Pharmacological potential of embryonic stem cells

Share Embed

Descrição do Produto

Pharmacological Research 47 (2003) 269–278

Pharmacological potential of embryonic stem cells Thorsten Gorba a,1 , Timothy E. Allsopp b,∗ a

Institute for Stem Cell Research, Kings Buildings, University of Edinburgh, Edinburgh EH9 3JQ, UK b Stem Cell Sciences Ltd., Kings Buildings, University of Edinburgh, Edinburgh EH9 3JQ, UK Accepted 14 January 2003

Abstract Established embryonic stem (ES) cell lines have been at the forefront of approaches to understand gene function during embryogenesis and in adult vertebrate organisms, principally due to exploitation of two essential attributes; their pluripotency or ability to contribute to all three germinal layers and germ line in mice and their ease of genetic modification. Endeavours to routinely establish ES cells from species other than mice have met with limited success, although with rapid progress being made in our understanding of their basic cell biology, the regular derivation of lines from pre-implantation embryos will become easier for many species including humans. With a recent growing awareness of how these cells can be made to grow in an unlimited, but regulated manner plus how their fate can be directed or manipulated into diverse, mature phenotypes in culture, it has become clear that the biological resource offers additional attractive features applicable for future biomedical research and therapy. Advanced mouse ES-based technologies are being used in the industry for pharmaceutical discovery and development, while it is also anticipated that human ES cell reagents will revolutionise aspects of regenerative medicine. This review will summarise the advantages, potential and great hope for ES cell based systems in these contexts. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Embryonic stem cells; Pluripotency; Pharmacology; Cell therapy; Differentiation

1. Introduction It is more than 20 years since the first embryonic stem (ES) cell lines were derived. Embryonic stem cell lines are established from the 3.5 day mouse blastocyst inner cell mass and can be expanded on mouse fibroblast feeders [1,2]. When injected into a host blastocyst, ES cells contribute to a variety of lineages of all three germ layers and the germ line, thereby creating normal chimeric animals [3,4]. This broad developmental potential has been termed pluripotency. Under the right conditions ES cells can replicate apparently without limit in culture, while remaining pluripotent [5]. Features of ES cells are high levels of telomerase activity [6], a short G1 cell cycle checkpoint, and initiation of DNA replication without external stimulation [7]. They are the only known truly immortal stem cells and most importantly maintain a normal diploid karyotype, which renders them a suitable model for developmental studies. Theoretically it should be possible to produce and isolate all stem cells of more restricted potency from these cells, and by ∗ Corresponding author. Tel.: +44-131-662-9829; fax: +44-131-662-9779. E-mail address: [email protected] (T.E. Allsopp). 1 Present address: NeuronZ Ltd., P.O. Box 9923, Newmarket, Auckland 1031, New Zealand.

now protocols for directed differentiation into a wide variety of cell types have been established (see the description later). In addition to that for mice, reports have described the derivation of pluripotent, embryonic stem cells from multiple vertebrate species, including fish [8], chicken [9], rabbit [10], pigs [11], cows [12], sheep [13], non-human primates [14,15], and most recently humans [16,17]. As only the established mouse lines have been so far shown to fulfill all the stringent criteria that define bona fide ES cells (see Ref. [18]), we describe these lines, particularly human lines as ES cell-like. A practically unlimited supply of cells and a variety of options for genetic manipulation, together with their differentiation capacity are clear advantages of ES cells as valuable tools in pharmacology. Thus ES cells and their differentiated progeny are being implemented increasingly in routine phases of the pharmaceutical pipeline of drug discovery and development.

2. Embryonic stem cells 2.1. Maintenance of ES cell proliferation and pluripotency When first isolated, co-culture with murine embryonic fibroblasts was considered essential for mouse ES cell

1043-6618/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-6618(03)00036-7


T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278

maintenance. Later it was discovered, that conditioned media preparations could substitute for the feeders [19] and the differentiation inhibiting activity in the medium was identified as the cytokine leukemia inhibitory factor (LIF), which is now, in combination with ␤-mercaptoethanol commonly used for feeder-free ES cell culturing [20,21]. This stimulation of ES cell self-renewal is restricted to LIF and related cytokines of the IL-6 family, which signal through the gp130 receptor via JAK kinase mediated STAT3 activation [22,23]. A study using a chimeric STAT3 molecule activated by estradiol suggests that STAT3 signalling alone can be sufficient to block differentiation [24]. In the contrary activation of the MEK/ERK pathway seems to enhance differentiation and its inhibition promotes self-renewal in ES-cells [25]. A role for PI3 kinase signalling in murine ES cell survival and cell cycle progression has also emerged [26]. Another property of all pluripotent cell types is the expression of the germ-line POU domain transcription factor Oct-4 [27]. Expression of Oct-4 in the zygote is essential for establishing the pluripotent stem cell population of the inner cell mass [28] and a fine balanced Oct-4 expression level is critical to maintain ES cell pluripotency [29]. It has to be noted, that the investigations of mechanisms maintaining pluripotency have been conducted on mouse ES cells and species-specific differences may possibly exist. Non-human primate and human ES-like cells do not appear to require exogenous LIF or other gp130 signalling cytokines for maintenance of the undifferentiated phenotype. Instead human ES-like cells are routinely supported by MEF feeder cells in the presence of bFGF to remain in an undifferentiated state [16], or a matrigel substrate in 100% MEF conditioned medium [30]. However, their apparent unresponsiveness to exogenous LIF makes it difficult to maintain them in an undifferentiated state and spontaneous differentiation hinders the culture and study of human ES-like cells. Despite this differences there are also important similarities. Human ES-like cells and mouse ES cells both express Oct-4 and downregulate its expression upon differentiation [31]. 2.2. Genetic engineering of ES cells Mouse ES cells are the most genetically flexible stem cells and after genetic manipulation by integration of purpose designed vectors, genetically altered cells can be easily selected and clonally expanded. The most widely used genetic manipulation technique for ES cells, is the generation of null mutation ‘knockout’ mice, through gene targeting by homologous recombination [32] to determine the role of genes in development and to create mouse models of human disease. As some mutations result in early embryonic death of homozygous embryos, it is not possible to study their effect on specific cell types in transgenic animals. In these cases in vitro differentiation of ES cells converted to a null genotype [33] has been employed to determine the effect on cell type-specific differentiation [34,35]. Nevertheless a gene knockout project is a time consuming experiment.

