in developmental
biology over the last decade, it is likely that in the next
five to ten years, it will be possible to routinely generate
clinically useful quantities of $-cells from human ES cells.
Second, integration into the body in a form that restores
function of the damaged tissue is essential. This is easier
for $-cells than for most cell types, as the function that
must be restored is secretion of insulin into the blood
stream in response to high glucose, and this function does
not require a complex physical connection between the transplanted
and host tissues. Indeed, the clinical trials using cadaver-derived
$-cells have transplanted the cells into the liver, and
the cells function in that site. Third, transplanted $-cells
must not be rejected by the immune system. Although the
transplantation of $-cells has been clinically successful,
the severe immunosuppressive therapy required may make the
procedure inappropriate for the average diabetic patient.
Importantly, $-cells derived from adult stem cells from
the patient, or even from ES cells derived through "therapeutic
cloning" using a nucleus from the patient, would not
solve the immune rejection problem for diabetes. Type 1
diabetes is an autoimmune process, and unless that immune
response is altered the very process that made the patient
diabetic in the first place would destroy transplanted $-cells
genetically identical to the patient's. Finally, neoplastic
transformation of the transplanted cells is a serious concern
for any cell-based therapy in which the cells are first
cultured extensively. All actively dividing cells accumulate
mutations over time, and the potential exists that enough
mutations could accumulate to make some cells tumor cells.
None of the challenges facing ES cell-based transplantation
therapies are insurmountable, and indeed, type 1 diabetes
is an excellent candidate for treatment using this approach.
However, the challenges do underscore both the importance
of careful preclinical testing, particularly in non-human
primates, and the amount of work still to be done before
people's lives will be improved by these therapies.
Since their initial derivation, there has been significant
progress in culture optimization, characterization, genetic
modification, and differentiation of human ES cells. However,
ethical and political controversy continues to impede progress
in human ES cell research. The decision by President George
W. Bush, restricting federal funding to human ES cell lines
derived before August 9, 2001, created a distribution bottleneck
that is just now beginning to be resolved. Although these
initial cell lines may support much of the basic research
now being conducted, the very first cell lines were originally
derived for research purposes, with the expectation that
future cell lines would more appropriately address legitimate
safety concerns for therapeutic applications. In spite
of the slow start, the diversity of investigators already
contributing to human ES cell research is, nonetheless,
promising and suggests that the initial lag phase for the
human ES cell field is already coming to an end and that
an exponential growth phase is beginning. During the next
year or two, it is likely that the purification of specific,
therapeutically useful human ES cell derivatives, such as
dopaminergic neurons, will be published, and that defined
culture conditions eliminating the need for both feeder
layers and non-human proteins will be developed. When these
events occur, President Bush's compromise will be particularly
damaging to the field, and there will be an even greater
need to derive new cell lines.
1. Thomson JA, Itskovitz-Eldor J, Shapiro
SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic
stem cell lines derived from human blastocysts. Science
1998; 282: 1145-1147.
2. Reubinoff BE, Pera MF, Fong CY, Trounson
A, Bongso A. Embryonic stem cell lines from human blastocysts:
somatic differentiation in vitro. Nat Biotechnol 2000; 18:
3. Lanzendorf SE, Boyd CA, Wright DL, Muasher
S, Oehninger S. Use of human gametes obtained from anonymous
donors for the production of human embryonic stem cell lines.
Fertility and Sterility 2001; 76: 132-137.
4. He Z, Huang S, Li Y, Zhang Q. Human embryonic
stem cell lines preliminarily established in China. Zhonghua
Yi Xue Za Zhi 2002; 82: 1314-1318.
5. Amit M, Itskovitz-Eldor J. Derivation
and spontaneous differentiation of human embryonic stem
cells. J Anat 2002; 200: 225-232.
6. Xu C, Inokuma MS, Denham J, Golds K,
Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated
human embryonic stem cells. Nat Biotechnol 2001; 19: 971-974.
7. Richards M, Fong CY, Chan WK, Wong PC,
Bongso A. Human feeders support prolonged undifferentiated
growth of human inner cell masses and embryonic stem cells.[comment].
