seminars in

CELL BIOLOGY, Vol 3, 1992 : pp 385-399

Mouse embryo stem cells : their identification, propagation and manipulation Austin G . Smith embryos . For example, several pre-implantation mouse embryos can be aggregated together and still generate a single, normally-sized, viable foetus . Conversely, it is possible to kill a large proportion of the cells in the murine blastocyst or egg cylinder and the embryo will still be able to adapt and form a normal conceptus . This interactive nature allows opportunity for the mammalian embryo to compensate for any damage or injury . Such a regulative capacity is dependent on two factors : the presence of pluripotential stem cells whose fate can be reprogrammed, and the operation of a flexible signalling system which mediates interactions between different embryonic tissues and in particular can modulate the behaviour of the stem cells .

The early mouse embryo contains a transient population of pluripotential stem cells which are responsible for generating both the foetal primordia and extraembryonic membranes . The characterisation of murine embryo stein cells and their isolation and propagation in culture provides the first instance in which pure populations of normal stem cells are directly accessible to the researcher. This marks a considerable advance in stem cell biology which may pave the way to the dissection of general stem cell control mechanisms and the identification of key regulatory factors . In addition, the genetic manipulation of embryo stem cells affords a unique avenue for experimental intervention in mammalian development and for controlled modification of the mouse germ line .

Key words : embryonic stem (ES) cell / mouse embryo / cytokine / differentiation inhibiting activity (DIA) / leukaemia inhibitory factor (LIF)

Pluripotential stem cells in the mouse embryo The initial stages of mammalian embryogenesis entail both the elaboration of extra-embryonic tissues and the expansion of the pluripotential founder cell lineage (Figure 1) . This process is generally presented as a series of bifurcations in which each differentiation event is considered to mark an irreversible segregation of developmental capacity . The evidence for this comes primarily from a series of elegant experiments in which the ability of different cell populations to contribute descendants to various tissues is analysed in chimaeras produced by combining embryonic cells from genetically distinct strains of mice (reviewed in ref 1) . Thus at the first stage at which there are two morphologically distinct cell types, ICM and trophectoderm in the blastocyst (Figure 1), it is found that ICM cells can contribute to all foetal and extra-embryonic tissues except the trophoblast (one of the embryonic components of the placenta), whereas the trophectoderm cells can only colonise the trophoblast . The second differentiation process which occurs prior to implantation divides the ICM into primitive endoderm and epiblast . This is accompanied by a further partitioning of developmental potential, such that the progeny of primitive endoderm are restricted to parietal and visceral yolk sac endoderm whilst

THE PROCESS OF embryonic development requires

the coordinated diversification of a multitude of specialised cell types from a single founder cell, the fertilised egg . In mammals there is no evidence that the initial regional specification of the developing embryo is directed by maternally deposited determinants, as occurs in some lower organisms . Instead the first cleavage divisions produce a population of identical cells known as blastomeres, each of which retains the potential to generate tissues of every lineage . Interactions within the embryo, or between the embryo and its environment, are then responsible for the development of cellular diversification . The organisation required of these interactions is reflected in the precise temporal and spatial orchestration of the subsequent differentiation events (depicted in Figure 1) and in the quantitative regulation of cell numbers in the different compartments . Paradoxically, however, the early mammalian embryo exhibits much greater plasticity than simpler From the AFRC Centre for Genome Research, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK ©1992 Academic Press Ltd 1043-4682/92/060385 + 15$8 .00/0

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8 . 5 days

Figure 1 . Development of the mouse embryo from fertilisation through gastrulation . Scale drawings of the first 9 days of mouse embryogenesis . Implantation occurs late on day 5 . ICM, inner cell mass ; E, epiblast ; PrE, primitive endoderm ; Ex .E, extraembryonic ectoderm ; PS, primitive streak . (Drawings by Rosa Beddington .) epiblast derivatives are found in all tissues except trophoblast and extra-embryonic endoderm . The hierarchical scheme of development which these and other studies have given rise to undoubtedly reflects the normal programme of murine embryogenesis . However, as discussed above, the early mammalian embryo is remarkably plastic, implying that there is considerable capacity for modulation within the system . Investigations into the timing of trophectoderm versus ICM restriction indicate that there is a degree of lability within the IC M . 1,2 ICM cores isolated from early blastocysts by immunosurgical or microsurgical removal of the trophectoderm can still regenerate a functional outer layer of trophectoderm, though this would not be their normal fate in an intact embryo . This ability declines in ICMs from more mature blastocysts, but it is apparent that the restriction of the ICM is not coincident with the initial differentiation of trophectoderm . Moreover, the eventual loss of developmental potential does not occur uniformly throughout the population, since both trophectoderm

and endoderm may differentiate simultaneously in culture from cells from an individual ICM . 3 The pluripotential ICM and epiblast tissues are formally distinguished by the inability of the latter to generate primitive endoderm . 4 Again, the precise timing of this restriction is indeterminate and may be subject to experimental perturbation . 1 These observations are consistent with the notion that the loss of developmental capacity is a progressive and heterogeneous process within a stem cell population rather than an all-or-nothing phenomenon (see also articles by Graham and Pragnell, pp 423-434, and Parkinson, pp 435-444, this issue) . The ICM and epiblast are the pluripotential tissues of the early embryo which harbour the germline and give rise to all somatic tissues of the embryo proper in addition to some of the extraembryonic components . The pluripotency and developmental lability of the epiblast is maintained throughout the egg cylinder stage of post-implantation development until the onset of organogenesis late on the eighth day of gestation (Figure 1) . The most dramatic

