Stem Cells, Vol. 19, No. 3, 193-204,
May 2001
© 2001 AlphaMed Press
Multilineage Differentiation from Human Embryonic Stem Cell Lines
Jon S. Odoricoa,
Dan S. Kaufmanb,
James A. Thomsonc,d
a Department of Surgery,
b Department of Medicine, and
c Department of Anatomy, University of Wisconsin School of Medicine, Madison, Wisconsin, USA;
d Wisconsin Regional Primate Research Center, Madison, Wisconsin, USA
Key Words. Embryonic stem cells • Transplantation • Human • Differentiation • Pluripotent
Jon S. Odorico, M.D., Assistant Professor of Surgery, Department of Surgery, University of Wisconsin Hospital, H4/756 Clinical Science Center, 600 Highland Ave., Madison, Wisconsin 53792, USA. Telephone: 608-265-6471; Fax: 608-262-5624; e-mail: jon{at}tx.surgery.wisc.edu
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ABSTRACT
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Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation. A wide variety of adult mammalian tissues harbors stem cells, yet "adult" stem cells may be capable of developing into only a limited number of cell types. In contrast, embryonic stem (ES) cells, derived from blastocyst-stage early mammalian embryos, have the ability to form any fully differentiated cell of the body. Human ES cells have a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferative potential, providing the possibility for unlimited expansion in culture. Furthermore, they can differentiate into derivatives of all three embryonic germ layers when transferred to an in vivo environment. Data are now emerging that demonstrate human ES cells can initiate lineage-specific differentiation programs of many tissue and cell types in vitro. Based on this property, it is likely that human ES cells will provide a useful differentiation culture system to study the mechanisms underlying many facets of human development. Because they have the dual ability to proliferate indefinitely and differentiate into multiple tissue types, human ES cells could potentially provide an unlimited supply of tissue for human transplantation. Though human ES cell-based transplantation therapy holds great promise to successfully treat a variety of diseases (e.g., Parkinson's disease, diabetes, and heart failure) many barriers remain in the way of successful clinical trials.
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INTRODUCTION
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Stem cells have the ability to choose between prolonged self-renewal and differentiation. This fate choice is highly regulated by intrinsic signals and the external microenvironment, the elements of which are being rapidly elucidated [1]. Stem cells can be identified in many adult mammalian tissues. In some tissues, such as epithelia, blood, and germline, stem cells contribute to replenishment of cells lost through normal cellular senescence or injury. Stem cells may also be present in other adult organs, such as the brain and pancreas, which normally undergo very limited cellular regeneration or turnover.
Although stem cells in adult tissues may have more "plasticity" than originally thought, they typically form only a limited number of cell types. Stem cells of the early mammalian embryo, in contrast, have the potential to form any cell type. In the unmanipulated blastocyst-stage embryo, stem cells of the inner cell mass (ICM) promptly differentiate to generate primitive ectoderm, which ultimately differentiates during gastrulation into the three embryonic germ (EG) layers. When removed from their normal embryonic environment and cultured under appropriate conditions, ICM cells give rise to cells that proliferate and replace themselves indefinitely. Yet, while in this undifferentiated state in culture, they maintain the developmental potential to form advanced derivatives of all three EG layers [2, 3]. ICM cells are the source cells from which pluripotent mouse, nonhuman primate, and human embryonic stem (ES) cells are generally derived, although there is evidence that mouse ES cells may be more closely related to primitive ectoderm [4-9].
The objective of this review is to describe the derivation and unique properties of human ES cells. Particular emphasis will be given to summarizing recent studies that focus on the potential of human ES cells for multilineage differentiation in vitro and in vivo. We will also outline key scientific questions that will need to be answered before the full therapeutic potential of human ES cells can be realized.
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DERIVATION OF HUMAN ES CELLS
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The origin of human ES cells from the pre-implantation embryo is the defining feature that distinguishes ES cell lines from other pluripotent human cell lines, namely human embryonal carcinoma (EC) cell and human EG cell lines [7, 8, 10, 11]. EC cell lines are pluripotent cell lines derived from the undifferentiated stem cell components of spontaneously arising germ cell tumors found occasionally in certain strains of mice and humans [12, 13]. Pluripotent EG cell lines have been derived from mouse and human primordial germ cells from the genital ridges of fetuses [11, 14]. Years before the isolation of human EG or ES cells, EC cell lines from both mouse and later human teratocarcinomas provided an important in vitro model of differentiation [15].
Human ES cells have been derived from the ICM of blastocyst-stage embryos in essentially the same manner as rhesus monkey ES cells [6, 7]. Cleavage-stage human embryos, produced by in vitro fertilization for clinical purposes, are donated by individuals after informed consent. After embryos are grown to the blastocyst stage, the ICM is isolated and plated onto mitotically inactivated murine embryonic fibroblast (MEF) feeder layers in tissue culture (Fig. 1
). The ICM cell outgrowths are propagated in the presence of serum, and colonies with the appropriate undifferentiated morphology are subsequently selected and expanded. After the initial derivation in serum, human ES cell lines can be maintained and propagated on feeder layers in medium containing serum alone or serum replacement medium and basic fibroblast growth factor (bFGF). Initially, human ES and EG cell lines were not clonally derived and so pluripotency could only be demonstrated for a population of cells. As such, the possibility existed that within a colony there were subpopulations of cells already committed to different lineages and no individual cell was capable of differentiating into derivatives of all three EG layers. Subsequently, clonally derived human ES cell lines, H9.1 and H9.2, were produced that retain all the properties of the parental ES cell line, including the ability to generate teratomas in vivo harboring derivatives of all three EG layers [16].
