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Stem Cells
®
Concise Review
Technical Assessment of the First 20 Years of Research
Using Mouse Embryonic Stem Cell Lines
Gregory J. Downing,a James F. Battey Jr.b
a
Office of Technology and Industrial Relations, National Cancer Institute, and bNational Institute on
Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA
Key Words. Mouse embryonic stem cell • Homologous recombination • Discovery • Technology
Abstract
This review assesses the effect that mouse embryonic
stem (ES) cells have had on biomedical research during
the 20 years that followed their isolation in 1981. Notable
scientific discoveries enabled by these cell lines—including insights into cell cycle regulation, spatial and temporal relationships during development, and the roles of
transcription factors and homeobox genes in developmental pathways—are discussed. The acceleration of
basic discovery of gene function and the genetic basis of
disease using a breakthrough technology (homologous
recombination between modified gene constructs and the
ES cell genome) became the principal enabling method to
establish transgenic laboratory animals with single targeted genetic change. This review also examines the widespread influence of mouse ES cells as an enabling technology by highlighting their effect on drug development
paradigms, directed differentiation to treat specific diseases, nuclear transfer protocols used in cloning, and
establishment of methodologies for isolating non-rodent
ES cells. This review concludes with a brief analysis of the
most influential mouse ES cell lines of the first 20 years as
viewed within the twin contexts of human disease application and contributions to the primary literature. Stem
Cells 2004;22:1168–1180
Introduction
These teratocarcinoma cell lines shared many morphological, biochemical, and immunological properties with pluripotent ES cells but had undergone transformation and karyotypic changes prior to establishment as cultured cell lines.
Concurrently, researchers such as Brinster et al. [8–10] and
Watanabe et al. [11] established the basic concepts for using
chimeras with a pluripotent cell line to introduce modifications to the mouse genome, thus guiding early experiments
with mouse ES cells in the following decade.
In 1981, building on experience with the teratocarcinoma cell lines and with the knowledge that early postimplantation embryos transferred to ectopic sites gave rise to
In the 1970s, scientists searched for a cell culture system that
could serve as a platform to conduct genetic studies focusing
on early embryonic development. Although it had been
established that post-implantation mouse embryos contained
pluripotent cells, early attempts to culture these cells in vitro
proved unsuccessful [1]. Prior to 1981, in vitro model systems to study the developing embryo used cell lines, such as
F9 and P10, derived from teratocarcinomas in vivo [2]. In
particular, Stevens [3–7] used these mouse embryonal carcinoma (EC) cell lines to establish many of the techniques that
would later be modified to isolate embryonic stem (ES) cells.
Correspondence: Gregory Downing, D.O., Ph.D., Director, Office of Technology and Industrial Relations, National Cancer
Institute, National Institutes of Health, Building 31, Room 10A-52, MSC 2580, 31 Center Drive, Bethesda, MD 20892-2580
USA. Telephone: 301-496-1550; Fax: 301-496-7807; e-mail: [email protected] Received May 3 2004; accepted for
publication July 16, 2004. ©AlphaMed Press 1066-5099/2004/$12.00/0 doi: 10.1634/stemcells.2004-0101
STEM CELLS 2004;22:1168–1180
www.StemCells.com
Downing, Battey
teratomas containing pluripotent stem cells [3, 12], two
groups of scientists successfully isolated and cultured
ES cells from the mouse. Two simple yet elegant studies
appeared that year that described the isolation and growth in
culture of proliferative cells derived from mouse embryos
[13, 14]. These cells were among the first to be shown to grow
in an undifferentiated state for long periods of time and to be
capable of differentiating into multiple cell types. With this
auspicious start, mouse ES cells soon became an indispensable tool for discovery in biomedical research.
There is no doubt that these cell lines have continued to
represent an important research tool for many areas of biomedical science. In the two decades since Martin [13] and
Evans and Kaufman [14] published their novel methods,
more than 1,200 scientific publications have included mouse
ES cells as a research emphasis. The era of molecular biology
and recombinant DNA technology was in its infancy in 1981,
making the discovery and application of this tool all the more
remarkable. In the late 1970s and early 1980s, there were no
genomic databases, polymerase chain reaction (PCR) techniques were only a concept, the application of homologous
recombination to gene-knockout technology had yet to be
proven, and applications of monoclonal antibody technology
in cell biology were just emerging. As such, the discoveries
enabled by mouse ES cells were remarkable from a historical
perspective.