Embryonic stem cells

demonstration of the plasticity of early embryo stem cells comes from the observation that over 80 of cells in the gastrulating egg cylinder can be killed by teratogenic insult or by transplacental delivery of mitomycin C5 , yet provided this occurs before organogenesis, a normal foetus can still be formed . This reveals the true potential for self-renewal and regulation within the egg cylinder. That pluripotentiality and lability resides in the epiblast is evidenced by the generation of multiple differentiated cell types on transplantation to an ectopic site, 6 by heterotopic grafting experiments which demonstrate that different regions of the epiblast are not irreversibly committed to formation of particular foetal primordia, 7 and by the ready generation of teratocarcinomas from epiblast tissue . The latter are multi-differentiated tumours which arise spontaneously in the gonads of certain mammals but can also be produced experimentally at high frequency by ectopic grafting of early mouse embryos . 8,9 The tumours may be fully differentiated and benign or may be malignant in which case they contain clearly identifiable stem cells known as embryonal carcinoma (EC) cells . Grafts from 8 .5 day embryos only generate benign teratomas, whereas approximately 50 % of grafts from 6 .5 or 7 .5 day egg cylinders give rise to malignant teratocarcinomas . 10 The epiblast origin of the tumours has been established by dissection of the embryos prior to grafting . 11 Teratocarcinomas can be produced from embryos of most, if not all, inbred mouse strains, although in certain cases host-related factors can influence the relative proportions of malignant versus benign growths . 12 The propensity of early mouse embryos to form continuously growing tumours indicates that a primary transformation event is unlikely to be involved . Instead teratocarcinomas appear to arise directly from disruption of the normal growth and differentiation programme of embryo stem cells . 13 In summary, the early mouse embryo contains a population of stem cells which undergo a highly organised series of differentiation events, initially to produce extraembryonic tissues and culminating in formation of the primary embryonic germ layers . This stem cell population expands rapidly immediately after implantation, but their capacity for self-renewal is curtailed within the embryo and pluripotential stem cells do not persist beyond gastrulation . Experimental manipulation, however, reveals firstly that the ICM/epiblast cells have a high degree of developmental lability and secondly that

387 without the embryonic microenvironment their propagation can apparently be sustained indefinitely . Thus, in common with somatic stem cell types, both differentiation and self-renewal of early embryo stem cells are determined by local regulatory factors . Derivation and characterisation of stem cell lines from early mouse embryos Teratocarcinoma stem cells can be propagated in culture and clonal EC cell lines established . 14-16 These cell lines often remain multipotent and can generate tumours containing a variety of endodermal, mesodermal and ectodermal tissues . EC cells exhibit a remarkable similarity to ICM/ epiblast cells in morphology, ultrastructure, cell surface antigen expression and protein synthesis profiles . 17,18 On aggregation some EC cells form complex differentiated structures known as embryoid bodies 16,19 whose development parallels that of ICMs cultured in suspension 20 and reflects certain aspects of egg cylinder differentiation . Most significantly, some lines of EC cells can incorporate into the host ICM if reintroduced into a blastocyst 2 l -23 or aggregated with morulae . 24,25 They may then participate in normal embryogenesis to produce chimaeric offspring . Such chimaeras can have extensive EC-derived contributions to many tissues and be quite healthy . This is consistent with the notion that transformation is not obligate for the establishment of an embryo-derived teratocarcinoma and subsequently of an EC line . However, conclusive evidence of both the normality and pluripotency of EC cells requires colonisation of the germ-line in chimaeras and transmission of the EC genotype to viable offspring . This has been achieved for EC cells maintained in vivo as ascites tumours, 22 though it has only been claimed once for a clonal EC line propagated in vitro . 26 In fact, most EC lines are aneuploid, which is expected to be incompatible with meiosis, and they often show reduced differentiation potential in vitro . It is therefore not surprising that many lines contribute poorly, if at all, to chimaeras and others give rise to tumour s or produce developmental abnormalities . These variable characteristics presumably reflect the acquisition of mutations during uncontrolled selection applied during tumour growth (teratocarcinomas are usually serially transplanted before derivation of cell lines) and/or during in vitro propagation . Consequently the EC cell system has not proven to be an effective route

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for the transgenic modification of the mouse germline and suffers serious limitations as a model system for the study of mouse embryogenesis . Nonetheless, the study of EC cells laid the experimental and intellectual groundwork that culminated in the isolation of normal stem cells directly from early embryos . Thus the characterisation of EC cells suggested that their embryonic counterparts were cells of the early epiblast . It was also apparent that pluripotency was best maintained by co-culture of the EC cells on a feeder layer of mitotically inactivated mouse embryo fibroblasts . 16 In 1981 two laboratories reported the derivation of cell lines by placing cells from late blastocysts directly into culture on feeder layers . 27,28 Continuously growing cells were established both from whole blastocysts and from immunosurgically isolated epiblast . These cells were originally termed EK or ESC cells, but are now known as embryonic stem (ES) cell lines . They possess a morphology typical of undifferentiated EC cells, growing as compact colonies of small cells with minimal cytoplasm and prominent nucleoli (Figure 2A) . They express the EC cell-specific antigen SSEA-1 29 and lack differentiation antigens . ES cells readily differentiate into multiple tissue types both in vitro in embryoid bodies30 and in vivo in teratocarcinomas .27 They generally contribute extensively to chimaeras in which they rarely, if ever, give rise to tumours . In further contrast to EC cells, ES cells often exhibit a normal diploid karyotype .

