This commissioned paper was prepared for
and discussed at the Council's July
2003 meeting. It was intended solely to aid discussion, and does
not represent the official views of the Council or of the United States
Current Progress in Human Embryonic Stem Cell Research
Tenneille E. Ludwig and James A. Thomson
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.
Wisconsin National Primate Research Center
University of Wisconsin-Madison
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 culture.
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
BRL® Knockout™ 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 ES cells.
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
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 are
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 for screening.
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
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 b-cells results in type 1 diabetes. b-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 b-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 b-cell specific markers, offering hope of a scalable
source of b-cells for transplantation .
The challenges for using human ES cell-derived b-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 b-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 b-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 b-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 b-cells have transplanted the cells into the liver,
and the cells function in that site. Third, transplanted b-cells
must not be rejected by the immune system. Although the transplantation
of b-cells has been clinically successful, the severe immunosuppressive
therapy required may make the procedure inappropriate for the average
diabetic patient. Importantly, b-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 b-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.
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