Among the unique aspects of the history of this biomedical research tool is that there is a clear lineage of the source
of the technology. In the 1980s, two strains of mice—129 and
C57BL/6—provided the most stable sources of ES cells. The
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pace of discovery grew slowly in the first few years, followed
by a rapid period of publication growth in the early 1990s
(Fig. 1, Appendix) that some have attributed to a Cold Spring
Harbor symposium and the use of the cell lines in homologous recombination, events that occurred several years earlier [15–20].
This article reviews the groundbreaking research capabilities and technologies enabled by mouse ES cells in the
previous two decades. From homologous recombination to
insight about cell cycle regulation, pathways for signal transduction and embryonic development, and spatial and temporal relationships during development, mouse ES cells have
become a fundamental discovery tool in biomedical research.
Many of the breakthroughs that will be discussed here are
highlighted in Table 1.
While mouse ES cells continue to inform basic research
discoveries, their true impact is felt through their far-reaching application as an enabling technology. Mouse ES cells
have influenced paradigms and methodologies for a variety
of disciplines during the previous two decades, providing
researchers with a tool that allows them to view and conduct
their experiments using approaches unavailable in 1980. We
highlight the part that mouse ES cells have played in breakthroughs with the potential to shape the future of medicine,
including drug development paradigms, directed differentiation to treat specific diseases, nuclear transfer protocols used
in cloning, and the establishment of methodologies for the
isolation of non-rodent ES cells. Remarkably, the wealth of
information enabled by mouse ES cells has largely resulted
from work using a few key cell lines. This article concludes
Figure 1. Number of publications per year that cite the use of mouse embryonic stem cell or germ cell lines. Details of search criteria are
provided in the Appendix.
Research Using Mouse Embryonic Stem Cell Lines
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Table 1. Twenty years of milestones in the application of mouse ES cells as a discovery tool
Year
Milestone
References
1981
Establishment of mouse ES cell lines
1985
ES cells shown capable of high level of organized development
68
1987
ES cells allow targeted mutagenesis via homologous recombination
17, 20
1988
Leukemia inhibitory factor regulates differentiation in ES cell cultures
59, 60
1989
First viable chimeric mice produced from homologous recombination
23
1990
Discovery of Oct-4 in mouse embryonal carcinoma cells
90
1998
High-throughput gene-trapping methodology established
99
2000
Directed differentiation of mouse ES cells into vascular progenitors,
neurons, and pancreatic islet-like cells
74–76
13, 14
Abbreviation: ES, embryonic stem.
by assessing the contributions of these lines in the twin contexts of their effect on the primary literature and the insights
that they have provided on the understanding of human diseases.
Mouse ES Cells Usher in the Genomic Era
Although mouse ES cells have supported discovery in myriad research fields, their value was established permanently
in the mid-1980s as a tool to enable targeted mutagenesis. In
1985, Oliver Smithies and colleagues [19] demonstrated
gene targeting using homologous recombination in a mammalian cell line. Two years later, several groups showed that
homologous recombination could produce targeted genetic
changes in the mouse ES cell genome that supported an animal model for Lesch-Nyhan syndrome, a rare neurological
disorder [17, 20]. While Thomas and Capecchi [17] modestly referred to their demonstration of site-directed mutagenesis in a mouse ES cell line as “a protocol [that] should be
useful for targeting mutations into any gene,” this gene-targeting technology would ultimately establish homologous
recombination as a means to systematically modify the
mouse genome. Several years earlier, it was demonstrated
that, when they were injected into mouse blastocysts, genetically altered ES cells could generate transgenic offspring
[21, 22]. The application of these techniques with homologous recombination technology thus provided scientists with
a controlled process to generate an unlimited variety of transgenic mice with engineered, predetermined genomes.
The technology developed rapidly, and the first viable
chimeric mice were reported in 1989 [23]. Inspired by
contemporary breakthroughs in PCR [24, 25], the development of positive-negative double selection [26], and geneknockout techniques [27, 28], researchers were able to
devise increasingly subtle and sophisticated targeted genetic
changes. ES cell culture was established as the first step in
creating knockout and, later, transgenic (knock-in) mice. As
a result of these groundbreaking efforts, a variety of protocols has been established for the precise introduction of
DNA and targeted changes into ES cells, and ES cells have
had a profound effect on the engineering of the mouse
genome [29, 30].