In consequence, they are capable of reproducibly generating functional gametes and transmitting the ES genotype to normal offspring . 31 ES cells thus fulfill the criteria for a non-transformed pluripotential stem cell . The precise relationship between ES cells and stem cells in the early embryo proper remains uncertain, however . E S cells can contribute functional differentiated progeny to all foetal tissue types and to extra-embryonic mesoderm, as expected for an epiblast-like cell (Figure 1), but they also colonise extraembryonic endoderm and even trophoblast,32 properties characteristic of early ICM cells . This apparent contradiction between the origin of the ES cells from late blastocysts and their developmental potential could indicate that ES cells should be readily established from earlier embryos and might be expected to exhibit characteristics of early ICM cells in vitro . ES cells have been obtained from dissociated morulae, 33 but since the stem cells are only isolated after a period of initial development in culture it is not possible to dissociate genesis of an ES cell from maturation of the embryonic tissues . Morphologically ES cells appear more like epiblast cells than the larger, more rounded cells of the ICM . In addition, they show very limited potential to generate trophectoderm and parietal endoderm cells in monolayer culture, which are the cell types readily produced by isolated ICM cells in vitro . Moreover, early post-implantation embryos can give rise to ES cells, 34,35 although their full developmental potential

Figure 2 . Undifferentiated and differentiated embryonic stem cells in monolayer culture . A. Undifferentiated ES cell colony after 4 days culture in the presence of DIA/LIF . B . Differentiated colony after 4 days culture in the absence of DIA/LIF . C . Differentiated colony

after 2 days exposure to 6 mM 3-methoxybenzamide . Bar= 10 µm .

Embryonic stem cells

has not been determined . A second explanation therefore is that establishment of epiblast cells in culture entails an epigenetic `loss of memory' or deprogramming (see article by Marvin and McKay, pp 401-411, this issue) . This may allow ES cells to dedifferentiate in response to aggregation in vitro or to signals provided by the host blastocyst . E S cells may thus possess greater developmental versatility than an embryo stem cell in situ . Alternatively the latter may have greater lability than has hitherto been revealed by transplantation experiments and an ES cell may represent an epiblast cell in a state of `suspended animation' . Unfortunately the developmental potential of ES cells and isolated postimplantation epiblast cells cannot be directly compared because efforts to produce chimaeras by blastocyst injection of the latter have not proved successful (ref 4; R . Beddington, personal communication) . At least in this respect the two cell types are clearly not identical . Another difference is in cell cycle time ; epiblast cells have been estimated to cycle every 6-8 h36,37 whilst ES cells divide every 12-18 h in culture . This could reflect a true distinction between the cell types, or the slower cycling time of ES cells could be an adaptation to the culture environment . It may also be directly relevant to the specific ability of ES cells to integrate into the ICM, since the rapid generation time of postimplantation epiblast cells simply may not allow time for them to reprogramme in response to the blastocyst environment . Regulation of ES cell self-renewal and differentiation From a developmental perspective, the complete integration of ES cells into host embryos implies that they are capable of responding to, and indeed of generating, the full repertoire of regulatory signals which direct murine embryogenesis . E S cells may therefore be exploited as an in vitro assay system for the identification and characterisation of such regulators . 38 ES cells were initially isolated and maintained by co-culture on `feeder' layers of mitotically inactivated mouse embryo fibroblasts . 16 Originally the feeders were considered essential to maintain stem cell viability . Subsequently it was appreciated that addition of 2-mercaptoethanol to the cultures was sufficient to sustain cell survival in the presence of foetal calf serum . 39 Under these conditions ES cells

389 underwent extensive differentiation when plated in the absence of feeders, implying that propagation of the stem cell phenotype is dependent on the active suppression of differentiation/promotion of selfrenewal . This was confirmed by the finding that various cell lines including Buffalo rat liver (BRL) epithelial cells secreted a potent Differentiation Inhibiting Activity (DIA) . 40 This macromolecular factor eliminates the differentiation of EC and ES cells almost entirely and enables their continuous propagation as monocultures . Purification of DIA revealed that activity resided in a basic, relatively hydrophobic, single chain glycoprotein of M r 43,000, 41 and it subsequently emerged that DIA is identical to a previously described myeloid regulator, D-Factor42 or Leukaemia Inhibitory Factor (LIF) . 43-45 Pure DIA/LIF is sufficient to maintain undifferentiated pluripotential ES cell populations in continuous culture in mercaptoethanol-supplemented serumcontaining medium . Significantly, the culture of ES cells in DIA/LIF does not compromise their developmental potential : the cells retain the capacity for differentiation, both in vitro on withdrawal from DIA/LIF, 40,41 and in chimaeras where germ-line transmission is readily obtained . 46,47 DIA/LIF can therefore be employed to provide simplified and experimentally advantageous conditions for the propagation and manipulation of ES cells . 40,4s It has also been demonstrated that DIA/LIF can replace feeder layers in the isolation of new ES cell lines directly from cultured embryos . 47'49 Although the use of feeder cells may confer some advantage under sub-optimal culture conditions, for example by detoxifying media contaminants it seems clear that DIA/LIF fulfills the essential function of a heterologous feeder layer in propagation of the stem cell phenotype . It remains possible that uncharacterised serum factors are required in addition to DIA/LIF to maintain the stem cell phenotype . The ability to propagate undifferentiated ES cells for short periods in serum-free media supplemented with DIA/LIF alone suggests that other cytokines may not be necessary (A .G. Smith, unpublished), but optimisation of conditions for serial cultivation in defined media is necessary to validate such a conclusion . The effect of DIA/LIF on ES cells apparently provides an example of deterministic control of stem cell behaviour . Thus at clonal density ES cells form colonies containing stem cells or consisting wholly of differentiated cells (Figure 2) depending entirely on the presence or absence respectively of DIA/LIF .