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Figure 1. Derivation of human ES cell lines. Human blastocysts were grown from cleavage-stage embryos produced by in vitro fertilization. ICM cells were separated from trophectoderm by immunosurgery, plated onto a fibroblast feeder substratum in medium containing fetal calf serum. Colonies were sequentially expanded and cloned.
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PROPERTIES OF HUMAN ES CELLS
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Human and nonhuman primate ES cells share a similar morphology that is distinct from human EG cells and mouse ES cells [4, 6, 7, 11]. Human ES cells form relatively flat, compact colonies that easily dissociate into single cells in trypsin or in Ca+2- and Mg+2-free medium, whereas human EG cells form tight, more spherical colonies that are refractory to standard dissociation methods, but which more closely resemble the morphology of mouse ES cell colonies. Moreover, human ES cells grow more slowly than mouse ES cells; the population-doubling time of mouse ES cells is ~12 hours, whereas the population-doubling time of human ES cells is about 36 hours [16].
Paralleling these differences in cellular morphology, human ES cells differ from their murine counterparts with regard to cell-surface antigen phenotype. Like undifferentiated primate ES cells and human EC cells, human ES cells express stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), high molecular weight glycoproteins TRA-1-60 and TRA-1-81, and alkaline phosphatase [7, 17]. Undifferentiated mouse ES cells do not express SSEA-3 or SSEA-4, but do express the lactoseries glycolipid SSEA-1, which is not expressed in human ES cells, rhesus ES cells, or human EC cells [7, 17]. The functional significance of these antigens is unknown.
Human ES cells also differ from mouse ES cells in their in vitro culture requirements for undifferentiated growth. Mouse ES cells require leukemia inhibitory factor (LIF) for undifferentiated proliferation. In contrast, LIF alone is not sufficient to prevent differentiation of human ES cells in vitro [7, 18]. Instead, continued undifferentiated propagation of human ES cells currently require feeder layers and either the presence of serum or, if cultured in serum-free medium, bFGF [7, 16]. Under conditions of low cell density, human ES cell lines are more difficult to propagate in serum, with a cloning efficiency of approximately 0.25% [16]. In contrast, culture in both serum replacement medium and supplemental bFGF significantly increases the cloning efficiency over culture in serum alone [16]. Fibroblast feeder layers are currently required to prevent differentiation of human ES cells. How undifferentiated proliferation can be sustained in the absence of feeder cells is an area of active investigation. The critical factors produced by fibroblast feeder layers, which prevent differentiation of human ES cells, are entirely unknown. Further work is clearly needed to clarify the mechanisms involved in sustaining human ES cell proliferation, including specific receptor-ligand interactions, downstream signaling events, and cellular target molecules. Ultimately, it would be essential to establish feeder-independent culture conditions, which permit large-scale propagation of human ES cells in culture.
Human ES cells have demonstrated remarkably stable karyotypes. Human ES cell lines demonstrate normal XX and XY karyotypes, similar to ES cell lines from other species, but distinct from human EC lines derived from teratocarcinomas [7]. This characteristic makes human ES cells more relevant as a model for the study of developmental biology mechanisms and for derivation of differentiated cells for transplantation therapy.
Human ES cells express high levels of telomerase. The expression of telomerase, a ribonucleoprotein that adds telomere repeats to chromosome ends, thereby maintaining their length, is highly correlated with immortality in human cell lines [19]. Most diploid somatic cells do not express high levels of telomerase and enter replicative senescence after a finite proliferative life span in tissue culture, usually after 50-80 population doublings. Unique among normal somatic cells, some populations of adult stem cells (i.e., hematopoietic stem cells) in vivo also constituitively express telomerase [20, 21]; however, telomerase activity is not sustained when cells are placed in culture. In contrast, cells of the early embryo have high telomerase activity levels [22, 23]. Likewise, human ES cell lines exhibit high telomerase activity levels even after more than 300 population doublings and passage for more than 1 year in culture [7, 16]. In summary, properties of cells of the early embryo, such as normal karyotype and high telomerase activity, are sustained for an extended period of time by human ES cell lines in culture. This unique property among human cell lines has important implications as a tool to study cellular senescence and mechanisms of stem cell renewal.
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MULTILINEAGE DIFFERENTIATION IN VITRO
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When removed from feeder layers and transferred to suspension culture, ES cells begin to differentiate into multicellular aggregates of differentiated and undifferentiated cells, termed embryoid bodies (EBs), which resemble early post-implantation embryos. Human EBs frequently progress through a series of stages beginning as simple, morula-like EBs eventually forming cavitated and cystic EBs between 7 and 14 days of post-differentiation development (Figs. 2A, B) [24]. As for mouse and nonhuman primate ES cells, differentiation in vitro is consistently disorganized and frequently variable from one EB to another within the same culture. A more comprehensive understanding of the morphology of human EBs and the relationships among different cell types comprising these complex embryo-like structures may yield important new information on early inductive events in human development.
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Figure 2. In vitro differentiation of human ES cells under a variety of conditions. In suspension culture, human ES cells differentiate to EBs or multicellular aggregates resembling early embryos. A) A single H9 EB in suspension culture for 8 days demonstrating that complex, cystic EBs can be formed by this time (phase contrast, 100x); B) a single complex H1 EB in suspension culture for 14 days; most human EBs, regardless of the cell line, display the formation of extraembryonic tissue structures. Insert shows probable blood islands (hematoxylin and eosin staining, B, 100x; insert 300x). Following 8-14 days of suspension culture, H9 EBs transferred to gelatinized tissue culture plastic for further differentiation grow into confluent cell sheets containing a variety of differentiated cell types including C) neural cells (phase contrast, 40x), and D) pigmented and non-pigmented epithelial cells (phase contrast, 100x). After initial differentiation on S17 bone marrow stromal cells, and subsequent replating in methylcellulose-based media with hematopoietic growth factors, H1 cells can differentiate to E) BFU-E (darkfield phase contrast, 50x), F) colony-forming unit-granulocyte, macrophage (CFU-GM) (darkfield phase contrast, 100x), and CFU-M (not shown) colonies.