The true influence of this breakthrough on scientific
research is difficult to quantify, as knockout and transgenic
mice have provided insight into the etiologies of hundreds of
diseases, from cancers to anxiety, from diabetes to addiction,
in approximately 15 years [29, 31–35] (for full listing, see the
Transgenic/Targeted Mutation Database, http://tbase.jax.
org). A Medline keyword search for “knockout mouse” in
publications from the previous 2 years produces more than
6,500 hits; perhaps a more useful measure of the impact of
homologous recombination is its almost instantaneous widespread use and application to numerous disciplines. Within 4
years of these breakthrough articles, homologous recombination in cultured ES cells, followed by reintroduction into
the mouse, provided insight into the roles of many developmental factors, including proto-oncogenes, homeobox
genes, specific growth factors, and transcription factors
[36–47]. By 1992, homologous recombination had outgrown
its initial application to Lesch-Nyhan syndrome, and mouse
ES cells had been used to develop useful animal models for
cystic fibrosis [48, 49] and muscular dystrophy [50].
The seminal homologous recombination experiments in
the late 1980s created null mutations in particular genes, and
analysis of the corresponding phenotypes generated by these
mutations provided insight into gene function. Researchers
soon realized that this approach could be modified slightly
by introducing a given cDNA in frame with the coding
sequence of the targeting gene, thereby engineering a modi-
Downing, Battey
1171
fied allele that expresses the cDNA insert in place of the
endogenous gene. Knock-in mice, created using this variant
of the null mutation approach, have extended capabilities to
study gene expression patterns [51], to monitor the fate of
cells that normally express the target gene in animals
homozygous for the mutation [52], to examine the function
of proteins coded by related genes from a multigene family
[53, 54], and to monitor neuronal activity at the synapse [55].
However, genetic diseases in humans rarely result from
null mutations. Therefore, sophisticated gene-targeting strategies that can produce point mutations and gene hypomorphs
are required to more precisely mimic the genetic bases of
many human diseases in mice. Directed mutagenesis via
mouse ES cells has been the central component of various
targeting strategies to engineer highly specific mutant alleles, as reviewed elsewhere [56, 57]. Many of these systems
can introduce subtle mutations with no interference in genetic expression. Moreover, there is evidence that several of
these targeted mutagenesis strategies may be amenable to
conditional mutagenesis, which makes them powerful tools
in situations in which the expression of the altered genes provokes embryonic lethality in an embryo or fetus homozygous
for the modified gene’s zygote, or renders a complex phenotype [57, 58].
of all of these factors, with a sharp increase in CDK4-associated kinase activity. These observations suggested that the
induction of differentiation converts ES cells to a CDK4dependent mode of control of G1-phase progression. Differentiation also resulted in an extended G1 phase and a slowed
rate of cell division, suggesting that signals that promote cell
division may inhibit differentiation.
Mouse ES cells have also played a part in elucidating
many of the signal transduction pathways that regulate progression through the cell cycle, such as the phosphatidylinositol 3-kinase pathway and the Ras/extracellular signal-regulated protein kinase pathway [65]. In addition, increases in
production of cyclin D1 have been shown to be affected by the
redox state within mouse ES cells, supporting the hypothesis
that a redox cycle within the mammalian cell cycle may be the
mechanistic link between the metabolic processes in early G1
and the subsequent activation of regulatory proteins that
signal entry into the S phase [66]. Furthermore, mouse ES
cells have proven useful for experimental confirmation
of mathematical models of regulation of the G1 phase [67].
The uniqueness of the mouse ES cell cycle has allowed
researchers to control the onset of differentiation, thereby
providing a framework for studying the integration of a differentiation program with cell cycle regulation.
Mouse ES Cells Provide Insight into
Cell Cycle Regulation Pathways
Configuring Spatial and Temporal
Relationships during Development:
A Case Study of Vasculogenesis
In 1988, two groups reported simultaneously that the presence of leukemia inhibitory factor (LIF) in the growth
medium allowed mouse ES cells to proliferate indefinitely in
vitro in an undifferentiated state in the absence of a feeder
layer [59, 60]. Upon reinjection into the blastocyst, ES cells
cultured in the presence of LIF contribute to the development
of the whole embryo [61]. Furthermore, withdrawal from
LIF induces cultured ES cells to differentiate into endodermlike cells. While this observation provided an alternative to
the use of feeder cells for ES cell culture protocols, it also
promoted the use of mouse ES cells as a discovery tool for
cell cycle regulation and signal transduction pathways.