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Detailed examination of the interaction between feeder cells and ES cells has revealed that DIA/LIF is produced in two distinct forms, one of which is freely diffusible and the other of which is immobilised by incorporation into the extracellular matrix . 50

A

This differential localisation originates from usage DIFF

B

of alternative first exons which alter the amino terminal sequence of the primary translation product from MKVLAAG- to MRCR-, whilst leaving the core hydrophobic secretory signal and the sequence for the mature protein unchanged . The modular organisation of the murine DIA/LIF gene constitutes

DIFF

a molecular strategy whereby the same regulatory factor can be targeted to alternative extracellular locations . 51 Consistent with a role in the generation of spatial diversity, the two DIA/LIF transcripts are differentially expressed in culture and during embryogenesis . 52 The potential significance of this is that there are important distinctions between the roles of soluble and immobilised factors . 50,53 Indeed

C

many cytokines are now known to be produced in both diffusible and cell- or matrix-associated forms . 51,54 One obvious benefit of matrix-association in the case of DIA/LIF is the potential for topographical restriction of the differentiation inhibitory DIFF

Figure 3 . Mechanisms of stem cell regulation . Alternative modes of DIA/LIF action . A . Autocrine expression by ES cells may regulate self-renewal at high cell density . B . Activation of DIA/LIF expression early in the differentiation process leads to feedback regulation of stem cell renewal . C . Paracrine modulation of DIA expression in differentiated cell types by cytokines and hormones (including induction of DIA/LIF by a heparinbinding growth factor secreted by ES cells 52 ) renders stem cell renewal dependent on the proximity and nature of differentiated cells .

signal ; stem cells will only be maintained where they remain in contact with DIA/LIF producing cells (see article by Parkinson, pp 435-444, this issue) . ES cells constitute a population of essentially normal early embryo cells which are capable of recapitulating many of the processes of embryonic tissue diversification . Such an experimental model is invaluable given the relative innaccessibility, very small size and cellular complexity of the early mammalian embryo . Not only can ES cells be used to identify candidate regulatory molecules such as DIA/LIF but they can also be employed to further characterise their potential roles . The analysis of

However, in the presence of DIA/LIF there is always a low and somewhat variable level of differentiation . This background differentiation is not affected by

DIA/LIF expression and activity in ES cultures has provided several insights into its possible modes of action in vivo (Figure 3) .51,52,55 Three observations

increasing the concentration of DIA/LIF and also occurs in serum-free medium (A .G . Smith, unpublished) . Such apparently random differentiation may be a culture artefact, or it could reflect an intrinsic property of stem cells . Since these two possibilitites cannot be distinguished at present, it appears equally valid to consider the activity of DIA/LIF either as a direct inhibition of cellular differentiation or as a survival/proliferative action specific for stem cells .

have been made . Firstly, undifferentiated ES cells express low but potentially significant levels of transcript for matrix-associated DIA/LIF . The reduced efficiency of ES cell differentiation in monolayer culture, unless cells are plated singly at low density48 could therefore reflect an autocrine activity of DIA/LIF . Secondly, expression of both forms of DIA/LIF is enhanced early during ES cell differentiation . This may provide a feedback mechanism to ensure that a stem cell population is

Embryonic stem cells maintained or even expanded during certain differentiation processes . Thirdly, the expression of DIA/LIF is induced as a primary response in certain cell types by specific cytokines, including a heparinbinding factor produced by ES cells, and is repressed by glucocorticoids . Thus a reciprocal paracrine signalling system (Figure 3C) may operate whereby in particular situations stem cells may be able to ensure their own expansion via stimulation of appropriate neighbouring cells . These interactions observed in differentiating ES cell cultures may mirror aspects of the expansion and differentiation of the ICM/epiblast in the early embryo . 19,30,51 Thus it seems possible that autocrine production of matrix-associated DIA/LIF could be a significant feature in the maintenance of pluripotency in vivo when the ICM and epiblast remain multi-layered, prior to and shortly after implantation, whilst the subsequent transition of the proliferating epiblast to an epithelial monolayer may render the stem cells more dependent on, and responsive to, the production of DIA/LIF and other cytokines by differentiated neighbours . DIA/LIF transcripts are present in the egg cylinder 52 and have also been detected in blastocysts by polymerase chain amplification of reverse-transcribed mRNA . 56 It remains to be demonstrated, however, that the protein is both functional and necessary for normal development at these stages . E S cells again provide a means of addressing these issues via their use as a transgenic system (see below) . Common regulatory components are often employed in multiple contexts during development . It therefore seems likely that factors operative on very early embryonic cells, as represented by ES cells, will also be active in later development . This appears to be the case for DIA/LIF to which a multitude of different activities have now been ascribed, both in vitro and in vivo (for reviews see refs 51, 57, 58) . Whether DIA/LIF serves a similar role in different systems is not yet clear . One possibility is that it has been recruited for multiple unrelated functions . However, an argument can also be made in favour of a more defined role as a stem cell factor . 51 Many of the actions of DIA/LIF appear to be mediated via stem cells or progenitor cells rather than direct effects on differentiated tissue . Thus the suppression of ES cell differentiation is a direct action on stem cells as are the survival/proliferative effects on primitive haematopoietic precursorsJ 9-61 and primordial germ cells . 62,63 In cultured kidney rudiments DIA/LIF specifically prevents nephrogenesis by reversible