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Human ES cells, like nonhuman primate ES cells, are able to differentiate into trophoblast in culture. Nonhuman primate ES cell lines spontaneously differentiate in vitro into extraembryonic endoderm lineages, including yolk sac, and into trophoblast, as evidenced by 
-fetoprotein and chorionic gonadotropin (CG) mRNA synthesis, and bioactive CG production [6]. Similarly, human EBs synthesize -fetoprotein transcripts and secrete -fetoprotein and hCG into the culture medium [7]. Human ES cells, therefore, represent a useful model in which to study human placental development and function.
Mouse ES cell lines are able to differentiate in vitro into a variety of embryonic and adult cell types from all three EG layers. These include cardiomyocytes, hematopoietic progenitors, yolk sac, skeletal myocytes, smooth muscle cells, adipocytes, chondrocytes, endothelial cells, melanocytes, neurons, glia, pancreatic islet cells, and primitive endoderm [25-39]. From these experiments it is clear that ES cells induced to differentiate in culture follow many of the critical developmental stages found in the normal embryo, and are ultimately able to generate post-mitotic terminally differentiated cell types depending on the particular growth factor conditions.
As a result of their ability to differentiate into many different cell types, ES cells have been recognized as a valuable model system for studying the mechanisms underlying lineage specification during the early stages of mammalian development [25, 40, 41]. For example, by comparing downstream gene expression profiles between null mutant and wild-type ES cells, one can dissect the complex network of transcription factor genes regulating tissue-specific gene expression [42]. Also, in vitro culture provides a unique setting enabling control of the extrinsic cytokine or growth factor environment to study how these factors influence cellular differentiation [28, 31, 43]. Furthermore, in vitro differentiation of ES cells transduced with gene trap vectors can be used to discover novel developmentally regulated genes that are important in tissue-specific differentiation programs [44-47]. Thus, developmental pathways of cell lineages, which can be derived from ES cells, can be studied using this in vitro model system.
Recent studies demonstrate that human ES cells differentiating in culture are able to activate the expression of genes restricted to each of the three EG layers [18, 24, 43]. Human EBs derived from the human ES cell line, H9, transcribed genes for -fetoprotein, neurofilament 68kDa subunit, 
-globin, and -cardiac actin marking primitive endoderm, neuroectoderm, and mesoderm derivatives [24]. Differentiating cells acquired morphologies characteristic of neurons and cardiomyocytes [18, 24]. We have also performed human ES cell in vitro differentiation experiments. We observed that during subsequent development of plated EBs, cultures showed a variety of different morphologies, including rhythmically contracting cardiomyocytes, pigmented and non-pigmented epithelial cells, and neural cells displaying an exuberant outgrowth of axons and dendrites (Figs. 2C
, D). Regions within the differentiated cultures also contained cells having a mesenchymal morphology.
Schuldiner et al. supplemented these data and showed that both undifferentiated human ES cells and differentiated EBs expressed receptors for a number of different soluble growth factors with established effects on developmental pathways in vivo [43]. Addition of each of these growth factors individually to the culture medium altered the expression profile of an array of tissue-restricted genes [43]. However, none of these growth factors directed differentiation exclusively to one particular cell type [43]. A better understanding of the epigenetic events regulating cell lineage commitment and differentiation should permit the focused use of soluble growth factors to achieve lineage-restricted differentiation of human ES cells.
In other studies using human ES cells, we have used a co-culture method to promote hematopoietic differentiation. If human ES cells are allowed to differentiate on MEFs, hematopoietic colony-forming cells are not seen. In striking contrast, when the H1, H9, or clonal H1.1 human ES cell lines are co-cultured with irradiated mouse bone marrow stromal cells (S17 cells) in medium containing fetal bovine serum but no other exogenously added growth factors, they differentiate into a variety of cell types. The cell types derived under these conditions include cystic structures that resemble yolk sac, and cobblestone-type cells that are typical for hematopoietic precursors. CD34+ cells can be identified by flow cytometry, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis shows induction of hematopoietic transcription factors. When human ES cells differentiated on S17 cells are harvested and placed in a methylcellulose colony-forming assay, a variety of hematopoietic colonies can be identified. These include colonies of erythroid, macrophage, granulocyte, and megakaryocyte cells (Figs. 2E, F). These data suggest that S17 bone marrow stromal cells either promote or support hematopoietic cell differentiation [48].
Endoderm lineages, such as pancreas and liver, are morphologically less distinct and more difficult to discern in ES cell cultures than blood or cardiac cells. This is one of the factors that has inhibited their detection among ES cell progeny. Differentiated ES cell progeny are able to express some endoderm and pancreas-restricted genes. Rhesus and mouse ES cells are capable of activating pancreatic and endoderm genes both in vitro and in vivo [38, 49]. Preliminary human ES cell gene expression studies show that differentiated derivatives of human ES cells can be induced to express endoderm genes, including hepatocyte nuclear factor 3 beta, and pancreatic islet genes including insulin, somatostatin, and glucagon (JSO, unpublished observations). Likewise, Schuldiner et al. recently presented RT-PCR gene expression data showing that differentiated progeny of human ES cells activate transcription of a variety of endoderm, liver, and pancreas-restricted genes [43]. However, it remains to be determined whether the initiation of pancreatic islet differentiation, as evidenced by gene transcription, will progress through a full differentiation program and lead to phenotypically adult islet cell populations with physiologic insulin secretion, as has been derived from mouse ES cells. Collectively, these data suggest that human ES cells can activate embryonic gene expression programs in culture and begin to differentiate into derivatives of all three EG layers.