An alternative approach to assessing gene function during development is to randomly create heterozygous phenotypes by insertional mutagenesis of genes using gene trapping. Gene trapping relies on the random integration of a promoterless reporter gene into the host cell. Cell clones, in which the expression of the reporter (usually the neomycin gene) is driven by a cellular promoter can be recovered by appropriate selection, resulting in a rate of single allele disruption approaching 100% [36]. Useful information can be obtained on the requirement of genes in ES cell differentiation through a systematic analysis of such in vitro libraries of genes trapped in ES cell clones. Besides ablating gene function with a knockout approach, a ‘knockin’ strategy targeting only one allele of a gene of interest with a tracer, like ␤-galactosidase or green fluorescent protein (GFP), has been extensively used to monitor gene expression during development. Another potent application is to transfect ES cells to express GFP either constitutively, or targeted under the control of a lineage-restricted gene. In the latter case instead of GFP a drug resistance gene can be inserted. An example for ubiquitous, constitutive expression is the study by Pratt et al., in which a fusion of GFP with a part of the tau protein provides excellent labelling of processes, especially axons of ES cell-derived neurons, enabling the study of axonal outgrowth and regeneration [37]. The targeted insertion of GFP or antibiotic resistance into a gene that is only expressed for a limited time period during development, or in specific differentiated progeny makes it possible to purify cell populations by fluorescence-activated cell sorting (FACS), or to positively select them with antibiotic treatment (Fig. 1). Of use to this strategy for genetically modifying a chosen cell lineage is the use of internal ribosome entry site (IRES) containing constructs that permit promoter dependent expression of bicistronic transcripts, including selectable markers. Examples how this technique has been used to efficiently isolate specific differentiated progeny from ES cells are given below. It can also be applied to maintain pure cultures of ES cells. For murine cells a neomycin resistance has been introduced into the Oct-4 gene, resulting in differentiating cells losing their Oct-4 expression and to die in the presence of G418 [38]. An approach like this can help to overcome the obstacle of spontaneous differentiation for human ES-like cells. Tackling this problem, Eiges et al. have developed a human ES line in which the stem cell-restricted gene Rex-1 drives expression of GFP, allowing FACS purification of pluripotent cells [39]. In gain-of-function experiments, the effect of overexpression of selected genes can be studied in differentiated progeny from ES cells clonally selected for high level expression, proving higher reproducibility of the results and a longer time window of expression than transient viral transfection of primary cultures. The interpretation of the results can nevertheless be hampered by position effects of random integration, which often leads to silencing after differentiation. An alternative could be extrachromosomal propagation

T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278


Fig. 1. Isolation of ES cell derivatives by lineage selection. A schematic depicting the proprietary process of lineage selection. A selectable marker (fluorescent protein or antibiotic resistance) is targeted to a gene locus (e.g. sox-1) using homologous recombination in undifferentiated ES cells. When the engineered cells are differentiated using standard protocols (see main text) and neural cells are specified, sox-1 promoter dependent expression of the selectable marker in neuroectoderm permits proprietary isolation of progenitor cells either by cell sorting or through cell survival due to antibiotic resistance. Purified ES cell progeny are then expanded with mitogen and used in screening assays for gene and drug discovery.

of plasmid expression vectors, utilising the polyoma virus replication system [40]. This system has been successfully implemented and can deliver a greater number of stable transfectants (1–5%, compared to less than 0.1% for stable integration) and higher expression levels of the transgene. 2.3. In vitro differentiation potential of mouse and human ES cells 2.3.1. Aggregation mediated differentiation: embryoid bodies ES cell differentiation in vitro as aggregate embryoid body (EB) cultures resembles processes of early embryonic development, which is indicated by a similar gene expression pattern. Outer cells acquire extraembryonic endoderm like features, coinciding with expression of early endodermal genes [41], while the inner pluripotent cells display a primitive ectoderm like gene expression. This feature has led to study them as a mammalian analogue of the amphibian animal cap [42] for the neural induction by default model [43]. In parallel, early mesoderm differentiation markers Brachyury and BMP-4 are expressed. As in the mouse embryo, where

it is transiently expressed during gastrulation, Brachyury expression is downregulated in later stages of EB differentiation [34]. Subsequently progenitor cells are formed, which differentiate in cell types of all three embryonic germ layers and protocols to enrich for many different specific cell types have been established, with endodermal derivatives being the most recent (Table 1). Despite the sequential expression of germ layer specific genes, the mixture of cells within the EB is far from resembling the precise patterning processes in the intact embryo. EBs form in confluent cultures of ES cells after withdrawal of LIF in untreated culture dishes, or in methylcellulose cultures [72]. Placing a known number of cells (typically 500–1000) in ‘hanging drop’ cultures [73] standardises the analysis of gene expression patterns. Varying from different protocols and depending on the desired differentiated cell types, EB are dissociated between day 4 and 8 and plated on an adhesive substrate for further differentiation. Often retinoic acid (RA) is used as an unspecific agent to induce differentiation. Variation in application time-points and concentrations of RA results in different induction of progeny, that is, a high concentration promotes