Proliferation in vitro without differentiation suggests
that cultured mouse ES cells exhibit an unusual cell cycle and
that this cycle is regulated by LIF. Based on observations of
the role of E- and D-type cyclins in the promotion of G1
phase progression and G1/S transition in cycling cells [62,
63], Savatier and colleagues [64] used mouse ES cells to
probe the cell cycle factors that lead to differentiation. These
researchers examined the expression of E- and D-type
cyclins, cyclin-dependent kinases (CDKs) 2 and 4, and several CDK inhibitors during the process of differentiation in
mouse ES cells induced by the removal of LIF from the culture medium. Withdrawal of LIF resulted in the upregulation
ES cells have become a powerful tool to study spatial and
temporal relationships that determine cell, tissue, and organ
development. This section profiles their role in understanding vasculogenesis and angiogenesis, processes that have
applications to many fields, from regenerative medicine to
cancer research.
The ES cell lines derived in the seminal 1981 experiments of Martin [13] and Evans and Kaufman [14] spontaneously differentiated to form embryoid bodies. However, it
was not until 1985 that researchers began to examine the
degree to which organized development within these embryoid bodies actually paralleled that within the developing
embryo. In that year, Doetschman and colleagues [68] used
cell lines derived from 129 and C57BL/6 blastocysts to show
that ES cells were capable of exhibiting a high level of organized development at a high frequency. From 8 to 10 days of
culture, approximately half of the embryoid bodies expanded
into cystic structures analogous to the visceral yolk sac of the
post-implantation embryo. Approximately one third of these
cystic embryoid bodies developed myocardium, and when
cultured in the presence of human cord serum, 30% developed blood islands. The authors also noted that this developmental potential and consistency of expression was observed
1172
in most ES lines, making the cells a useful tool to investigate
early embryogenesis.
In 1992, Wang and coworkers expanded the work of
Doetschman [68] and others [69] to show that a primitive vasculature forms in these embryoid bodies, establishing them as
a useful model for the study of developmental blood vessel
formation [70]. Not only were the blood islands lined by
endothelial cells, but also the blood island matured to form
connections and channels that resemble blood vessels. Thus,
the ES cell culture system retained the cellular signals for both
the primary differentiation of pluripotent stem cells into
endothelial cells and for some of the cellular processes involved in subsequent vascular development.
Based on these discoveries, ES cells became a viable tool
for developing in vitro systems to investigate vasculogenesis
as it occurs in embryos. However, cell types from multiple
lineages were generated when using these systems, thus
hampering clear understanding of the interactions between
endothelial cells and other essential cells, such as mural cells,
in vascular development and maintenance [71, 72]. Interested in dissecting the cellular events that lead to formation
of a primitive vasculature, Hirashima and colleagues [73]
developed a system to isolate endothelial cell progenitors in
vitro. From mouse ES cells, they derived and isolated
endothelial cell progenitors that expressed fetal liver kinase 1
(Flk1), a marker essential for vasculogenesis. In subsequent
experiments, they showed that these Flk1+ cells can differentiate into both endothelial and mural cells and can reproduce
the vascular organization process [74]. As a result, the Flk1+
cells function as vascular progenitor cells capable of differentiating to form mature blood vessels. Recently, similar
strategies have been employed to direct differentiation of
mouse ES cells into pancreatic islet-like cells [75] and neurons [76]. As with many applications of mouse ES cells, the
potential achievements enabled by stem cells extend far
beyond researchers’initial visions; what began initially as a
quest to understand mechanisms of development thus produced an enabling methodology for tissue engineering of the
vascular system.
Understanding pathways in Early Embryonic
Development: Homeobox Genes and
Transcription Factors
Genes that contain the homeobox motif, a highly conserved,
180–base pair DNA sequence, are expressed in a temporal
and tissue-specific pattern during embryogenesis [77]. Using
breakthroughs in homologous recombination, researchers
rapidly identified the presence, and later the identity and location, of many of the homeobox genes implicated in early
development [78–82]. In 1987, Harald Eistetter [80] demonstrated that several homeobox-containing DNA sequences
Research Using Mouse Embryonic Stem Cell Lines
were stage-specifically regulated during in vitro differentiation of mouse ES cells, prompting a flurry of research activity
that would ultimately characterize numerous murine homeobox genes, including the Hox family [36, 83–85], Csx [86],
Nkx2-5 [82], and Pem [87, 88].