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MULTILINEAGE DIFFERENTIATION IN VIVO
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Although ES cells can differentiate to multiple embryonic and adult cell types in vitro, pattern formation or organogenesis does not occur to a significant degree. Differentiation in the context of an in vivo environment, such as following injection into a host blastocyst or implantation into mice, unveils the full developmental potential of undifferentiated ES cell lines [3]. In this context, many of the normal features of tissue architecture are reproduced. For example, epithelia exhibit polarity, are enveloped by a basement membrane, and are surrounded by mesenchyme; complex tissue structures such as hair follicles, teeth, and gut are also formed. Human ES cells injected into severe combined immunodeficient mice form benign teratomas, with advanced differentiated tissue types representing all three EG layers (Fig. 3) [7]. Easily identifiable differentiated cells in human ES cell teratomas include smooth muscle, striated muscle, bone, cartilage, fetal glomeruli, gut, respiratory epithelium, keratinizing squamous epithelium, hair, neural epithelium, and ganglia (Fig. 3). Compared with human EC cell lines, human ES cell lines exhibit both more advanced and more consistent developmental potential. For example, the human EC cell line NTERA2 c1.D1 injected into immunocompromised mice forms teratocarcinomas containing simple tubular structures resembling primitive gut, neural rosettes, and tissue resembling neuropile [10].
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Figure 3. Tissue derivatives of all three EG layers differentiated from human ES cells in vivo. Human ES cells injected into immunocompromised mice form benign teratomas. Present within these teratomas are advanced derivatives of ectoderm, such as A) neural epithelium (100x), of mesoderm, such as B) bone (100x), C) cartilage (40x), D) striated muscle (200x), and E) fetal glomeruli and renal tubules (100x; insert, 200x), and of endoderm, such as F) gut (40x). To some degree micro-architectural tissue relationships of complex organs can be reproduced in human ES cell teratomas. H1, H7C, H9, H13, and H14 cell lines, which produced the above teratomas, exhibit a similar range of differentiation. All photomicrographs are of hematoxylin- and eosin-stained sections.
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Embryonic inductive events and complex epithelial-mesenchymal interactions control the formation of organized tissue structures during normal embryogenesis. These events and interactions begin to occur in teratomas but are less pronounced during in vitro differentiation. Unfortunately, the precise inductive events regulating embryonic pattern formation are still being elucidated and cannot yet be reliably reproduced in vitro. Because in vivo differentiation of human ES cells is more complete than in vitro differentiation, it would be useful to explore means to extract the cells or tissue of interest from the heterogeneous mix of tissues comprising teratomas or to direct differentiation in vivo to a particular lineage. Possible methods of achieving this include: A) adding specific combinations of chemical morphogens or growth factors [43, 50]; B) co-culturing or co-transplanting ES cells with inducer tissues or cells; C) implanting ES cells into specific organs or regions of animals; D) overexpressing tissue-specific homeobox transcription factor genes [51, 52]; E) selecting cells that activate a particular lineage-specific gene expression program [38, 53, 54], and/or F) isolating cells of interest based on fluorescence-activated cell sorting [55, 56]. Some of these methods have been explored to enrich in vitro ES cell cultures for a cell type of interest, but the application of these methods in combination with in vivo differentiation awaits future studies.
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DEVELOPING TRANSPLANTATION THERAPIES
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Diseases that result from the destruction and/or dysfunction of a limited number of cell types, such as diabetes mellitus, in which pancreatic islet cells have been selectively destroyed, or Parkinson's disease, which results from the destruction of dopaminergic neurons within a particular region of the brain, could be treated by the transplantation of differentiated derivatives of ES cells. Studies in animal models show that transplantation of either pluripotent stem cell derivatives, or fetal cells, can successfully treat a variety of chronic diseases, such as, diabetes, Parkinson's disease, traumatic spinal cord injury, Purkinje cell degeneration, liver failure, heart failure, Duchenne's muscular dystrophy, and osteogenesis imperfecta [38, 57-64].
Although considerable progress in human transplantation medicine has been achieved in recent years, several major obstacles still restrict more widespread application of cellular transplantation in the routine treatment of these conditions. The chief obstacles that face this field are the need for massive doses of immunosuppressive drugs to prevent rejection of the transplanted tissue and the scarcity of organs from human cadaver donors. In light of these obstacles, a human ES cell-based strategy could permit the generation of an unlimited supply of cells or tissue from an abundant, renewable, and readily accessible source. Moreover, by virtue of their permissiveness for stable genetic modification, ES cells could be engineered to escape or inhibit host immune responses.
