Technical Assessment of the First 20 Years of Research Using Mouse Embryonic Ste - 《stemcells》2004-2005文献干细胞之家



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a Office of Technology and Industrial Relations, National Cancer Institute, and. Y( M9 C, l/ G8 K' R0 A/ `
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b National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA

Key Words. Mouse embryonic stem cell ? Homologous recombination ? Discovery ? Technology' b( l/ b4 [) o! O" Z2 q

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: downingg@mail.nih.gov2 L0 O/ ?8 K- S( c8 E" X

ABSTRACT6 y2 C8 O) w2 h; w  U; q9 D  w/ J  ?
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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.
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INTRODUCTION/ p1 O$ _* h9 E& z: q, U( V

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 . 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 . In particular, Stevens  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.
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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.  and Watanabe et al.  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.7 d- z+ g9 d+ M3 P: l: R8 J
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In 1981, building on experience with the teratocarcinoma cell lines and with the knowledge that early post-implantation embryos transferred to ectopic sites gave rise to teratomas containing pluripotent stem cells , 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 . 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  and Evans and Kaufman  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.. O. ^& I5 e! Y* T* M
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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 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 .

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.

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.$ a- S7 P. w5 a: K8 k
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Table 1. Twenty years of milestones in the application of mouse ES cells as a discovery tool8 {; V4 a* A) n5 P

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 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.
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MOUSE ES CELLS USHER IN THE GENOMIC ERA# T) K' M$ O& H8 G
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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  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 . While Thomas and Capecchi  modestly referred to their demonstration of site-directed mutagenesis in a mouse ES cell line as "a protocol  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 . 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 . Inspired by contemporary breakthroughs in PCR , the development of positive-negative double selection , and gene-knockout techniques , 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 .* [$ v( U/ e% N0 e8 }
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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  (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 . 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  and muscular dystrophy .

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 modified 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 , to monitor the fate of cells that normally express the target gene in animals homozygous for the mutation , to examine the function of proteins coded by related genes from a multigene family , and to monitor neuronal activity at the synapse .
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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 . 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 .

MOUSE ES CELLS PROVIDE INSIGHT INTO CELL CYCLE REGULATION PATHWAYS
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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 . Upon reinjection into the blastocyst, ES cells cultured in the presence of LIF contribute to the development of the whole embryo . Furthermore, withdrawal from LIF induces cultured ES cells to differentiate into endoderm-like 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., p$ M* y/ |8 ~2 D9 v+ U

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 , Savatier and colleagues  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 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 CDK4-dependent 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.& Y2 j3 N# o% K4 _# ]& w8 N4 M

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 . 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 . Furthermore, mouse ES cells have proven useful for experimental confirmation of mathematical models of regulation of the G1 phase . 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.
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CONFIGURING SPATIAL AND TEMPORAL RELATIONSHIPS DURING DEVELOPMENT: A CASE STUDY OF VASCULOGENESIS

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  and Evans and Kaufman  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  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 in most ES lines, making the cells a useful tool to investigate early embryogenesis.$ X2 u# |- h  y5 W; n- g
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In 1992, Wang and coworkers expanded the work of Doetschman  and others  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 . 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.
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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 . Interested in dissecting the cellular events that lead to formation of a primitive vasculature, Hirashima and colleagues  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 . 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  and neurons . 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
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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 . 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 . In 1987, Harald Eistetter  demonstrated that several homeobox-containing DNA sequences 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 , Csx , Nkx2-5 , and Pem .0 y+ b3 X0 \) }- K

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 . In 1990, LeMouellic and colleagues  used homologous recombination to replace the murine Hox-3.1 gene with lacZ, a reporter gene for ?-galactosidase, from Escherichia coli. Because lacZ expression is easily detected in situ with high sensitivity, these researchers were able to observe localized areas of ?-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 . 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 .
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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 . 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  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.$ f6 W: U, t/ j! r, }; y& G

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 . 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  and primate  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.* g) p6 Z! g" o( r3 o! A
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Mouse ES cells have also recently enabled the identification of the homeobox gene, nanog, a unique pluripotency-sustaining factor in ES cells . 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 CELLS6 P1 s# D8 v, {% |% G; v0 y
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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 Paradigms5 `( `' F! f% Y8 a$ q7 J# [
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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 . 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 . Gene-trap vectors can be introduced into mouse ES cells by retroviral integration  or by physical methods such as electroporation and transfection . The technique provides great promise for the 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 . Using this approach, researchers created a library of mouse ES cells that contains sequence-tagged mutations in 2,000 genes.; b8 I2 [) n& _" }  X( S+ ]

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 . 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.& ^" j# v  j; r5 _

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 . 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 gene-trapping programs. Questions about the utility of the knockout mouse have now been supplanted by concerns related to scale such as cost, labor intensity, and timeliness. As the 21st century unfolds, more demands will be made on high-throughput platforms and screening processes, and mouse ES cells therefore will influence another level of biotechnology development.$ c% ?5 ^6 c4 k; [5 C' R2 M5 t
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Directed Differentiation for Specific Disease Applications# N% i! U/ R; Q# Q

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 .; K+ \( o" P4 @  U
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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 . 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 . Dopaminergic neurons produced from mouse ES cells share the electrophysiological and behavioral properties expected of neurons from the midbrain , and transplantation of ES cell–derived dopaminergic neurons has been shown to partially correct the phenotype of a murine model of Parkinson’s disease .

Techniques to induce efficient neuronal differentiation of mouse ES cells have recently been extended to derive dopaminergic neurons from primate ES cells . 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.1 H2 u! `$ N0 n: ]) Z( G" o
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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 . Although currently a relatively inefficient process, nuclear transfer using embryo-derived nuclei  and fetal and adult cell nuclei  has produced viable sheep, including the well-known "Dolly." SCNT has been used successfully in the genetic modification of cattle , mice , goats , and pigs , 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 non-rodent knockout animal, a sheep with deletion of the prion protein (PrP) gene .
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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  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.( d0 U. d4 C$ A# j5 ^0 J9 o

Furthermore, novel mouse ES cell lines have been created via nuclear transfer from adult mouse somatic cells from inbred, hybrid, and mutant strains . These ES cells can be induced to differentiate into numerous cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo.3 _5 k; B& [9 u) C
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Non-Mouse Stem Cells
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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  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 .7 X; N. n2 e2 t, d0 j

Just as studies of mouse teratocarcinoma-derived stem cells preceded the isolation and characterization of mouse 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)  prepared the way for Thomson and coworkers to isolate human ES cells from human blastocysts in 1998 . 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.
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FROM ONE TO MANY: THE TRAJECTORY OF MOUSE ES CELL LINES IN THE FIRST TWO DECADES7 B$ D* T& k1 h0 r5 a* [

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).- [% U, @) ?& l! U: `6 f! g

Table 2. Publications and disease insights provided by mouse ES cell lines% J2 M0 {4 j7 g' t1 h% s

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.% O9 o0 n4 f3 f1 e9 |1 S7 v' i7 n5 V

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.
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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 high-throughput 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.1 q# M, e2 B) i; |0 L! \2 G2 o

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 regulation 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.
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APPENDIX9 K1 R* v. o1 O5 d

Criteria for the Selection of Scientific Publications That Feature Mouse Embryonic Stem Cell Lines( N: v2 j+ h) z
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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.2 X& ?1 i+ G7 E- r
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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/.
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