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) [1] 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

1

Area of interest	Publications to date	Reference(s)
Derivation	5	[1-5]
Culture Optimization	6	[6-11]
Feeder Layer Alternatives/Replacements	4	[6-9]
Media Analysis	1	[10]
Freezing	1	[11]
Characterization	5	[12-16]
Modification	6	[17-22]
Differentiation into multiple lineages	3	[23-25]
Differentiation into specific lineages	15	[26-40]
Neural	4	[26-29]
Cardiac	5	[30-34]
Endothelial (Vascular)	1	[35]
Hematopoietic (Blood)	2	[36, 37]
Pancreatic (Islet-like)		[38]
Hepatic (liver)	1	[39]
Trophoblast	1	[40]

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 [41] .  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^Ò Knockoutä Serum Replacer), it supports human ES cells and significantly reduces the batch variability associated with serum [12] .  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 [6] .  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 [9] , fetal muscle or skin [7] , adult human fallopian tube epithelial cells [7] , or human foreskin [8] 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 [7] .  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 [17] and stable [18] 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" [20] .

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 [22] .  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 [12] . 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 [45] ; 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 [46] .  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 [47] .  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 are inadequate.

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 [50] . 

Spontaneously differentiated rhesus monkey [51] or human ES cells [1] do secrete modest amounts of chorionic gonadotropin, indicating the differentiation of trophoblast cells [52] .  Recently it was discovered that a single growth factor (BMP4) would induce human ES cells to differentiate to a pure population of early trophoblast [40] .  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 [40] . 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 Differentiation

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 [53] .  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 [34] , and 5-aza-2'-deoxycytidine or density gradient separation allows some enrichment of cardiomyocyte populations [32] . 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 [30] .  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 [55] .  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 [35] , and such cells may be useful in supporting co-transplanted heart cells. 

Neural Differentiation

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 [28] .  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 [28] .  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 carefully.

Hematopoietic Differentiation

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 [36] . Enrichment of ES cell-derived hematopoietic precursors is accomplished by treatment with cytokines or BMP-4 [37] .  Cell sorting using hematopoietic-specific cell surface markers yields myeloid, erythroid, and megakaryocyte precursors [36] .  

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] .

Pancreatic Differentiation

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 [59] .  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 [38]

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.

Conclusions

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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