The first step toward successful development of a stem cell-based therapy for human diseases is to establish that human ES cells are capable of differentiating to a particular cell type of interest and to purify this lineage from the mixed population (Fig. 4). Unfortunately, the heterogeneous nature of development in culture has hampered the use of ES cell derivatives in transplantation studies. Rarely have specific growth factors or culture conditions led to establishment of cultures containing a single cell type [43, 65]. In fact, human pluripotent cell lines retain a broad pattern of multilineage gene expression despite the addition of specific growth factors [43, 65]. Furthermore, there is significant culture-to-culture variability in the developments of a particular phenotype under identical growth factor conditions. Given the broad range of lineages to which ES cells commit, derivation of a relatively homogeneous cell population will ultimately depend on selection from a mixed population of cells. One approach might involve using a tissue-specific promoter to drive a selectable marker such as an antibiotic resistance gene [38, 53, 54]. An alternative approach could involve transduction of a gene construct containing a tissue-specific promoter/enhancer controlling expression of a green fluorescence protein gene [66]. In this way, cells activating a lineage differentiation program of interest could be selected by fluorescence-activated cell sorting in much the same way that CD34+ hematopoietic stem cells are selected and sorted for stem cell transplantation. Both approaches would rely on the development of efficient gene transfer methodologies for human ES cells. A potential pitfall of these approaches is the concern for rejection of the transplanted cells which now express a foreign protein and the potential malignant transformation of genetically manipulated cells [67].
Second, it will be critical to test and demonstrate that the differentiated cellular derivatives function in a normal physiologic way; i.e., that pancreatic islet cells exhibit normal glucose-responsive insulin secretion. Prior studies have demonstrated that many differentiated cell types derived from murine ES cells in vitro (e.g., cardiomyocytes and dopaminergic neurons) display a terminally differentiated, physiologically mature phenotype and do exhibit normal physiologic functioning in vitro and in vivo [36, 38, 68]. However, differentiated ES cell cultures can contain multipotent progenitors as well as terminally differentiated cells [69]. Because many fetal or embryonic tissues and multipotent progenitor cells are functionally immature, one cannot assume that all ES cell progeny will subserve normal cellular physiologic functions.
A third major milestone on the road to clinical trials will be to demonstrate efficacy in rodent and large animal models of disease. Rhesus ES cells and the rhesus monkey provide an excellent preclinical model for developing ES cell-based transplantation therapies and for testing strategies to prevent immune rejection. Indeed, for Parkinson's disease and diabetes mellitus, good models are already available in the rhesus monkey [70, 71]. Achieving a therapeutic result will mandate integration of the transplanted cells into the host tissue in a functionally useful form. For example, replacing infarcted heart muscle or scar tissue with ES cell-derived cardiomyocytes will require that new muscle cells integrate with the existing muscle, contract in a coordinated and mechanically useful manner, and develop a new blood supply. Although complex structural integration would be essential for some cell transplants (e.g., neurons and cardiomyocytes), normal functioning of other ES cell-derived transplants will be more independent of such complex tissue interactions (e.g., islet cells and hematopoietic cells).
Fourth, the possibility arises that transplantation of differentiated human ES cell derivatives into human recipients may result in the formation of ES cell-derived tumors. From in vivo differentiation studies, it is clear that if undifferentiated rhesus or human ES cells are not rejected after implantation into host recipient animals, then a benign teratoma can result [6, 7]. These tumors are not metastatic, and do not rapidly kill the host animals. Tumor growth in immunodeficient animals appears to be dependent on the presence of a stem cell population in undifferentiated cultures. Thus, as ES cells are allowed to fully differentiate into post-mitotic, terminally differentiated derivatives, they should deplete the undifferentiated stem cell pools, thereby reducing the probability of uncontrolled tumor growth. In fact, from a limited number of short-term studies, it appears that transplanting differentiated progeny derived from murine ES cells into adult rodents does not result in significant tumor formation [38, 53, 57, 72]. However, these studies were not specifically designed to address this question, and as such, they lack sufficient animal numbers and long-term surveillance to allow firm conclusions. If tumor formation does depend on persistence of a stem cell population, then one could design a transgenic methodology to eliminate residual minority stem cells from differentiated ES cell cultures, possibly based on negative selection of Oct 4-expressing cells. Use of a positive selection transgene to achieve lineage-directed differentiation would also reduce the risk of tumor formation by selecting against the remaining undifferentiated, proliferating stem cell population. Irrespective of the persistence of stem cells, the possibility for malignant transformation of the derivatives will also need to be addressed. Ultimately, as the potential for tumor growth is a major safety consideration, a fail-safe method to prevent tumor growth may need to be developed. Once devised, strategies could be tested in a preclinical rhesus monkey model, using rhesus ES cell-derived cells, prior to embarking on human clinical trials.
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PREVENTING IMMUNOLOGIC REJECTION OF TRANSPLANTED CELLS
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A fifth consideration is the prevention of immune-mediated rejection of the human ES cell-derived cellular graft (Figs. 4 and 5) [73]. Currently available multidrug immunosuppressive regimens are effective in preventing rejection in most recipients of solid organ transplants. Even with a modest level of efficacy, a therapy brought to clinical trials for a disease with no other available treatment might still be considered a viable option, particularly if there were few repercussions of a failed transplant for the patient other than being returned to their baseline disease state. Unfortunately, however, currently used immunosuppressive drugs are far from ideal and are associated with numerous complications including wound healing, opportunistic infections, drug-related toxicities, skin malignancies, and low-grade lymphomas called post-transplant lymphoproliferative disorders. Instead, human ES cells could be genetically manipulated to reduce or eliminate immune-mediated rejection so that lifelong pharmacologic immunosuppression would not be required.
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Figure 5. Possible ways to circumvent immune-mediated rejection of tissue transplants derived from human ES cells. A) ES cells can be genetically altered to either eliminate foreign MHC genes or replace foreign MHC genes with ones specifically matched to a particular transplant recipient. B) In nuclear reprogramming, a nucleus from a somatic cell of an individual can be reprogrammed by transfer to an enucleated oocyte. An embryo established by nuclear transfer could be used to derive an ES cell line that expresses all histocompatibility antigens and other nuclear genes identical to those of the person from whom the somatic cell was obtained. C) Hematopoietic stem cells and a second tissue generated from the same parental ES cell line could be transplanted simultaneously or successively to the same recipient after administration of a nonmyeloablative conditioning drug regimen. This would create a hematopoietic chimera, thereby establishing immunologic tolerance to the second tissue.