391 inhibition of the differentiation of mesenchymal progenitor cells . 64 In vivo DIA/LIF stimulates the production of ectopic bone, suggesting that it acts on osteogenic precursors . 65 Indeed the complex phenotype produced by ectopic expression of DIA/LIF in the whole animal is consistent with activity on a range of progenitor cell populations . 65,66 It will be of considerable interest to investigate the involvement of DIA/LIF in well-characterised stem cell systems such as the intestinal crypts and epidermis . If DIA/LIF is a generalised stem cell factor, then autocrine production by stem cells and/or expression by subsets of differentiated progeny may be key components of the specialised microenvironment or 'niche' 67 required for homeostatic maintenance of stem cells, whilst modulation of DIA/LIF expression by other cytokines and hormones may play a significant role in stem cell responses to wounding and trauma (Figure 3) . Cellular responses to DIA/LIF are initiated by binding to specific cell surface receptors which have been detected on a variety of responsive cell types . 41,45,68-70 A DIA/LIF receptor cDNA has been isolated by expression cloning . 71 The encoded protein is a member of a group of structurally-related receptors for functionally diverse ligands, the haematopoietin receptor superfamily . 72 An unexpected property of many of the haemotopoietin receptors is that they can be generated as secreted extracellular proteins via alternative splicing . 71,11 The DIA/LIF receptor falls into this class, as murine cDNAs have been identified which do not include transmembrane and cytosolic domains . 71 Secreted receptors can act as natural antagonists of cytokine action by directly competing for ligand with transmembrane receptors and/or by promoting the systemic clearance of secreted cytokines . 74 If the extracellular DIA/LIF receptor functions in this manner to reduce DIA/LIF activity it could be an important determinant of early embryonic differentiation . However, there is also evidence that extracellular receptors may facilitate the appropriate presentation of a ligand to a signaltransduction complex . 75,76 Clearly the extracellular receptor is likely to be an important parameter in DIA/LIF action and further investigation is required to clarify its role . The cloning of a DIA/LIF receptor cDNA provides fresh impetus to the investigation of the molecular events downstream of receptor binding which mediate propagation of the undifferentiated ES cell phenotype . Interaction with at least one other subunit appears to be required for formation of a high affinity

3 92 DIA/LIF-receptor complex, which is presumed to be the active signalling entity . 55,71 Recently it has been reported that high affinity DIA/LIF binding sites can be generated by association of the cloned DIA/LIF receptor with a transmembrane protein known as gp130 . 77 The latter was originally characterised as the 13-subunit of the interleukin-6 receptor. 75,78 This implication of gp130 in DIA/ LIF action is particularly striking in view of the overlapping biological activities of DIA/LIF and interleukin-6 . The two factors exhibit only 10-12 homology at the protein level and the respective actions of DIA/LIF on ES cells and IL-6 in immune regulation appear quite distinct, but they have similar effects in several other systems . Thus both cytokines induce differentiation of the myeloid M 1 cell line and are active in acute phase reactions, bone remodelling, haematopoietic stem cell proliferation and thrombopoiesis (for review of IL-6 see ref 79) . These shared activities are indicative of a convergence of signalling mechanisms which may be accounted for by the common involvement of gp 130 . A third cytokine, oncostatin M, which has limited homology to both DIA/LIF and interleukin-6, 80 has been reported to bind directly to gp130 and also to the high affinity DIA/LIF receptor complex . 77,81 It has been postulated that oncostatin M shares all the biological activities of DIA/LIF, 81 including inhibition of ES cell differentiation, but this has yet to be rigourously determined . The suggestion that there may be a family of cytokines capable of regulating ES cell self-renewal is particularly intriguing in view of the increasing evidence, both from in vitro assays and gene knock-out experiments, for a considerable degree of functional redundancy amongst cytokines . Further characterisation of the structural and functional relationships between the DIA/LIF, oncostatin M and interleukin-6 receptors should pave the way for an analysis of signalling systems in stem cells . At present the mechanism of signal transduction from this receptor family remains obscure . Eventually, however, this may prove a route to the identification of stem cell determinants i.e . the trans-acting factor(s) whose continuous expression is necessary and sufficient for maintenance of the stem cell phenotype . A more direct approach is to attempt to isolate DIA/LIF-responsive or stem cell-specific cDNA clones directly from ES cells . A complementary strategy is the identification of gene products which are specifically induced or repressed during the initial stages of ES cell differentiation,