Nature Biotechnology 2002; 20: 933-936.
8. Amit M, Margulets V, Segev H, Shariki
K, Laevsky I, Coleman R, Itskovitz-Eldor J. Human Feeder
Layers for Human Embryonic Stem Cells. Biol Reprod 2003;
9. Cheng L, Hammond H, Ye Z, Zhan X, Dravid
G. Human adult marrow cells support prolonged expansion
of human embryonic stem cells in culture. Stem Cells 2003;
10. Lim JW, Bodnar A. Proteome analysis
of conditioned medium from mouse embryonic fibroblast feeder
layers which support the growth of human embryonic stem
cells. Proteomics 2002; 2: 1187-1203.
11. Reubinoff BE, Pera MF, Vajta G, Trounson
AO. Effective cryopreservation of human embryonic stem cells
by the open pulled straw vitrification method. Hum Reprod
2001; 16: 2187-2194.
12. Amit M, Carpenter MK, Inokuma MS, Chiu
C, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA.
Clonally derived human embryonic stem cell lines maintain
pluripotency and proliferative potential for prolonged periods
of in vitro culture. Developmental Biology 2000; 227: 271-278.
13. Henderson JK, Draper JS, Baillie HS,
Fishel S, Thomson JA, Moore H, Andrews PW. Preimplantation
human embryos and embryonic stem cells show comparable expression
of stage-specific embryonic antigens. Stem Cells 2002; 20:
14. Drukker M, Katz G, Urbach A, Schuldiner
M, Markel G, Itskovitz-Eldor J, Reubinoff B, Mandelboim
O, Benvenisty N. Characterization of the expression of MHC
proteins in human embryonic stem cells. Proc Natl Acad Sci
U S A 2002; 99: 9864-9869.
15. Sathananthan H, Pera M, Trounson A.
The fine structure of human embryonic stem cells. Reprod
Biomed Online 2002; 4: 56-61.
16. Moore FL, Jaruzelska J, Fox MS, Urano
J, Firpo MT, Turek PJ, Dorfman DM, Pera RA. Human Pumilio-2
is expressed in embryonic stem cells and germ cells and
interacts with DAZ (Deleted in AZoospermia) and DAZ-like
proteins. Proceedings of the National Academy of Sciences
of the United States of America 2003; 100: 538-543.
17. Lebkowski JS, Gold J, Xu C, Funk W,
Chiu CP, Carpenter MK. Human embryonic stem cells: culture,
differentiation, and genetic modification for regenerative
medicine applications. Cancer J 2001; 7: S83-93.
18. Eiges R, Schuldiner M, Drukker M, Yanuka
O, Itskovitz-Eldor J, Benvenisty N. Establishment of human
embryonic stem cell-transfected clones carrying a marker
for undifferentiated cells. Current Biology 2001; 11: 514-518.
19. Pfeifer A, Ikawa M, Dayn Y, Verma I.
Transgenesis by lentiviral vectors: Lack of gene silencing
in mammalian embryonic stem cells and preimplantation embryos.
PNAS 2002; 99: 2140-2145.
20. Ma Y, Ramezani A, Lewis R, Hawley R,
Thomson JA. High-level sustained transgene expression in
human embryonic stem cells using lentiviral vectors. Stem
Cells 2003; 21: 111-117.
21. Gropp M, Itsykson P, Singer O, Ben-Hur
T, Reinhartz E, Galun E, Reubinoff BE. Stable genetic modification
of human embryonic stem cells by lentiviral vectors. Mol
Ther 2003; 7: 281-287.
22. Zwaka TP, Thomson JA. Homologous recombination
in human embryonic stem cells. Nat Biotechnol 2003; 21:
23. Itskovitz-Eldor J, Schuldiner M, Karsenti
D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N. Differentiation
of human embryonic stem cells into embryoid bodies compromising
the three embryonic germ layers. Mol Med 2000; 6: 88-95.