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One potential method for limiting the immune response is to decrease the immunogenicity of transplanted cells. Homologous recombination has been used to "knock-out" major histocompatibility complex (MHC) class I and class II molecules in mouse ES cells [74]. However, MHC class I- and class II-deficient skin grafts are still rejected, possibly on the basis of indirect allo-recognition-mediated rejection and/or natural killer cell-mediated destruction [74]. Thus, in addition to deleting foreign MHC genes, it might be necessary to "knock-in" the desired MHC genes, so that ES cell-derivative transplants are seen as "self" by the prospective recipient [75, 76]. Alternatively, genes for immunosuppressive molecules such as Fas-ligand could be inserted into ES cells, or important immune-stimulating proteins, such as B7 antigens or CD40-ligand, could be deleted from ES cells [77, 78]. Irrespective of the method used, the ability to stably integrate genetic modifications into ES cells provides an advantage over using adult somatic cells, which are less reliably genetically altered.
Precisely how immunogenic is a cellular or tissue transplant from human ES cells? This may be a relevant question to ask as scientists attempt to engineer ES cells to reduce their immunogenicity. The immunogenicity of tissues is generally correlated with the MHC antigen expression profile and the relative abundance of antigen-presenting cells (e.g., dendritic cells) within the tissue. The MHC expression profile of human ES cell derivatives will depend on the degree of differentiation and/or the specific cell type derived. For example, adult somatic cells normally only express MHC class I antigens, whereas B cells, macrophages, and dendritic cells typically express both class I and class II antigens. Furthermore, whereas most adult organs and tissues harbor immunostimulatory dendritic cells and vascular endothelial cells, these normal tissue components would be expected to be absent from ES cell-derived cellular or tissue transplants. The specific removal of antigen-presenting cells from solid organ transplants generally increases graft survival [79, 80]. Consequently, some ES cell-derived tissues may be rather inert immunologically, while others, like hematopoietic stem cells, may be as immunogenic as normal adult tissues. Therefore, human ES cell-derived transplants may in some cases provide an inherent immunologic advantage compared to human cadaver tissue transplants.
Nuclear transfer technology may provide a more precise means to prevent rejection of transplanted cells (Fig. 5). This technique would lead to ES cell-derived cells that are an exact genetic match to the recipient. In this way, there should be minimal host immune response since all nuclear genes, including major and minor histocompatibility loci, would be seen as "self." Here, a nucleus would be extracted from a normal somatic cell of a patient, say from a skin biopsy, and then injected into an enucleated oocyte. Oocyte cytoplasm has the ability to reprogram differentiated nuclei, and as such, would reestablish an embryonic gene expression program in the chromatin of the somatic cell nucleus. A blastocyst developing from this oocyte would be a source for the derivation of a new ES cell line, which would be genetically matched for each nuclear gene of the patient. In this setting, the potential immune-mediated destruction of the graft would be limited to minor antigen differences derived from mitochondrial genes or to autoimmune processes, such as diabetes. Extending nuclear transfer technology to achieve the fusion of an entire cell with an enucleated oocyte might eliminate some of the remaining genetic differences that would exist between ES cell derivatives of nuclear transferred embryos and the prospective patient. In fact, in cows and mice, investigators have successfully combined nuclear transfer technology and ES cell derivation to establish transgenic ES cell lines from reprogrammed somatic cell nuclei [81]. Clearly though, the generation of human embryos by nuclear reprogramming to create novel human ES cell lines would be exceptionally controversial. Furthermore, the poor availability of human oocytes, the low efficiency of the nuclear transfer procedure, and the long population-doubling time of human ES cells make it difficult to envision this becoming a routine clinical procedure even if ethical considerations were not a significant point of contention. By studying how oocyte cytoplasm mediates nuclear reprogramming in these animal models, it might be likely that nuclear reprogramming could be achieved by other methods, thereby obviating the need for human oocytes.
Establishing hematopoietic chimerism is another potential means of preventing rejection of transplanted cells (Fig. 5). There are now many patients who have undergone bone marrow transplantation and subsequently received a solid organ transplant (typically a kidney) from the same donor as the bone marrow [82, 83]. In these circumstances, no immunosuppression is required for the solid organ transplant because the recipient's lymphocytes are from the same donor and have rendered the recipient immunologically tolerant. However, success of a bone marrow or hematopoietic stem cell transplant has generally required highly toxic immunosuppression. More recently, strategies have been designed which are significantly less myelotoxic while still permitting engraftment of donor hematopoietic stem cells [84]. This relatively mild treatment can permit long-term engraftment and could potentially allow successful solid organ transplantation in humans without prolonged immunosuppressive therapy. By using the same ES cell lines to derive both hematopoietic stem cells and other lineages, it may be possible to initially achieve hematopoietic chimerism followed by engraftment of a second cell type. Based on the principles described above, a second lineage should not be rejected as it would be regarded as "self" by the chimeric patient's bone marrow and immune system, which were derived, in part, from the same ES cell line. No long-term treatment with potentially toxic drugs would then be required. Ultimately, the rhesus monkey model will be essential for testing these and other strategies to prevent immune rejection.
Human ES cells fulfill all of the criteria of pluripotent ES cells. They are capable of indefinite self-renewal and multilineage differentiation, both in vivo and in vitro. This dual property provides the rationale for developing human ES cells as a basis for therapeutic tissue replacement. Not only do transplantation therapies based on ES cell-derived tissues offer the potential of overcoming limited tissue supplies, but they also present the exciting possibility of being able to perform tissue transplants with minimal or no immunosuppression.