A . G . Smith

either following withdrawal from DIA/LIF or on exposure to inducing agents such as retinoic acid 4'0 '82 or 3-methoxybenzamide . 48 The latter is completely effective even in the presence of DIA/LIF and rapidly produces an apparently uniform population of differentiated cells with a similar morphology to the predominant cell type obtained on withdrawal of DIA/LIF (Figure 2) . This suggests that 3-methoxybenzamide may block DIA/LIF signalling in ES cells and it will be of interest to determine the specificity of this effect in comparison with responses to DIA/LIF in other cell types . ES cells represent a unique resource for dissecting in vitro the cellular and molecular mechanisms which govern stem cell decisions . As such they will undoubtedly make a major contribution to the analysis of early mouse development, whilst the extent to which they reveal some principles and mechanisms of stem cell regulation should prove of general interest . Applications of ES cells in transgenesis and developmental genetics The capacity for germ-line colonisation means that ES cells can be exploited as vehicles for transgenic manipulation of the mouse genome, 83 either via the addition of new genetic information or via the alteration of host gene sequences (Figure 4) . The utility of ES cells as transgenic vectors is enhanced by the fact that the majority of ES cell lines have a male chromosome complement (40 XY) . The reason for this is poorly understood but is probably related to the presence of two active X chromosomes in female ES cells which is an unstable and presumably unfavourable combination . 84 However, as a result ES cells pass through the male germ-line with its greater reproductive capacity . Moreover, the introduction of XY ES cells into XX host blastocysts can result in a distortion in the sex ratio of chimaeric offspring such that there are more males than females . This phenomenon of sex conversion arises because if XY cells become the dominant population in the indifferent gonad they induce testis formation and subsequent transformation of the nascent reproductive tract from female to male .83 XX cells appear to be incapable of forming functional sperm . Consequently, any gametes produced will be derived from the ES cells and, if fertile, these animals exhibit 100% transmission of the ES genotype . A major and increasing use of ES cells is in the production of mice carrying predetermined genetic

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Genetic manipulation I . Introduce marker genes 2 . Gain of function mutant 3 . Loss of function mutant 4. Altered function mutant

T ES cells

Figure 4 . Transgenic applications of embryonic stem cells . modifications generated via homologous recombination . Gene targeting strategies have been reviewed extensively elsewhere 85-87 so will not be covered in detail here . Briefly, the technology is based on the observation that recombination can occur between plasmid DNA and homologous chromosomal sequences ." In mammalian cells this event is generally very rare compared with illegitimate recombination at random sites in the genome . Therefore a cell culture system is required in which the rare homologous integrations can be isolated . This is provided by the ES cells, which can then convey the modified gene into the mouse germline . The ability to introduce defined mutations into mice is having a revolutionary impact on many areas of mammalian biology, including developmental genetics . Although the long generation time of mammals and the complications posed by in utero development preclude the mouse becoming as tractable an organism as Caenorhabditis or Drosophila for the genetic dissection of embryogenesis, gene targeting does afford new possibilities for the investigation of developmental mechanisms in

mammals . This is illustrated, for example, by the recent definitive demonstrations of the requirement for homeobox genes in mammalian embryogenesis .89-91 To date applications of gene targeting have focused on the production of null alleles, but attention is increasingly moving towards strategies for introducing more subtle mutations into genes 86,92,93 in order to explore structure-function relationships . The transgenic applications of ES cells are not restricted to gene targeting procedures . A potentially very powerful use of ES cells is as a means of identifying novel developmentally regulated genes via transfection with enhancer or gene trap vectors . 77,94,95 The latter are constructs in which expression of a reporter gene, typically lac Z, is respectively dependent on integration either in the vicinity of an active enhancer or into an active transcription unit . Screening for temporal and spatial expression patterns of interest is achieved by histochemical staining of whole embryos for the reporter gene product (/(3-galactosidase) . Enhancer traps have been successfully employed, both in ES cells 77 and in conventional transgenics produced via pronuclear injection, 96,97 to visualise regulated patterns of expression . However, the distances over which mammalian enhancers operate mean that it is not trivial to exploit such integrations to isolate the relevant enhancer or an endogenous responsive gene . By contrast, gene trap vectors are only functional following integration directly into a host gene which gives them two advantages . Firstly, they are mutagenic and therefore have the potential to generate an informative phenotype in addition to an expression pattern . Secondly, production of a fusion transcript facilitates isolation of the host gene by PCR cloning of the disrupted cDNA . The disadvantage of gene trap constructs is that the vast majority of integrations are non-productive . Therefore pre-sceening is required and as with gene targeting this is provided by the ES cell system . ES cell transfectants expressing 13-galactosidase can easily be identified and isolated . These are used for blastocyst injections and patterns of j3galactosidase staining determined either in chimaeras or, following germ-line transmission, in heterozygotes and homozygotes which may also yield phenotypic information . From the limited number of gene traps so far analysed, it appears that the expression pattern of lac Z reflects quite accurately that of the endogenous gene . 98 Although labourintensive, with improvements in vector design (ref 95, W . Skarnes, personal communication)