In particular, ES cells have enabled investigators to follow the developmental outcomes of mutations in the family
of Hox genes, which are expressed in defined domains along
the anterior–posterior axis of the developing embryo [89]. In
1990, LeMouellic and colleagues [83] used homologous
recombination to replace the murine Hox-3.1 gene with lacZ,
a reporter gene for β-galactosidase, from Escherichia coli.
Because lacZ expression is easily detected in situ with high
sensitivity, these researchers were able to observe localized
areas of β-galactosidase activity during development. Mice
homozygous for the targeted Hox-3.1 allele demonstrated
phenotypic characteristics, such as the adjusted placement of
the rib cage, that reflected similar segmentary transformations in Drosophila associated with loss-of-function homeotic mutations [84]. By contrast, mice deficient in Hox-1.6
exhibit profound defects in the ears, cranial nerves, and ganglia that result from a loss, rather than a homeotic transformation, of tissue [85].
In 1990, mouse EC cells enabled the identification of a
novel DNA octamer-binding transcription factor, Oct-4 (also
called Oct-3 and Oct 3/4), widely studied as a prototypical
marker of undifferentiated, dividing cells [90]. A transcription factor capable of activating or repressing gene expression, Oct-4 is necessary for maintaining cells in an undifferentiated state. Ten years after this key discovery, Niwa and
colleagues [91] shifted previous paradigms of binary transcriptional control by demonstrating that the precise level of
Oct-4, rather than its mere presence, governs the fate of ES
cells. In their groundbreaking experiments, these researchers
showed that a less than twofold increase in Oct-4 expression
caused cells to differentiate into primitive endoderm and
mesoderm, while repression of Oct-4 induced loss of
pluripotency and dedifferentiation into trophectoderm. As
such, the transcription factor has been termed a “master regulator” of pluripotency.
Oct-4 has been shown to regulate at least seven genes,
activating some and repressing others. It has been postulated
that the transcription factor exerts its overall impact by preventing the expression of genes that are required for differentiation [92]. Investigation of the role of Oct-4 in mouse ES
cells has prompted researchers to speculate that it may have a
similar role in human and primate development, as suggested
by its presence in human [93] and primate [94] ES cells.
However, complexities in cell culture conditions have meant
that the vast majority of information about Oct-4 has been
made possible by the study of mouse ES cells in vitro.
Downing, Battey
Mouse ES cells have also recently enabled the identification of the homeobox gene, nanog, a unique pluripotencysustaining factor in ES cells [95, 96]. Nanog mRNA is present
in pluripotent mouse and human ES cells yet absent in differentiated cells. The nanog gene is capable of maintaining
mouse ES cell self-renewal independent of the LIF/Stat3
pathway associated with transcription factors such as Oct-4,
Sox2, and FoxD3. Elevated nanog expression from transgene
constructs is sufficient for clonal expansion of ES cells, and
nanog-deficient ES cells differentiate into extraembryonic
endoderm lineage. Understanding of the molecular mechanisms that govern pluripotency is a key step toward generating pluripotent cells from somatic stem and other cells. The
identification and characterization of nanog demonstrates
the central role of mouse ES cells as an enabling technology
to understand the pathways that govern early embryonic
development.
The Widespread Effect of Technologies
Enabled by Mouse ES Cells
The true influence of mouse ES cells can be seen in their
widespread application as an enabling technology. In both
subtle and overt ways, mouse ES cells have provided
researchers with a tool that allows them to view their experiments through a set of lenses unavailable in 1980. Mouse ES
cells have influenced paradigms and methodologies for a
diverse set of applications during their first 20 years, leading
to refinements in existing protocols and offering the basis for
breakthroughs with wide future application. This section
highlights the effect of mouse ES cells on areas that will
shape the future of medicine, including drug development,
directed differentiation to treat specific diseases, nuclear
transfer protocols used in cloning, and the establishment of
methodologies for the isolation of non-rodent ES cells.
Discovering Molecular Targets and
Drug Development Paradigms
In the post-genomic era, strategies that use homologous
recombination to target single genes have become augmented by a battery of gene-trapping approaches with the
potential for probing the entire genome simultaneously [97].