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ACKNOWLEDGMENTS
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The authors wish to sincerely thank A. Schmick and J. Adsit for their editorial assistance, D. Hullett for critical review of the manuscript, and L. Sager for manuscript preparation.
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References
|
|---|
-
Watt FM, Hogan BL. Out of Eden: Stem cells and their niches. Science 2000;287:1427-1430.
-
Nagy A, Gocza E, Diaz EA et al. Embryonic stem cells alone are able to support fetal development in the mouse. Development 1990;10:815-821.
-
Bradley A, Evans M, Kaufman MH et al. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 1984;309:255-256.
-
Evans M, Kaufman M. Establishment in culture of pluripotent cells from mouse embryos. Nature 1981;292:154-156.
-
Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 1981;78:7634-7638.
-
Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995;92:7844-7848.
-
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-1147.
-
Bongso A, Fong CY, Ng SC et al. Isolation and culture of inner cell mass cells from human blastocysts. Hum Reprod 1994;9:2110-2117.
-
Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci USA 1997;94:5709-5712.
-
Andrews PW, Damjanov I, Simon D et al. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro. Lab Invest 1984;50:147-162.
-
Shamblott MJ, Axelman J, Wang S et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci USA 1998;95:13726-13731.
-
Andrews PW, Bronson DL, Benham F et al. A comparative study of eight cell lines derived from human testicular teratocarcinoma. Int J Cancer 1980;26:269-280.
-
Mintz B, Illmensee K. Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc Natl Acad Sci USA 1975;72:3585-3589.
-
Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992;70:841-847.
-
Andrews PW, Oosterhuis J, Damjanov I. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: IRL Press, 1987.
-
Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271-278.
-
Thomson JA, Marshall VS. Primate embryonic stem cells. Curr Top Dev Biol 1998;38:133-165.
-
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro [published erratum appears in Nat Biotechnol 2000;18:559]. Nat Biotechnol 2000;18:399-404.
-
Kim NW, Piatyszek MA, Prowse KR et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011-2015.
-
Yui J, Chiu CP, Lansdorp PM. Telomerase activity in candidate stem cells from fetal liver and adult bone marrow. Blood 1998;91:3255-3262.
-
Chiu CP, Dragowska W, Kim NW et al. Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow. STEM CELLS 1996;14:239-248.
-
Betts DH, King WA. Telomerase activity and telomere detection during early bovine development. Dev Genet 1999;25:397-403.
-
Brenner CA, Wolny YM, Adler RR et al. Alternative splicing of the telomerase catalytic subunit in human oocytes and embryos. Mol Hum Reprod 1999;5:845-850.
-
Itskovitz-Eldor J, Schulding M, Karsenti D et al. Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol Med 2000;6:88-95.
-
Rathjen PD, Lake J, Whyatt LM et al. Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy. Reprod Fertil Dev 1998;10:31-47.
-
Doetschman TC, Eistetter H, Katz M et al. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985;87:27-45.
-
Baker RK, Lyons GE. Embryonic stem cells and in vitro muscle development. Curr Top Dev Biol 1996;33:263-279.
-
Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 1995;7:862-869.
-
Rohwedel J, Maltsev V, Bober E et al. Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev Biol 1994;164:87-101.
-
Drab M, Haller H, Bychkov R et al. From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model. FASEB J 1997;11:905-915.
-
Dani C. Embryonic stem cell-derived adipogenesis. [Review] [34 refs]. Cells Tissues Organs 1999;165:173-180.
-
Poliard A, Nifuji A, Lamblin D et al. Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways. J Cell Biol 1995;130:1461-1472.
-
Risau W, Sariola H, Zerwes HG et al. Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development 1988;102:471-478.
-
Yamane T, Hayashi S, Mizoguchi M et al. Derivation of melanocytes from embryonic stem cells in culture. Dev Dyn 1999;216:450-458.
-
Brustle O, Jones KN, Learish RD et al. Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science 1999;285:754-756.
-
Bain G, Kitchens D, Yao M et al. Embryonic stem cells express neuronal properties in vitro. Dev Biol 1995;168:342-357.
-
Brustle O, McKay RD. Neuronal progenitors as tools for cell replacement in the nervous system. Curr Opin Neurobiol 1996;6:688-695.
-
Soria B, Roche E, Berna G et al. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000;49:157-162.
-
Abe K, Niwa H, Iwase K et al. Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp Cell Res 1996;229:27-34.
-
Doetschman T, Shull M, Kier A et al. Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders. Hypertension 1993;22:618-629.
-
Dinsmore J, Ratliff J, Jacoby D et al. Embryonic stem cells as a model for studying regulation of cellular differentiation. Theriogenology 1998;49:145-151.
-
Duncan SA, Navas MA, Dufort D et al. Regulation of a transcription factor network required for differentiation and metabolism. Science 1998;281:692-695.
-
Schuldiner M, Yanuka O, Itskovitz-Eldor J et al. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 2000;97:11307-11312.
-
Thorey IS, Muth K, Russ AP et al. Selective disruption of genes transiently induced in differentiating mouse embryonic stem cells by using gene trap mutagenesis and site-specific recombination [published erratum appears in Mol Cell Biol 1998;18:6164]. Mol Cell Biol 1998;18:3081-3088.
-
Salminen M, Meyer BI, Gruss P. Efficient poly A trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos. Dev Dyn 1998;212:326-333.
-
Gossler A, Joyner AL, Rossant J et al. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 1989;244:463-466.