3 94 entrapment strategies seem likely to prove a highly effective means of exploring the genetic basis of murine development and of identifying important regulatory elements and genes . Interestingly, unique lac Z expression patterns produced by entrapment constructs are readily distinguishable in chimaeras. This confirms that ES cells do not preferentially colonise particular tissues but that chimaerism is relatively consistently and evenly distributed regardless of the quantitative variability in overall ES cell contribution . The intermingling of ES cells with host embryo cells and their contribution to all tissues creates new possibilities for mosaic analyses of mammalian development . For example, gene products which induce dominant embryonic lethality can be systemically analysed in ES cell chimaeras with the aim of revealing specific effects on early development . To date this approach has seen only limited application to viral oncogenes, 99,1 °° but it should prove more fruitful as a means of exploring the functions of signalling molecules such as DIA/LIF which have been directly implicated in developmental control processes . Such `gain of function' studies are potentially a valuable complement to the production of `loss of function' mutants via gene targeting (Figure 4) . Chimaeric analysis can also be employed in conjunction with homologous recombination to investigate the phenotypic consequences of cellular null mutations . The latter are obtained either via targeting of an X-linked gene or via inactivation of both copies of an autosomal gene .'°1-'°3 Chimaeras produced from such null ES cells can reveal specific defects which are not apparent from breeding null animals, either because of early and/or complex lethality in the latter or because the effect is only seen in competition with wild-type cells . For example, chimaeras made with ES cells bearing a targeted disruption of the erythroid-specific transcription factor GATA-1 exhibit a specific exclusion of ESderived progeny from the erythroid lineage . 104 This strategy is likely to be particularly informative for gene products which play essential roles at different stages of development . The utility of chimaeric analysis is not confined to transgenic manipulations . For instance, the phenomenon of non-equivalence of maternal and paternal genomes, or genomic imprinting, 105 is being investigated using androgenetic ES cell lines . 106 These originate from eggs in which the maternal pronucleus has been replaced by a paternal

A . G . Smith

pronucleus and they therefore contain only paternally derived chromosomes . Production of chimaeras has established that the androgenetic cells are capable of appropriate differentiation into a range of foetal tissue types . However, the great majority of the chimaeras exhibit severe skeletal abnormalities indicating that the cell lines retain at least some aspects of paternal imprinting and pointing to a possible key role for an imprinted gene(s) in chondrogenesis . The resource of permanent cell lines should facilitate detailed phenotypic characterisation and identification of the underlying cellular defects . In a similar manner, ES cells can provide an invaluable route for the mosaic analysis of classical mouse developmental mutants. Thus cell lines have been derived which are homozygous for the embryonic lethal Brachyury (T) mutation .98 A high proportion of chimaeras exhibit specific defects in the neural tube, posterior mesoderm and allantois . This precise reproduction of the Brachyury phenotype establishes the cell autonomous nature of T function (ref 98, R . Beddington, personal communication) . Further analysis of the distribution of the mutant cells should lead to an increased understanding of the mechanistic basis of the disruption to mesoderm development consequent on loss of T function . The strength of this general approach of chimaeric analysis is that combination and competition with normal embryo cells can reveal information about the nature of mutant phenotypes which may not be apparent in homozygous embryos . Limitations of the ES cell system and future prospects The versatility of ES cells is resulting in their increasing exploitation for both in vitro and in vivo studies in many areas of mammalian biology . There are limitations, however, to our current abilities to manipulate ES cells and apply them as analytical tools . Foremost amongst these is the present inability to establish ES lines from species other than the mouse and Chinese hamster . 107 There is considerable academic, industrial and commercial interest in the isolation of ES cells from other animals, particularly rats and agricultural livestock . However, it is often overlooked that even in mice the ability to derive a stem cell line is highly strain-dependent . E S cells are most readily established from strain 129 mice in which reproducibly 20-30% of embryos can give rise to cell lines . Lines have been obtained from some

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other inbred and outbred strains and from F 1 embryos28,1 o8,109 but usually at much lower frequency . To the author's knowledge, only the 129 lines consistently pass through the germ-line . The reason(s) for this strain variation is obscure . It may reflect some subtle difference in the timing of developmental

of germ-line chimaeras, although against this should be weighed the poor fertility often exhibited by inbred mice . An alternative strategy is to employ host embryos whose ability to contribute to foetal tissues has been compromised so as to give a competitive advantage to the ES cells . The most extreme

restrictions or in adaptation to culture, in which case minor modifications of existing protocols should

demonstration of this approach is provided by the

enable isolation of stem cells from any mouse strain . On the other hand, a specific genetic component could be essential for the establishment of an ES cell line . This could be a mutation, for example in a

use of tetraploid recipients which have a very limited capacity to participate in foetal development . 113 Introduction of ES cells into such embryos results in foetuses which are essentially entirely ES cell-

proto-oncogene or anti-oncogene, which is borne by those strains which do produce cell lines . Alternatively, it could arise from strain-specific imprinting at a

derived . These embryos develop to term with no overt abnormalities . This indicates that only relatively short-term interactions are required to `normalise' ES cells within the developing embryo

particular locus . It may be significant that the effects of paternal imprinting appear to be less extreme for foetal development in strain 129 compared with other mouse strains . 110 In either case, ES cell technology

and that they can then generate the entire programme of embryogenesis autonomously . Unfortunately, however, to date these embryos have all died post-natally so it remains to be determined whether

is unlikely to be extended to other mice, let alone other species, using current protocols until the relevant gene(s) have been identified . One option

this will prove to be a useful method for ensuring germ-line colonisation . Full exploitation of the potential of ES cells for

which may be worthy of investigation would be to return to the derivation of lines from embryoderived teratocarcinomas and determine whether the

chimaeric analyses of developmental processes demands detailed knowledge of the distribution of the ES cell progeny . This requires the generation

superior conditions developed for ES cell maintenance might allow the isolation and propagation of `normal' EC cells . A second limitation of the ES cell system is the efficiency of germ-line transmission . Notwithstanding

of ES cells bearing a marker which will enable in situ localisation . Only limited success has been achieved with attempts to introduce a lac Z gene into ES cells