Table 1 Differentiated cell types derived from mouse ES and human ES-like cells in vitro Germ layer







Mouse ES

Neurons Astrocytes Oligodendrocytes Keratinocytes Melanocytes Dendritic cells

[44,45] [46] [47] [48,49] [50] [51]

[61,62] [63,64] [65]



[52,53] [54,55] [56] [49] [57] [58] [59] [60] [31,66,69] [70]

Hepatocytes Pancreatic islet cells Pneumocytes

Human ES-like

Hematopoietic cells Cardiomyocytes Skeletal muscle Smooth muscle Mast cells Adipocytes Osteoblasts Chondrocytes Cardiomyocytes Hematopoietic cells

Pancreatic islet cells



T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278

formation of neurons and a lower concentration formation of cardiomyocytes [74]. 2.3.2. Differentiation in monolayer culture Only recently the differentiation in monolayer culture is starting to be used as an alternative to differentiation in EB. When deprived of LIF ES cells in monolayer readily differentiate. However, the arising phenotypes have not been carefully defined. Pioneering work on aggregation-free monolayer protocols was performed by Nishikawa’s group. They proved the production of specific mesodermal cell types and were able to FACS clonogenic endothelial and hematopoietic progenitors by this method [53], that were even able to form vasculature [49]. More recently, the derivation of a primitive neural stem cell from monolayer cultured ES cells was described [75]. Neural progeny can be generated from ES cells easily in monolayer protocols as has recently been documented [76]. This technique will permit a more regulated manipulation of the overall differentiation procedure and could yield abundant supply of a purified single phenotype if used in combination with lineage selection technology. 2.4. Directed in vitro differentiation and lineage selection 2.4.1. Growth factor directed differentiation The majority of directed differentiation protocols utilise an initial EB aggregation step. Therefore, the early-acting differentiation promoting activities inside the EB are largely unknown. Subsequently, the EB were plated in medium containing a cocktail of specific growth and differentiation factors, sometimes to be applied sequentially, to promote generation of a particular cell type. This often happens in a defined minimal medium, which does not permit survival of other cell types. With this kind of techniques a variety of cell and tissue types have been derived from ES cells (Table 1). Reports exist of the efficient generation of hepatocytes and dendritic cells from human ES-like cells, but these have not yet been published. In the case of neuronal differentiation it was possible to enrich for specific neurotransmitter phenotypes. The McKay group has developed a protocol to derive a high percentage (34% of all neurons, 20% of all cells) of dopaminergic midbrain neurons, which is important, due to the prospect of transplants to treat Parkinson’s disease [77]. They exposed neural precursors selected in minimal medium and expanded with bFGF, shh, FGF-8 (ventralising activity in the developing brain [78]) and ascorbic acid. Surprisingly, with a very similar protocol, which omitted FGF-8 and shh and used nicotinamide instead, the same group was able to generate insulin-secreting pancreatic islet cells [64], although these cells are of endodermal and not ectodermal origin.

‘knockin’ approach to obtain highly purified populations either by FACS, or by drug selection. Klug et al. introduced a neomycin resistance under the control of the cardiac ␣ myosin heavy chain promoter and were able to obtain over 99% pure preparations of cardiomyocytes, compared to less than 1% in unselected cultures [79]. The same sort of approach was first used by Li et al. to purify neural precursors, by targeting a neomycin resistance under the control of the sox-2 promoter, which is active in the early neural plate [80]. This selection procedure enriched for 94% nestin-positive precursors cells, which could be expanded in bFGF for at least 3 weeks. Withdrawal of bFGF in serum free medium resulted in more than 90% of the cells in the culture to express neuronal markers, compared to about 50% without selection. Instead of selecting for neural precursors, Wernig et al. used GFP expression under the control of the tau promoter to select for post-mitotic neurons and obtained cultures, which contained more than 90% neurons [81]. The GFP remains expressed in mature neurons, allowing the identification of transplanted neurons, integrated into the host tissue. 2.4.3. Forced differentiation by overexpression A third approach to derive particular cell lineages is the forced expression of transcription factors, which govern the transfected cells to adopt a specific phenotype. This has only been attempted a couple of times so far, probably to a lack of knowledge of phenotype-defining transcription factors, or to complex interactions between several transcription factors together with epigenetic differentiation factors. Examples in which a single transcription factor determines differentiation towards a specific cell type, like the bHLH factor MyoD for skeletal muscle [82] are rare. Indeed for this well characterised control gene of differentiation, overexpression of MyoD in ES cells resulted in effective formation of skeletal myocytes, which fused to multinuclear, contractile myotubes [83]. In a similar experiment to promote hepatocyte differentiation, the transcription factor HNF3 was overexpressed in ES cells, which led to induction of early but not late endodermal markers [84]. Hepatocyte generation from ES cells could only be achieved by growth and differentiation factor application [61]. It is reasonable to expect, that the combination of forced overexpression with growth factor differentiation protocols will lead to a greater efficiency for the generation of specific cell types, which have been hard to obtain until now. McKay’s group were the first to successfully use such a combined strategy. Applying their dopaminergic differentiation protocol [85] to ES cells overexpressing the midbrain differentiation inducing transcription factor Nurr1, they more than doubled the percentage of dopaminergic neurons in their cultures [85]. 3. Applications of embryonic stem cells