When inserted into a gene, these gene-trap vectors generate
fusion RNA transcripts that facilitate identification of the
trapped endogenous gene from each clone of interest by
sequencing. Gene trapping is therefore advantageous in
mammalian cells with complex genomic organization, and
the technique provides a convenient method for tagged random mutagenesis in mice [98]. Gene-trap vectors can be
introduced into mouse ES cells by retroviral integration [99]
or by physical methods such as electroporation and transfection [100]. The technique provides great promise for the
1173
large-scale screening efforts associated with drug discovery
and development. For example, one recent report describes a
high-throughput gene-trapping method that allows the automated identification of sequence tags from the mutated genes
[99]. Using this approach, researchers created a library of
mouse ES cells that contains sequence-tagged mutations in
2,000 genes.
Genetically engineered mice have become an enabling
technology in the private sector, spurring the processes of
drug discovery and development. High-throughput construction and evaluation of genetically engineered mice have
become standard practices for investigating molecular
mechanisms of disease, evaluating prospective therapeutic
targets, and screening candidate agents for efficacy and
safety [101]. For example, a drug thought to work by modifying the activity of a specific protein should behave differently
in a mouse model where the gene encoding the specific protein is targeted. In this manner, scientists can determine if a
drug is working as predicted or by modulating a different cellular pathway. Looking forward, phenotypes of genetically
engineered mice may reveal potential target organs and
uncover toxic effects of potential therapies. Furthermore,
constructs in which the engineered gene does not result in
significant therapeutic potential may provide insight into
molecular mechanisms and signaling pathways with numerous other applications.
The widespread use of knockout mice has shifted paradigms in drug development, enabling a supporting biotechnology industry in the process. A recent retrospective evaluation of the 100 best-selling drugs demonstrates that knockout
phenotypes for the drugs’ targets correlate well with known
drug efficacy, thus supporting continued use of the technology as a tool for target discovery and validation [102]. For
example, specific inhibitors of cyclooxygenase-2 (COX2),
such as celecoxib and rofecoxib, have achieved high sales as
treatments for arthritis. The role of COX2 in the inflammatory process has been elucidated in part from extensive study
of Cox2 knockout mice, which exhibit reduced inflammation
and a significant reduction in collagen-induced arthritis. The
observation that Cox2 knockout mice also have decreased
polyp formation has suggested additional uses for currently
marketed COX2 inhibitors in areas such as cancer.
Furthermore, new molecular targets for drug discovery
can be identified by systematically mutating genes and determining the phenotypic consequences, such as obesity, arthritis, and cardiovascular disease. The sequencing of the human
genome will provide the pharmaceutical industry with a
plethora of potential new drug targets, thereby encouraging
the development of large-scale mouse knockout or genetrapping programs. Questions about the utility of the knockout mouse have now been supplanted by concerns related to
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scale such as cost, labor intensity, and timeliness. As the 21st
century unfolds, more demands will be made on highthroughput platforms and screening processes, and mouse
ES cells therefore will influence another level of biotechnology development.
Directed Differentiation for Specific
Disease Applications
The ability to induce stable genetic modifications in ES cells
offers the potential to direct the course of differentiation in
vivo through a series of in vitro manipulations. Human ES
cells may one day serve as a source of specialized cells useful
in numerous regenerative medicine applications. One of the
most widely studied examples of directed differentiation, the
formation in vivo of putative dopaminergic and serotonergic
neurons from mouse ES cells, has great potential for the
understanding and treatment of neurodegenerative diseases
such as Parkinson’s disease [103].
Breakthroughs with neuronal differentiation in vivo have
largely been realized within the last decade, and advances
could not have been possible without the wealth of basic
research on the culture and modification of mouse ES cells in
vitro. Using established techniques, researchers have shown
that, when they are transplanted into the mouse brain, mouse
ES cells can be induced to differentiate into dopaminergic and
serotonergic neurons [104]. Advances in cell culture techniques have also provided efficient methods for generating
dopaminergic and serotonergic neurons in high yields from
mouse ES cells in vitro [76, 105]. Dopaminergic neurons produced from mouse ES cells share the electrophysiological and
behavioral properties expected of neurons from the midbrain
[106], and transplantation of ES cell–derived dopaminergic
neurons has been shown to partially correct the phenotype of a
murine model of Parkinson’s disease [107].
Techniques to induce efficient neuronal differentiation
of mouse ES cells have recently been extended to derive
dopaminergic neurons from primate ES cells [108]. While
the application of these breakthroughs to the treatment of
motor disorders in humans has yet to be realized, mouse ES
cells are pivotal tools that enable researchers to investigate
factors that govern differentiation and to apply this knowledge to putative models of human disease.