24. Schuldiner M, Yanuka O, Itskovitz-Eldor
J, Melton DA, Benvenisty N. Effects of eight growth factors
on the differentiation of cells derived from human embryonic
stem cells. Proc Natl Acad Sci U S A 2000; 97: 11307-11312.
25. Goldstein RS, Drukker M, Reubinoff BE,
Benvenisty N. Integration and differentiation of human embryonic
stem cells transplanted to the chick embryo. Dev Dyn 2002;
26. Zhang SC, Wernig M, Duncan ID, Brustle
O, Thomson JA. In vitro differentiation of transplantable
neural precursors from human embryonic stem cells. Nat Biotechnol
2001; 19: 1129-1133.
27. Reubinoff BE, Itsykson P, Turetsky T,
Pera MF, Reinhartz E, Itzik A, Ben-Hur T. Neural progenitors
from human embryonic stem cells. Nat Biotechnol 2001; 19:
28. Carpenter MK, Inokuma MS, Denham J,
Mujtaba T, Chiu CP, Rao MS. Enrichment of neurons and neural
precursors from human embryonic stem cells. Exp Neurol 2001;
29. Schuldiner M, Eiges R, Eden A, Yanuka
O, Itskovitz-Eldor J, Goldstein RS, Benvenisty N. Induced
neuronal differentiation of human embryonic stem cells.
Brain Research 2001; 913: 201-205.
30. He JQ, Ma Y, Lee Y, Thomson JA, Kamp
TJ. Human Embryonic Stem Cells Develop Into Multiple Types
of Cardiac Myocytes. Action Potential Characterization.
Circ Res 2003; 5: 5.
31. Kehat I, Kenyagin-Karsenti D, Snir M,
Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor
J, Gepstein L. Human embryonic stem cells can differentiate
into myocytes with structural and functional properties
of cardiomyocytes. J Clin Invest 2001; 108: 407-414.
32. Xu C, Police S, Rao N, Carpenter MK.
Characterization and enrichment of cardiomyocytes derived
from human embryonic stem cells. Circ Res 2002; 91: 501-508.
33. Kehat I, Gepstein A, Spira A, Itskovitz-Eldor
J, Gepstein L. High-resolution electrophysiological assessment
of human embryonic stem cell-derived cardiomyocytes: a novel
in vitro model for the study of conduction. Circ Res 2002;
34. Mummery C, Ward-Van Oostwaard D, Doevendans
P, Spijker R, Van Den Brink S, Hassink R, Van Der Heyden
M, Opthof T, Pera M, De La Riviere AB, Passier R, Tertoolen
L. Differentiation of human embryonic stem cells to cardiomyocytes:
role of coculture with visceral endoderm-like cells. Circulation
2003; 107: 2733-2740.
35. Levenberg S, Golub JS, Amit M, Itskovitz-Eldor
J, Langer R. Endothelial cells derived from human embryonic
stem cells. Proc Natl Acad Sci U S A 2002; 99: 4391-4396.
36. Kaufman DS, Hanson ET, Lewis RL, Auerbach
R, Thomson JA. Hematopoietic colony-forming cells derived
from human embryonic stem cells. Proc Natl Acad Sci U S
A 2001; 98: 10716-10721.
37. Chadwick K, Wang L, Li L, Menendez P,
Murdoch B, Rouleau A, Bhatia M. Cytokines and BMP-4 promote
hematopoietic differentiation of human embryonic stem cells.
Blood 2003; 17: 17.
38. Assady S, Maor G, Amit M, Itskovitz-Eldor
J, Skorecki KL, Tzukerman M. Insulin production by human
embryonic stem cells. Diabetes 2001; 50: 1691-1697.
39. Rambhatla L, Chiu CP, Kundu P, Peng
Y, Carpenter MK. Generation of hepatocyte-like cells from
human embryonic stem cells. Cell Transplant 2003; 12: 1-11.
40. Xu RH, Chen X, Li DS, Li R, Addicks
GC, Glennon C, Zwaka TP, Thomson JA. BMP4 initiates human
embryonic stem cell differentiation to trophoblast. Nat
Biotechnol 2002; 20: 1261-1264.