2.4.2. Embryonic lineage selection The lineage selection strategy relies on less strict differentiation protocol, but instead utilises the earlier described

Due to their fundamental attributes of unlimited expansion and pluripotency embryonic stem cells have gained

T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278

considerable interest from the biopharmaceutical sector for their use in drug discovery and development and cell replacement therapy. The current pharmaceutical discovery process is universally accepted as being time consuming, inefficient and a high financial burden. It is anticipated that improvements could be made to the discovery phase of new compounds through the application of more appropriate, disease-orientated cellular screens not only for therapeutic target validation but also for optimisation of target-acting compounds following their discovery via high-throughput screens. Furthermore, the development phase of newly discovered compounds can be advanced or achieved earlier in the overall process once suitable cellular assays are available for initial studies of drug action and toxicology. Advanced technology platforms utilising established, robustly growing mouse ES cells are currently being validated in these key areas with a view to improving the information and diversity of content available from cellular assays. Crucial to the success of the reagents in this context has been the verification of the following: that purified, mouse ES-derived progeny display pharmacological phenotypes entirely appropriate for the major classes of therapeutic target; that the reagents can generate fully mature pharmacological phenotypes in many of those lineages studied (though improvements for certain cell types is needed); and that certain ES-derived progeny display cellular functions that are superior to those that can be obtained by existing or competing cellular technologies. In the near future some of these assays currently being validated will be replaced with human ES cell equivalents, but this will be dependent on substantial improvements being made in our understanding of how to make the human cells grow as well as their existing mouse counterparts. Combining human somatic cell reprogramming with the derivation of cloned ES lines will facilitate the design of advanced screening assays for complex, genetic, human diseases. Finally, concomitant with the necessary improvements in cell lines, assays and basic genetic engineering capability, human ES offer hope as a resource for cell replacement in the treatment of diseases associated with cellular dysfunction, degeneration and as therapeutic delivery vehicles for gene based therapy.


Fig. 2. Mouse neural cells purified by lineage selection from differentiating ES cells express ␤- and ␥-secretase molecular components. An immunoblot using antibodies appropriate for the detection of enzymes catabolising the amyloid precursor protein. Embryonic stem cells engineered for lineage selection at the sox-2 locus were differentiated using standard protocols and purified neural progeny were grown for a further 7 days in vitro. Protein samples were extracted, resolved by SDS–PAGE and immunoblotted using antibodies specific for ␤-secretase (BACE) and presenilin-1 (PS1). Embryonic stem cell samples were compared with extracts prepared from post-natal day 1 mouse brain (P1). Numbers indicate molecular size markers (kDa).

3.1. Drug discovery

dustry is the finding that the neural cells express the functional secretase components required to catabolise human amyloid precursor protein into ␤-amyloid peptide (Fig. 2), forming the basis of an ES cell based Alzheimer’s disease screening assay. Cell types representative of other lineages have also been partially characterised at the functional level in vitro [49,55,56,59,65,73]. A few examples have been reported concerning the functional characterisation of lineages derived from each of the three germinal layers obtained from human ES-like cells [67,69,71]. In particular, categories of pharmacological phenotypes existing in neuronal derivatives from human ES-like differentiation procedures include dopaminergic, GABAergic, glutamatergic, glycinergic, cholinergic and purinergic [86].

3.1.1. Cellular assays Distinct populations of terminally differentiated phenotypes from the existing mouse ES and to a lesser extent human lines have been characterised with respect to their functional properties. Pivotal to the success of such an approach is the ability through directed differentiation or lineage selection to obtain diverse but pure cell populations. Mouse ES-derived neural cells obtained by these procedures express neurotransmitter receptors, ionic channels, kinases and proteases representative of many of the major classes [44–46,90]. Of particular interest for the pharmaceutical in-

3.1.2. Compound identification and optimisation Although it is desirable in certain circumstances for an assay to consist of a single, physiologically relevant, ES-derived lineage phenotype, there are also occasions when a mixture of cell types is required in order to simplistically replicate tissue histology typical of a disease. A broader depth of information is obtainable from screens utilising such assays when the natural complement of cell types can be faithfully generated from differentiating ES cells, but the appropriateness of therapeutic target location to a single cell type is also maintained. The possibility to


T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278

generate these two alternative formats from a single starting resource, in combination with the ability to engineer cells to express a human therapeutic target, make mouse ES cells particularly relevant to screen designs for drug validation and optimisation. Thus, mouse neural cells purified by lineage selection utilising an engineered sox-1 or -2 loci provide an ideal starting resource for neurological screens as not only can such reagents generate a range of mature, functional neuronal phenotypes, but also multipotent stem cells and progenitors can be obtained. Common to all applications of stem cells in pharmaceutical discovery and development is a requirement in the reagents to be provided at sufficient scale for reproducible, robust screening. For pluripotent ES cells, this necessitates utilisation of the basic process of symmetrical self-renewal for biomass expansion while maintaining the potential of the cells to yield consistent differentiation. Although significant research progress has been made for feeder-free mouse ES cells concerning the understanding of the mechanisms underlying these issues, they have yet to be fully developed and exploited in relation to issues of scale-up. Straightforward pharmacological screens based on established mouse ES cells are being complemented with assay designs based on derivatives of human ES-like cells. Considerable advantages, however, can also be provided to pharmacological assays via the use of cloned human ES cell lines derived from reprogrammed, disease-specific somatic cell nuclei (Fig. 3). These reagents will be used to emulate the genetic background, in terms of disease susceptibility loci, that contribute to disease tissue pathology. The anticipated benefits to assay design also relate to the prospects of obtaining diverse, disease representative cell types that are