Somatic Cell Nuclear Transfer Protocols
and Cloning
Recent improvements in somatic cell nuclear transfer
(SCNT) methodologies have suggested numerous potential
applications for cloning in basic research, medicine, and
agriculture [109]. Although currently a relatively inefficient
process, nuclear transfer using embryo-derived nuclei [110]
Research Using Mouse Embryonic Stem Cell Lines
and fetal and adult cell nuclei [111] has produced viable
sheep, including the well-known “Dolly.” SCNT has been
used successfully in the genetic modification of cattle [112],
mice [113], goats [114], and pigs [115], suggesting that, once
optimized, the technique may have a central role in research
on disease models, xenotransplantation, and the genetic
engineering of livestock. The technique has recently been
used with homologous recombination to create the first nonrodent knockout animal, a sheep with deletion of the prion
protein (PrP) gene [116].
While this technology does not require mouse ES cells
per se, the results of gene-modification strategies using
mouse ES cells are a clear motivation for these techniques in
mammals. Understanding of developmental processes in
mouse ES cells provides a guidepost to researchers as they
develop state-of-the-art SCNT methodologies. Mouse ES
cells are commonly used as a comparative tool in SCNT protocols to assess the characteristics of the newly modified cells.
For example, in their seminal papers on the creation of cloned
sheep, Campbell and colleagues [110] often describe their
embryonic cells in terms of the morphology and expression of
markers of differentiation present in mouse ES cells at complementary stages of development. When Campbell suggests
that somatic cell nuclear transfer will offer opportunities for
genetic manipulation of livestock analogous to gene modification in knockout mice, it is evident that mouse ES cells have
not only enabled a developing technology but also inspired a
vision for the technology’s future direction and potential.
Furthermore, novel mouse ES cell lines have been created via nuclear transfer from adult mouse somatic cells from
inbred, hybrid, and mutant strains [105]. These ES cells can
be induced to differentiate into numerous cell types, including dopaminergic and serotonergic neurons in vitro and germ
cells in vivo.
Non-Mouse Stem Cells
Although mouse ES cells offer a versatile tool to study mechanisms that control differentiation, significant differences
between early human and early mouse development suggest
that other models may be more appropriate for the study of
human development and the application to human disease. By
modifying cell culture techniques developed for mouse ES
cells, Thomson and colleagues [117] successfully isolated the
first primate ES cell line from rhesus monkeys in 1995.
Because of their similarity to humans, rhesus monkeys may
provide an appropriate model to investigate strategies to prevent immune rejection of transplanted cells and to demonstrate the safety and efficacy of ES cell–based therapies [118].
Just as studies of mouse teratocarcinoma-derived stem
cells preceded the isolation and characterization of mouse
Downing, Battey
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ES cells, studies of human teratocarcinoma-derived stem
cells preceded the isolation of human ES cells. Definition of
human teratocarcinoma stem cell morphology and expression of the stage-specific embryonic antigens SSEA-3 and
SSEA-4 (but not SSEA-1) [119–121] prepared the way for
Thomson and coworkers to isolate human ES cells from
human blastocysts in 1998 [118]. These cells demonstrated
the ability to form trophoblasts and derivatives of all three
embryonic germ layers in teratomas after injection into
immunodeficient mice. Ethical and practical concerns are
raised by experiments that employ human ES cell lines, thus
suggesting that some future applications of primate and
human ES cells will be preceded by mouse models and complementary experiments that use mouse ES cells.
From One to Many: The Trajectory
of Mouse ES Cell Lines in the First
Two Decades
In the early years of mouse ES cell research, a substantial
body of knowledge was obtained using relatively few cell
lines. Between 1981 and 2001, approximately two thirds of
published mouse ES cell original research articles feature
combinations of six key cell lines (Table 2), and subclones
and modifications of these lines continue to spawn new cell
lines and research directions. From their early application to
Lesch-Nyhan syndrome, these seminal cell lines have contributed to the creation of transgenic animal models of many
human diseases, including osteoporosis, Huntington’s disease, and myeloid leukemia. In addition, transgenic mice
derived using ES cells have provided insight into complex
human disorders from diabetes to cystic fibrosis. The
dynamic, expanding application of mouse ES cells as a discovery tool is reflected in the steady annual increase in the
number of publications that feature mouse ES cells (Fig. 1,
Appendix).