41. McKiernan SH, Bavister BD. Different
lots of bovine serum albumin inhibit or stimulate in vitro
development of hamster embryos. In Vitro Cell Dev Biol 1992;
42. Smithies O, Gregg RG, Boggs SS, Koralewski
MA, Kucherlapati RS. Insertion of DNA sequences into the
human chromosomal beta-globin locus by homologous recombination.
Nature 1985; 317: 230-234.
43. Thomas KR, Capecchi MR. Site-directed
mutagenesis by gene targeting in mouse embryo-derived stem
cells. Cell 1987; 51: 503-512.
44. Zwaka TP, Thomson JA. Homologous recombination
in human embryonic stem cells. In: Lanza R (ed.) The handbook
of embryonic stem cells. Vol 2: Embryonic stem cells. San
Deigo: Elsevier science; 2003: (in press).
45. Odorico JS, Kaufman DS, Thomson JA.
Multilineage differentiation from human embryonic stem cell
lines. Stem Cells 2001; 19: 193-204.
46. Rideout WM, 3rd, Hochedlinger K, Kyba
M, Daley GQ, Jaenisch R. Correction of a genetic defect
by nuclear transplantation and combined cell and gene therapy.
Cell 2002; 109: 17-27.
47. Finger S, Heavens RP, Sirinathsinghji
DJ, Kuehn MR, Dunnett SB. Behavioral and neurochemical evaluation
of a transgenic mouse model of Lesch-Nyhan syndrome. Journal
of Neurological Science 1988; 86: 203-213.
48. Fisher SJ. The placenta dilemma. Semin
Reprod Med 2000; 18: 321-326.
49. Cross JC. Genetic insights into trophoblast
differentiation and placental morphogenesis. Semin Cell
Dev Biol 2000; 11: 105-113.
50. Beddington RS, Robertson EJ. An assessment
of the developmental potential of embryonic stem cells in
the midgestation mouse embryo. Development 1989; 105: 733-737.
51. Thomson JA, Kalishman J, Golos TG, Durning
M, Harris CP, Becker RA, Hearn JP. Isolation of a primate
embryonic stem cell line. Proceedings of the National Academy
of Sciences, U.S.A. 1995; 92: 7844-7848.
52. Muyan M, Boime I. Secretion of chorionic
gonadotropin from human trophoblasts. Placenta 1997; 18:
53. American Heart Association. Heart disease
and stroke statistics -- 2003 update. Dallas, Tex: American
Heart Association; 2002.
54. Mummery C, Ward D, van den Brink CE,
Bird SD, Doevendans PA, Opthof T, Brutel de la Riviere A,
Tertoolen L, van der Heyden M, Pera M. Cardiomyocyte differentiation
of mouse and human embryonic stem cells. J Anat 2002; 200:
55. Soonpaa MH, Daud AI, Koh GY, Klug MG,
Kim KK, Wang H, Field LJ. Potential approaches for myocardial
regeneration. Ann N Y Acad Sci 1995; 752: 446-454.
56. Gandy KL, Weissman IL. Tolerance of
allogeneic heart grafts in mice simultaneously reconstituted
with purified allogeneic hematopoietic stem cells. Transplantation
1998; 65: 295-304.
57. Spitzer TR, Delmonico F, Tolkoff-Rubin
N, McAfee S, Sackstein R, Saidman S, Colby C, Sykes M, Sachs
DH, Cosimi AB. Combined histocompatability leukocyte antigen-matched
donor bone marrow and renal transplantation for multiple
myeloma with end-stage renal disease: The induction of allograft
tolerance through mixed lymphohematopeoietic chimerism.
Transplantation 1999; 68: 480-484.
58. Wekerle T, Sykes M. Mixed chimerism
as an approach for the induction of transplantation tolerance.
Transplantation 1999; 68: 459-467.
59. The effect of intensive treatment of
diabetes on the development and progression of long-term
complications in insulin-dependent diabetes mellitus. The
Diabetes Control and Complications Trial Research Group.
N Engl J Med 1993; 329: 977-986.