currently in sparse supply and being able to screen for compounds that act at earlier stages of disease progression and by virtue are likely to be preventative rather than palliative. 3.2. Functional gene discovery Pharmaceutical screens to identify new therapeutic targets are performed frequently in systems that posses an inadequate combination of throughput and clinical relevance. The establishment of a system involving extrachromosomal propagation of plasmid expression vectors in ES cells [40] as described previously, provides the basic resource required for building relevant screens to identify or validate gene function and combines both high throughput capacity and clinical relevance. Furthermore, the mouse polyoma virus method of independent DNA replication and cDNA expression when used in tandem with ES cells can be utilised to identify the function of genes assignable to a comprehensive range of basic cellular features (cell division, fate and death) and for the broadest collection of protein families (structural proteins, kinases, phosphatases). Due to the high transfection rates obtained with subsequent homogenous gene expression, the technology is routine to perform using a straightforward, readout of basic cellular function in undifferentiated ES cells. Progress is also being made in the combined use of mouse ES cells and screens for gene ‘knock-down’ (siRNA, RNAi). Additional refinements to these technologies will enable successful translation of the basic approach onto a human ES resource. Novel functions have been assigned recently to a number of genes through the use of this cDNA library screening technology in undifferentiated ES cells, thereby validating

Fig. 3. Design of advanced human disease assays using cloned ES cells. Schematic depicting the basic options for the generation of either normal or disease specific cells for discovery processes. Using human ES cells derived from donated, excess IVF embryos, cells of a chosen lineage are generated by a combination of directed differentiation and lineage selection. Disease-specific cells are generated by: (1) the reprogramming of donated nuclei from susceptible patients using unfertilised eggs; (2) the derivation of cloned ES cell lines; (3) engineering of lines to perform promoter-dependent lineage selection; (4) directed differentiation with progenitor purification.

T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278

the approach. For example the neural determination gene sFRP2, a soluble, secreted form of the wnt receptor frizzled, was identified following its subtraction cloning and expression profiling in differentiating ES cells [87]. The wnt antagonist exacerbated the production of neural progeny, whereas forced expression of wnt-1 in ES cells and treatment with lithium chloride both inhibited neural differentiation. This recent example illustrates the usefulness of the technology for the identification of novel stem cell induction and growth factors. Furthermore, conditional expression profiling when combined with techniques to generate and purify ES-derived progeny is a powerful method for the functional assessment of new therapeutic entities in differentiated phenotypes, but this approach remains to be validated. Isolation of target, library candidates should be straightforward to obtain by careful design of a specific end-point assay in which molecular function (or dysfunction) is modelled in an appropriate mature phenotype. 3.3. Toxicology Within the toxicological field ES cells have been utilised in two approaches, both of which are still being validated; established mouse lines are used in the Embryonic Stem cell Test (EST) for developmental toxicology and both mouse and human ES-like derivatives of particular lineages are being used for early stage assessment of drug adsorption, metabolism and toxicity. The EST is an in vitro embryotoxicity test in which test reagents are assessed according to three endpoints: inhibition of the differentiation of ES cells into contracting myocardium; cytotoxicity in ES cells; and cytotoxicity of mouse 3T3 fibroblasts which serve as a comparison for differentiated cells [88]. Recent results from a prevalidation assessment of the standardised protocol for the test, performed in several different laboratories, indicated that the assay outperformed alter-


native embryotoxicity assays (foetal limb micromass and post-implantation whole rat embryo) and was extremely accurate in predicting cellular toxicity. Gene profiling from candidate compound exposure experiments using the EST are now being performed (predictive toxicogenomics) with the anticipation that after analysis, descriptive assignments to the requirement of the expression of key genes will be correlated with endpoints from the assay. Furthermore, it can be imagined that functional gene screening in mouse ES cells will prove useful for the determination of the mechanistic features associated with changes in gene expression in the existing and additional endpoint assessments in the assay. The derivation of all varieties of fully functional cardiomyocytes, hepatocytes and hematopoietic cellular subtypes, via directed differentiation protocols from mouse ES cells is currently arguable and it is therefore anticipated that the derivation of related progeny from human ES-like cells will be afforded the same level of close scrutiny. Of clear necessity for toxicological studies is the generation of pure populations of appropriate cell lineages for the key systems of kidney, liver, heart, blood, brain and reproductive organs and a combination of regulated differentiation and purification by lineage selection using mouse ES cells should achieve this prospect. Although reports now describe the provisional generation from human ES-like cells of cardiomyocytes, hematopoietic, neural and hepatic progeny [68–70], it will be crucial in the near future to further validate the full functional characteristics of these cell types. For example, whether human ES-like hepatic derivatives adsorb compounds and up regulate members of the CYP family of proteins following experimental compound exposure. The design of optimised protocols for the specification of endothelial (vascular and brain) and glomerular epithelial cells from either mouse or human ES-like cells are also keenly awaited.

Table 2 Transplantation of differentiated cells or precursors derived from murine ES cells into animal models for human disease Cell type and pre-treatment/selection

Nervous system RA induced neurons and precursors GRP expanded with bFGF and PDGF EB differentiated 4 days, no RA shh, FGF-8 induction of Nurr1 overex-pressing cells [82,84] RA induced neurons and precursors Non-nervous system Lineage-selected cardiomyocytes Lineage-selected insulin-secreting cells a

Not determined.