the advantages of an XY karyotype, the goal of reproducible germ-line transmission has proved elusive for many researchers . A major contributory factor is that the karyotypic stability of ES cells is only relative and it has not always been appreciated how sensitive the cells are to environmental perturbation and in particular to any change in the culture regimen . Recent successes with gene targeting indicate that the technology of ES cell manipulation is now becoming more widespread, though protocols for maintenance of germ-line competent ES cells vary from laboratory to laboratory and optimal conditions have yet to be rigourously defined . 48,111 A second factor which influences the frequency of germ-line transmission is the strain of mouse used to provide host blastocysts . Whilst chimaeras can readily be produced in many inbred and outbred strains, the relative contribution of the ES cells varies considerably and colonisation of the germ-line appears to be particularly dependent on the host . 112 Certain inbred strains, notably C57131/6 and Balb-c, have been found to give a consistently high proportion

under the control of various house-keeping promoters (ref 114 ; R . Beddington, personal communication) . The marker gene must be expressed constitutively and ubiquitously to be generally useful . In practice, however, the majority of integrations of such constructs seem to give widespread expression patterns in early development which become more restricted during organogenesis . Modification of the expression constructs, for example via the inclusion of additional regulatory sequences, may overcome this problem or it may be more effective to integrate the lac Z gene into a chromosomal house-keeping locus via homologous recombination or serendipitously with an entrapment vector . Alternatively, a repeated DNA sequence could be employed as a marker which could be visualised by in situ DNA hybridisation without any requirement for expression . The principal attribute of ES cells as transgenic system is that they enable pre-selection for desired events . Their utility for genetic manipulation would be greatly enhanced, however, if it were possible to isolate and maintain haploid cell lines . Unfortunately, although ES cells can be derived from haploid parthenogenetic embryos, they undergo

3 96 diploidization at a very early stage during establishment in culture . 108 One future application for which ES cells may prove invaluable is in attempts to introduce very large pieces of DNA into mice via yeast or mammalian artificial chromosomes . There is currently growing interest in this area as a means for the co-ordinate transfer of several genes and for the generation of transgenics in which the desired specificity and level of expression can be assured due to the inclusion of a battery of structural and regulatory elements . The transfer and maintenance of intact artificial chromosome sequences is likely to be a rare occurrence, however . In this case the ES cell route will be more appropriate than pronuclear injection . E S cells may also prove practically more amenable to techniques for the introduction of megabase DNA fragments . The great unrealised potential of ES cells is in dissection of the process by which a puripotential stem cell either self-renews or becomes committed to a particular lineage and a specific differentiation programme . The power of in vitro approaches is exemplified by the advances in understanding of haemopoietic regulatory factors which have flowed from the availability of a variety of culture systems . 115,116 The haemopoietic stem cell itself remains elusive, however (see article by Graham and Pragnell, pp 423-434, this issue) . The unique advantage of ES cells is that they are a pure population of normal stem cells which can be propagated indefinitely . This provides an experimental system both for the identification and characterisation of cytokines such as DIA/LIF and for the analyses of signalling mechanisms and transcriptional regulation in stem cells . There are limitations, however, to the use of ES cells for studying lineagespecification and cellular differentiation . These relate partly to the paucity of markers for establishing the identity of early embryonic cell types and partly to the complexity of differentiation in embryoid bodies . In monolayer culture it is possible to obtain relatively homogeneous initial differentiation of ES cells 48 (Figure 2), but the relationship between the differentiated progeny and normal embryonic cell types has not been determined at present . Following aggregation and the formation of embryoid bodies, mature differentiated cell types which are readily identifiable, such as beating muscle, red blood cells and neurons, are generated . However, this process requires days or even weeks and occurs within fairly disorganised, multi-differentiated

A . G. Smith

structures . 30 The degree of differentiation observed in embryoid bodies also varies considerably and unpredictably with culture conditions and serum batch . Nonetheless, reproducible effects of particular cytokines have been observed on the quantitative differentiation obtained from embryoid bodies . For example, erythropoietin can significantly increase the proportion of red cells . 117 This is likely to be an effect on viability and/or maturation of the red cells or their immediate precursors . However, the fact that mature cell types from different lineages are obtained from embryoid bodies indicates that lineage-specific progenitor cells are present, if only transiently . The generation and amplification of such cells are key events in differentiation processes . The development of simplified culture regimens in which ES cell differentiation can be directed down particular routes would constitute a considerable experimental advance in providing a tractable system for the production, characterisation and manipulation of committed embryonic progenitor cells . Ultimately this may result in the establishment of lineage-specific stem cell lines directly from differentiating ES cultures and/or facilitate their isolation from primary tissue .

Conclusion Over the last 10 years ES cells have risen from a position of relative obscurity as a rather esoteric embryological phenomenon to become a major investigative tool for many aspects of pure and applied biology . They are having a transforming impact on genetic manipulation in mammals whilst their potential for unravelling stem cell control mechanisms has only just begun to be realised . The power of this system owes much in both practice and concept to those pioneers of teratocarcinoma biology 9,16,118 whose vision of a totipotent stem cell is finally being appreciated . Acknowledgements I would like to acknowledge the support and patient understanding of my wife Angela . I am indebted to many colleagues in Edinburgh, Oxford and Adelaide for their encouragement, advice and sharing of ideas . I am particularly grateful to Rosa Beddington for her critical comments on the manuscript and for generously providing Figures 1 and 4 . Photographic reproduction was carried out by Frank Johnston and Graeme Brown .

Embryonic stem cells

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