Although novel mouse ES cell lines continue to be created, cell lines developed in the 1980s, such as CCE, D3, and
E14, have remained useful. In a sense, these cell lines are the
engines that have driven the first two decades of mouse ES
cell research. It must be noted, however, that these lines were
created by modifying techniques and conditions established
for murine EC cell lines such as F9 and P19. The varied and
continual subcloning of these fundamental lines has resulted
in a traceable lineage for each mouse ES cell line. A diagram
showing selected subclones that have been developed from
the E14 line is provided in Figure 2.
While mouse ES cells have contributed to the development of transgenic animal models of human disease, they cannot be linked directly to the development of a well-known
drug or diagnostic tool. However, ES cell–based breakthroughs, such as homologous recombination, underscore
many patented technologies, including disease-specific
mouse ES cell lines, gene-trapping techniques, and highthroughput platforms. In the two decades since they were first
created, murine ES cell lines have proved to be a versatile
enabling tool, and trends in the literature suggest that new
applications of this technology continue to proliferate. As
the 21st century unfolds, it is clear that emerging stem cell–
based technologies will continue to create new breakthroughs
and establish new milestones in biomedical research and
application.
Conclusion
The isolation of murine ES cell lines in 1981 enabled a
plethora of research opportunities. From gene targeting by
homologous recombination to new insights on cell cycle reg-
Table 2. Publications and disease insights provided by mouse ES cell lines
Cell line
Year
established
[reference]
F9a
1973 [122]
77
1982 [123]
51
P19
a
No. of featured
publications
(1981–2001)
Key transgenic animal models / human
disease applications [reference]
CCE
1981 [14]
147
Lesch-Nyhan syndrome [18], Hemophilia B [124], Myeloid leukemia [125]
D3
1985 [68]
279
Muscular dystrophy [50], Human B0 thalassemia [126], Inflammatory bowel
disorders [127]
E14 lines
1987 [128]
195
Lesch-Nyhan syndrome [128], Cystic fibrosis [48]
AB lines
1990 [40]
76
Osteoporosis [44], Hypercholesterolemia [129], Citrullinemia [130]
J1
1992 [131]
75
Tay-Sachs disease [132], Sandhoff disease [132]
R1
1993 [133]
129
Diabetes [134], Rickets [135], Paroxysmal nocturnal hemoglobinuria [136]
a
Embryonal carcinoma cell lines.
1176
Research Using Mouse Embryonic Stem Cell Lines
Figure 2. Selected subclones and cell lines derived from E14. Key cell lines such as E14 have produced numerous embryonic stem cell
lines cited in the literature.
ulation and development, these ES cells provided researchers
with the toolkit needed to better understand the genetic basis
of many diseases. This review demonstrates the diverse influence of mouse ES cells on molecularly based therapeutic
development, cloning, directed differentiation, and the establishment of mammalian ES cell lines. Moreover, the understandings of the pluripotent and proliferative nature of these
cells provide the foundations for pioneering work with their
human counterparts, including the overall frontier of stem
cell biology that aims to regenerate and restore form and
function to patients. As applications of ES cell biology progress from a discovery tool to applied technologies for medicine, research with mouse ES cells continues to enable many
extraordinary achievements.
Acknowledgments
The authors thank Charles A. Goldthwaite Jr. for his assistance in preparing this manuscript. The database referenced
here is available as a searchable Web-based resource on the
NIH stem cell Website at http://stemcells.nih.gov/research/
mouselit/.
Appendix
Criteria for the Selection of Scientific
Publications That Feature Mouse Embryonic
Stem Cell Lines
A PubMed search of the scientific literature from 1981 to
2001 was conducted using the keyword “mouse embryonic
stem cells” and yielded over 2,700 citations. Although the
literature includes publications that feature cell lines developed from other species (e.g., rat, hamster, cow, pig), our
search focused only on cell lines developed from mice
because of the ubiquitous nature in which they are used. Publications were collected and reviewed to determine if they
met the entry criteria for the analysis. There were 1,338 original papers deemed acceptable for review. Thus, more than
1,400 review papers, methods papers, publications describing work on isolated cell preparations (not cultures), and
incorrectly coded PubMed citations were excluded. Thirty
other eligible publications were identified that were not captured via the key words in the PubMed search, and these were
included in the analysis.
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