Disease model

Site of transplant

Survival and marker expression

Functional recovery


Quinolinic acid injection in rats Myelin-deficient mutant rats

Striatum Intraventricular injection

+ +

nda nda

[89] [47]

6-OHDA injection in rats 6-OHDA injection in rats

Median forebrain bundle Striatum

+ +

Reduced rotation Reduced rotation

[90] [85]

Spinal cord contusion in rats

Spinal cord


Hindlimb motor activity


Dystrophic mdx mice Streptozotocin-induced diabetes in mice

Ventricular myocard Spleen

+ +

nda Normalised blood glucose levels

[79] [63]


T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278

3.4. Embryonic stem cells as therapeutic tools 3.4.1. Tissue transplantation The use of ES cell derivatives as reagents for restorative or regenerative therapy is a realistic solution that addresses current, acute problems of tissue shortage for the treatment of many degenerative diseases, including Parkinson’s disease, but discussion of which is beyond the scope of this review (but see Lindvall O., this edition). However, a brief synopsis concludes that experiments so far have involved the transplantation of ES-derived cells in a number of animal models for human disease (Table 2). A potential risk is the possibility of tumour formation by undifferentiated ES cells still present in transplanted ES-derived cells, which could give rise to teratomas and teratocarcinomas. A solution for this problem would be to genetically engineer a safety mechanism that would selectively eliminate persisting ES cells from cell preparations. For mouse ES cells this approach has been demonstrated to function by targeting the Oct-4 locus with a hygromycine-thymidine-kinase construct [92] and subsequent elimination of Oct-4 expressing pluripotent cells from ES-derived neural precursor populations [93]. To overcome the problem of immunological incompatibility of allografts of ES-derived cells, the generation of autologous ES cells for every patient by therapeutic cloning has been suggested [94]. The technology has been successfully validated in mice, with the generation of fully pluripotent ES cells from somatic cell nuclear transfer [95,96]. 3.4.2. Cell-based drug delivery and immunotherapy There are also two other potential applications for the use of ES cell derivatives as therapeutic tools. Embryonic stem cell derived progenitors could be used as delivery vehicles for the regulated release of drugs or therapeutic proteins. Transplantation of cells expressing the therapeutic gene or protein at the site of tissue trauma or damage would also overcome biological efficacy limitations associated with incomplete drug accessibility. A cell-based delivery of proteins may be at a steady level and released at more physiological concentrations if the transplanted cells are functionally integrated into the transplant site. For grafting of insulin-producing cells derived from murine ES cells there already have been encouraging results in a mouse diabetes model [63]. Stem cells have also been used previously as ‘cellular bullets’ in an approach where they were transfected for expression of tumouricidal genes and transplanted at sites of tumour growth. Promising results indicated that the cells caused a marked reduction in tumour size or even regression [97,98]. A second potential application is in the use of ES cell derivatives as immunotherapies, with the aim to instruct a patient’s immune system to recognise and attack tumour cells. The cellular basis for immunotherapy is dendritic cells, a population of antigen-presenting cells responsible for identifying non-self antigens and initiating an immune reaction of cytotoxic T and NK cells. Anti-cancer vaccines

have been developed using dendritic cells primed in vitro to elicit an immune response against tumour cells [99] and human ES-like cells have been reported to generate dendritic cells.

Acknowledgements Work at the Institute for Stem Cell Research is supported by the Medical Research Council (UK). We thank Dr. P. Mountford for advice on the compilation of Fig. 3.

References [1] Martin G. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma cells. Proc Natl Acad Sci USA 1981;78:7634–8. [2] Evans MJ, Kaufman MH. Establishment in culture of pluripotential stem cells from mouse embryos. Nature 1981;291:154–6. [3] Bradley A, Evans M, Kaufman MH, Robertson E. Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines. Nature 1984;309:255–6. [4] Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. Derivation of completely cell-culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 1993;90:8424–8. [5] Suda Y, Suzuki M, Ikawa Y, Aizawa S. Mouse embryonic stem cells exhibit indefinite proliferative potential. J Cell Physiol 1987;133:197– 201. [6] Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271–8. [7] Savatier P, Lapillonne H, van Grunsven LA, Rudkin BB, Samarut J. Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells. Oncogene 1996;12:309–22. [8] Sun L, Bradford CS, Ghosh C, Collidi P, Barnes DW. ES-like cell cultures derived from early zebrafish embryos. Mol Mar Biol Biotechnol 1995;4:193–9. [9] Pain B, Clark ME, Shen M, Nakazawa H, Sakurai M, Samarut J, et al. Long-term in vitro culture and characterization of avian embryonic stem cells with multiple morphogenetic potentialities. Development 1996;122:2339–48. [10] Graves KH, Moreadith RW. Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos. Mol Reprod Dev 1993;36:424–33. [11] Wheeler MB. Development and validation of swine embryonic stem cells: a review. Reprod Fertil Dev 1994;6:563–8. [12] Cherny RA, Stokes TM, Merei J, Lom L, Brandon MR, Williams L. Strategies for the isolation and characterization of bovine embryonic stem cells. Reprod Fertil Dev 1994;6:569–75. [13] Handyside A, Hooper ML, Kaufman MH, Wilmut I. Towards the isolation of embryonic stem cell lines from sheep. Roux’s Arch Dev Biol 1987;196:185–90. [14] Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995;92:7844–8. [15] Thomson JA, Marshall VS. Primate embryonic stem cells. Curr Top Dev Biol 1998;38:133–65. [16] Thomson JA, Itskovits-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–7.