-
Baker RK, Haendel MA, Swanson BJ et al. In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse. Dev Biol (Orlando) 1997;185:201-214.
-
Kaufman DS, Lewis RL, Auerbach R et al. Directed differentiation of human embryonic stem cells into hematopoietic colony forming cells. Blood 1999;94(suppl 1, part 1 of 2):34a.
-
Jacobson L, Kahan B, Djamali J et al. Differentiation of endoderm derivatives, pancreas and intestine, from rhesus embryonic stem cells. Transplant Proc 2001;33:674
-
Bonner-Weir S, Taneja M, Weir GC et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA 2000;97:7999-8004.
-
Levinson-Dushnik M, Benvenisty N. Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells. Mol Cell Biol 1997;17:3817-3822.
-
Ferber S, Halkin A, Cohen H et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptzotocin-induced hyperglycemia. Nat Med 2000;6:568-572.
-
Klug MG, Soonpaa MH, Koh GY et al. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 1996;98:216-224.
-
Li M, Pevny L, Lovell-Badge R et al. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr Biol 1998;8:971-974.
-
Li A, Simmons PJ, Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 1998;95:3902-3907.
-
Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100:157-168.
-
McDonald JW, Liu XZ, Qu Y et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med 1999;5:1410-1412.
-
Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309-313.
-
Li RK, Weisel RD, Mickle DA et al. Autologous porcine heart cell transplantation improved heart function after a myocardial infarction. J Thorac Cardiovasc Surg 2000;119:62-68.
-
Studer L, Tabar V, Mckay RDG. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1998;1:290-295.
-
Brustle O, Choudhary K, Karram K et al. Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat Biotechnol 1998;16:1040-1044.
-
Zhang W, Lee WH, Triarhou LC. Grafted cerebellar cells in a mouse model of hereditary ataxia express IGF-I system genes and partially restore behavioral function. Nat Med 1996;2:65-71.
-
Lilja H, Arkadopoulos N, Blanc P et al. Fetal rat hepatocytes: isolation, characterization, and transplantation in the Nagase analbuminemic rats. Transplantation 1997;64:1240-1248.
-
Kobayashi N, Miyazaki M, Fukaya K et al. Transplantation of highly differentiated immortalized human hepatocytes to treat acute liver failure. Transplantation 2000;69:202-207.
-
Shamblott MJ, Axelman J, Littlefield JW et al. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci USA 2001;98:113-118.
-
Hadjantonakis AK, Nagy A. FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis 2000;27:95-98.
-
Heim DA, Hanazono Y, Giri N et al. Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model. Mol Ther 2000;1:533-544.
-
Wobus AM, Wallukat G, Hescheler J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomycocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 1991;48:173-182.
-
Okabe S, Forsberg-Nilsson K, Spiro AC et al. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech Dev 1996;59:89-102.
-
Burns RS, Chiueh CC, Markey SP et al. A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Natl Acad Sci USA 1983;80:4546-4550.
-
Jones CW, Reynolds WA, Hoganson GE. Streptozotocin diabetes in the monkey: plasma levels of glucose, insulin, glucagon, and somatostatin, with corresponding morphometric analysis of islet endocrine cells. Diabetes 1980;29:536-546.
-
Brustle O, Spiro AC, Karram K et al. In vitro-generated neural precursors participate in mammalian brain development. Proc Natl Acad Sci USA 1997;94:14809-14814.
-
Kaufman DS, Odorico JS, Thomson JC. Transplantation therapies from human embryonic stem cells—circumventing immune rejection. e-biomed 2000;1:11-15: available at http://www.liber/pub.com/EBI/defaultstatic.asp.
-
Grusby MJ, Auchincloss H Jr, Lee R et al. Mice lacking major histocompatibility complex class I and class II molecules. Proc Natl Acad Sci USA 1993;90:3913-3917.
-
Westphal CH, Leder P. Transposon-generated ‘knock-out’ and ‘knock-in’ gene-targeting constructs for use in mice. Curr Biol 1997;7:530-533.
-
Hardy RR, Malissen B. Lymphocyte development. The (knock) ins and outs of lymphoid development [Editorial]. Curr Opin Immunol 1998;10:155-157.
-
Harlan DM, Kirk AD. The future of organ and tissue transplantation: can T-cell costimulatory pathway modifiers revolutionize the prevention of graft rejection? [Review]. JAMA 1999;282:1076-1082.
-
Walker PR, Saas P, Dietrich PY. Role of Fas ligand (CD95L) in immune escape: the tumor cell strikes back. [Review]. J Immunol 1997;158:4521-4524.
-
Silvers WK, Bartlett ST, Chen HD et al. Major histocompatibility complex restriction and transplantation immunity. A possible solution to the allograft problem. Transplantation 1984;37:28-32.
-
Briscoe DM, Dharnidharka VR, Isaacs C et al. The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID mouse model of skin rejection. Transplantation 1999;67:1590-1599.
-
First NL, Thomson J. From cows stem therapies? [News; Comment]. Nat Biotechnol 1998;16:620-621.
-
Spitzer TR, Delmonico F, Tolkoff-Rubin N et al. Combined histocompatibility leukocyte antigen-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease: the induction of allograft tolerance through mixed lymphohematopoietic chimerism. Transplantation 1999;68:480-484.
-
Butcher JA, Hariharan S, Adams MB et al. Renal transplantation for end-stage renal disease following bone marrow transplantation: a report of six cases, with and without immunosuppression. Clin Transplant 1999;13:330-335.
-
Sykes M, Szot GL, Swenson KA et al. Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning. Nat Med 1997;3:783-787.
Received February 6, 2001;
accepted for publication March 14, 2001.
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Abstract
Introduction