T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278 [17] Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci USA 1998;95:13726–31. [18] Smith AG. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 2001;17:435–62. [19] Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity, which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev Biol 1987;121:1–9. [20] Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988;336:688–90. [21] Williams RL, Hilton DJ, Pease S, Wilson TA, Stewart CL, Fenring DP, et al. Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336:684–7. [22] Niwa H, Burdon T, Chambers I, Smith AG. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–60. [23] Burdon T, Chambers I, Niwa H, Stracey C, Smith AG. Signalling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells. Cells Tissues Organs 1999;165:131–43. [24] Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999;18:4261–9. [25] Burdon T, Stracey C, Chambers I, Nichols J, Smith A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol 1999;210:30–43. [26] Burdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol 2002;12:432–8. [27] Pesce M, Gross MK, Schöler H. In line with our ancestors: Oct-4 and the mammalian germ. BioEssays 1998;20:722–32. [28] Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct-4. Cell 1998;95:379–91. [29] Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–6. [30] Xu C, Inokumi MS, Denham J. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–4. [31] Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404. [32] Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 1987;51:503–12. [33] Mortensen RM, Conner D, Chao S, Geisterfer-Lowrance AAT, Seidman JG. Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol 1992;12:2391–5. [34] Rohwedel J, Guan K, Zurschatter W, Jin S, Ahnert-Hilger G, Fürst DO, et al. Loss of ␤1 integrin function results in a retardation of myogenic, but an acceleration of neuronal differentiation of embryonic stem (ES) cells in vitro. Dev Biol 1998;201:167–84. [35] Metzler M, Chen N, Helgason CD, Graham RK, et al. Life without huntingtin: normal differentiation into functional neurons. J Neurochem 1999;72:1009–18. [36] Evans MJ, Carlton MBL, Russ AP. Gene trapping and functional genomics. Trends Genet 1997;13:370–4. [37] Pratt T, Sharp L, Nichols J, Price DJ, Mason JO. Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein. Dev Biol 2000;228:19–28. [38] Mountford P, Nichols J, Zevnik B, O’Brien C, Smith A. Maintenance of pluripotential embryonic stem cells by stem cell selection. Reprod Fertil Dev 1998;10:527–33. [39] Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 2001;11:514–8.


[40] Gassmann M, Donoho G, Berg P. Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells. Proc Natl Acad Sci USA 1995;92:1292–6. [41] Abe K, Niwa H, Iwase K, Takiguchi M, Mori M, Abe SI, et al. Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp Cell Res 1996;229:27–34. [42] Weinstein DC, Hemmati-Brivanlou A. Neural induction. Annu Rev Cell Dev Biol 1999;15:411–33. [43] Muñoz-Sanjuán I, Brivanlou AH. Neural induction, the default model and embryonic stem cells. Nat Rev Neurosci 2002;3:271–80. [44] Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. Embryonic stem cells express neuronal properties in vitro. Dev Biol 1995;168:342–57. [45] Strübing C, Ahnert-Hilger G, Shan J, Wiedemann B, Hescheler J, Wobus AM. Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech Dev 1995;53:275–87. [46] Fraichard A, Chassande O, Bilbaut G, Dehay C, Savatier P, Samarut J. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J Cell Sci 1995;108:3181–8. [47] Brüstle O, Jones KN, Learish R, Karram K, Choudhary K, et al. Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science 1999;285:754–6. [48] Bagutti C, Wobus AM, Fässler R, Watt FM. Differentiation of embryonal stem cells into keratinocytes: comparison of wild-type and ␤1 -integrin deficient cells. Dev Biol 1996;179:184–96. [49] Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S, et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 2000;408:92–6. [50] Yamane T, Hayashi SI, Mizoguchi M, Yamazaki H, Kunisada T. Derivation of melanocytes from embryonic stem cells in culture. Dev Dyn 1999;216:450–8. [51] Fairchild PJ, Brook FA, Gardner RL, Graca L, Strong V, et al. Directed differentiation of dendritic cells from mouse embryonic stem cells. Curr Biol 2000;10:1515–8. [52] Nakano T, Kodama H, Honjo T. In vitro development of primitive and definitive erythrocytes from different precursors. Science 1996;272:722–4. [53] Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama N. Progressive lineage analysis by cell sorting and culture identifies FLK1(+)ve-cadherin(+) cells at a diverging point of endothelial and hemopoietic lineages. Development 1998;125:1747–57. [54] Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. 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. [55] Maltsev VA, Rohwedel J, Hescheler J, Wobus AM. Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodol, atrial and ventricular cell types. Mech Dev 1993;44:41–50. [56] Rohwedel J, Maltsev V, Bober E, Arnold HH, Hescheler J, Wobus AM. 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. [57] Tsai M, Wedemeyer J, Ganiatsas S, Tam SY, Zon LI, Galli SJ. In vivo immunological function of mast cells derived from embryonic stem cells: an approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo. Proc Natl Acad Sci USA 2000;97:9186–90. [58] Dani C, Smith AG, Dessolin S, Leroy P, Staccini L, et al. Differentiation of embryonic stem cells into adipocytes in vitro. J Cell Sci 1997;110:1279–85. [59] Buttery LD, Bourne S, Xynos JD, Wood H, Hughes FJ, Hughes SP, et al. Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng 2001;7:89–99. [60] Kramer J, Hegert C, Guan K, Muller PK, Hughes FJ, Rohwedel J. Embryonic stem cell-derived chondrogenic differentiation in vitro:







[66] [67]







[74] [75]






T. Gorba, T.E. Allsopp / Pharmacological Research 47 (2003) 269–278 activation by BMP-2 and BMP-4. Mech Dev 2000;92:193– 205. Hamazaki T, Iboshi Y, Oka M, Papst PJ, Meacham AM, Zon LI, et al. Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS Lett 2001;497:15–9. Jones EA, Tosh D, Wilson DI, Lindsay S, Forrester LM. Hepatic differentiation of murine embryonic stem cells. Exp Cell Res 2002;272:15–22. Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000;49:157–62. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001;292:1389–94. Ali NN, Edgar AJ, Samadikuchaksaraei A, Timson CM, Romanska HM, Polak JM, et al. Derivation of type II alveolar epithelial cells from murine embryonic stem cells. Tissue Eng 2002;8:541–50. Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. Stem Cells 2001;19:193–204. Schuldiner M, Eiges R, Eden A, Yanuka O, Itskovits-Eldor J, Goldstein RS, et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res 2001;913:201–5. Zhang SC, Wernig M, Duncan ID, Brüstle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–33. Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001;108:407–14. Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 2001;98:10716–21. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–7. Wiles MV, Keller G. Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development 1991;111:259– 67. Wobus AM, Wallukat G, Hescheler J. Pluripotent embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotopic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 1991;48:173–82. Rohwedel J, Guan K, Wobus A. Induction of cellular differentiation by retinoic acid in vitro. Cells Tissues Organs 1999;165:190–202. Tropepe V, Hitoshi S, Sirard C, Mak TW, Rossant J, van der Kooy D. Direct neural fate specification from embryonic stem cells: a primitive mammalian stem cell stage acquired through a default mechanism. Neuron 2001;30:65–78. Ying QL, Staviridis M, Griffiths D, Li M, Smith A. Direct and quantitative conversion of embryonic stem cells to neuroectodermal precursors. Nat Biotechnol 2003;21:182–6. Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000;18:675–9. Ye W, Shimamura K, Rubenstein JLR, Hynes MA, Rosenthal A. FGF and shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998;93:755–66. Klug MG, Soonpa MH, Koh GY, Field LG. Genetically selected cardiomyocytes from differentiating embryonic stem cells. J Clin Invest 1996;98:216–24. Li M, Pevny L, Lowell-Badge R, Smith A. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr Biol 1998;8:971–4.

[81] Wernig M, Tucker KL, Gornik V, Schneiders A, Buschwald R, Wiestler OD, et al. Tau EGFP embryonic stem cells: an efficient tool for neuronal lineage selection and transplantation. J Neurosci Res 2002;69:918–24. [82] Weintraub H, Davis R, Tapscott S, Thayer M, Krause M, Benezra R, et al. The myoD gene family: nodal point during specification of the muscle cell lineage. Science 1991;251:761–6. [83] Dekel I, Magal Y, Pearson-White S, Emmerson CP, Shani M. Conditional conversion of ES cells to skeletal muscle by an exogenous myoD1 gene. New Biol 1992;4:217–24. [84] Levinson-Dushnik M, Benevisty N. Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells. Mol Cell Biol 1997;17:3817–22. [85] Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 2002;418:50–6. [86] Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu C-P, Rao MS. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 2001;172:383–97. [87] Aubert J, Dunstan H, Chambers I, Smith A. Functional gene screening in embryonic stem cells implicates wnt antagonism in neural differentiation. Nat Biotechnol 2002;20:1240–6. [88] Scholz G, Genschow E, Pohl I, Bremer S, Paparella M, Raabe H, et al. Prevalidation of the embryonic stem cell test EST—a new in vitro embryotoxicity test. Toxicol In Vitro 1999;13:675–81. [89] Dinsmore J, Ratliff J, Deacon T, Pakzaban P, Jacoby D, Galpern W, et al. Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation. Cell Transplant 1996;5:131– 43. [90] Björklund LM, Sanchez-Pernautem R, Chung S, Andersson T, Chen IYC, McNaught KSP, et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci USA 2002;99:2344–9. [91] McDonald JW, Liu XZ, Qu Y, Liu S, Turetsky D, Mickey SK, et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med 1999;5:1410–2. [92] Lupton SD, Brunton LL, Kalberg VA, Overell RW. Dominant positive and negative selection using a hygromycin phosphotransferasethymidine kinase fusion gene. Mol Cell Biol 1991;11:3374–8. [93] Billon N, Jolicoeur C, Ying QL, Smith A, Raff M. Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells. J Cell Sci 2002;115:3657– 65. [94] Lanza RP, Cibelli JB, West MD. Human therapeutic cloning. Nat Med 1999;5:675–9. [95] Munsie MJ, Michalska AE, O’Brien CM, Trounson AO, Pera MF, Mountford PS. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse cell nuclei. Curr Biol 2000;10:989– 92. [96] Wakayama T, Tabar V, Rodriguez I, Perry ACF, Studer L, Mombaerts P. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 2001;292:740–2. [97] Benedetti S, Pirola B, Magrassi L, Bruzzone MG, Rigamonti D, Galli R, et al. Gene therapy of experimental brain tumours using neural progenitor cells. Nat Med 2000;6:447–50. [98] Abody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci USA 2000;97:12846–51. [99] Reinhard G, Märten A, Kiske SM, Feil F, Bieber T, Schmidt-Wolf IG. Generation of dendritic cell-based vaccines for cancer therapy. Br J Cancer 2002;86:1529–33.

Lihat lebih banyak...


Copyright © 2017 DADOSPDF Inc.