Pluripotential Stem Cells from Vertebrate Embryos: Present Perspective and Future
Challenges
Richard L. Gardner
INTRODUCTION
Many have contributed to the various developments that brought recognition of the enormous potential of cells of early embryonic origin for genetic modification of organ-isms, regenerative medicine, and in enabling investiga-tion of facets of development that are difficult to explore in vivo. However, historically, this field is firmly rooted in the pioneering work of Roy Stevens and Barry Pierce on mouse teratomas and teratocarcinomas, tumors which continued for some time after these workers had embarked on their studies to be regarded with disdain by many main-stream pathologists and oncologists. While Stevens devel-oped and exploited mouse strains with high incidences of such tumors to determine their cellular origins, Pierce focused his attention on the nature of the cell that endowed teratocarcinomas with the potential for indefinite growth which the more common teratomas lacked. Conversion of solid teratocarcinomas to an ascites form proved a signifi-cant advance in enabling dramatic enrichment of the mor-phologically undifferentiated cells in such tumors which their stem cells were expected to be included among. Then, in an experiment of heroic proportions, Kleinsmith and Pierce showed unequivocally that, on transplantation to histocompatible adult hosts, individual morphologically undifferentiated cells could form self-sustaining teratocar-cinomas that contained as rich a variety of differentiated tissues as the parent tumor. Hence, the embryonal carci-noma EC cell, as the stem cell of teratocarcinomas has come to be known, was the first self-perpetuating pluripo-tential cell to be characterized. Though teratocarcinomas were obtained initially as a result of genetically-determined aberrations in the differentiation of male or female germ cells, it was found later that they could also be established in certain genotypes of mice by grafting early embryos
Stem Cell Anthology
Copyright . 2009, Elsevier Inc. All rights of reproduction in any form reserved.
ectopically in adults. Adaptation of culture conditions to enable EC cells to be perpetuated in an undifferentiated state or induced to differentiate in vitro soon followed. Although the range of differentiation detected in these cir-cumstances was more limited than in vivo, it could never-theless be quite impressive. Research on murine EC cells, in turn, provided the impetus for obtaining and harnessing the human counterpart of these cells from testicular tumors for in vitro study.
One outstanding question regarding the use of murine EC cells as a model system for studying aspects of develop-ment remained, namely the basis of their malignancy. Was this a consequence of genetic change or simply because such “embryonic” cells failed to relate to the ectopic sites into which they were transplanted? The obvious way of addressing this was to ask how EC cells behave when placed in an embryonic rather than an adult environment. This was done in three different laboratories by inject-ing the cells into blastocyst stage embryos. The results from each laboratory led to the same rather striking con-clusion. EC cells, which if injected into an adult would grow progressively and kill it, were able to participate in entirely normal development following their introduc-tion into the blastocyst. Using genetic differences between donor and host as cell markers, EC cells were found to be able to contribute to most if not all organs and tissue of the resulting offspring. Most intriguingly, according to reports from one laboratory, this could very exception-ally include the germline. The potential significance of this finding was considerable in terms of its implications for possible controlled genetic manipulation of the mam-malian genome. This is because it raised the prospect of being able to select for very rare events, and thus bring the scope for genetic manipulation in mammals closer to that in microorganisms.
PART | I An Introduction to Stem Cell Biology
There were problems, however. One was that the EC contribution in chimeric offspring was typically both more modest and more patchy than that of cells transplanted directly between blastocysts. The chimeras also not infre-quently formed tumors, with those that proved to be terato-carcinomas often being evident already at birth. Therefore, it seems likely that regulation of growth of at least some of the transplanted EC cells failed altogether. Other chi-meras developed more specific tumors such as rhabdomy-osarcomas as they aged which were also clearly of donor origin, thereby revealing that the transplanted EC cells had progressed further along various lineages before their dif-ferentiation went awry. In extreme cases the transplanted EC cells disrupted development altogether so that fetuses did not survive to birth. Although the best EC lines could give very widespread contributions throughout the body of chimeras, they did so only very exceptionally. Finally, the frequency with which colonization of the germ line could be obtained with EC cells was too low to enable them to be harnessed for genetic modification. It seemed likely, therefore, that the protracted process of generating teratocarcinomas in vivo and then adapting them to cul-ture militated against retention of a normal genetic consti-tution by their stem cells. If this was indeed the case, the obvious way forward was to see if such stem cells could be obtained in a less circuitous manner. This prompted investigation of what happens when murine blastocysts are explanted directly on growth-inactivated feeder cells in an enriched culture medium. The result was the derivation of lines of cells that were indistinguishable from EC cells in both morphology and expression of various antigenic and other markers, as well as in the appearance of the colonies they formed during growth. Moreover, like EC cells, these self-perpetuating blastocyst-derived stem cells could form aggressive teratocarcinomas in both syngeneic and immu-nologically compromised non-syngeneic adult hosts. They differed from EC cells principally in giving much more frequent and widespread somatic chimerism following reintroduction into the pre-implantation conceptus and, if tended carefully, also in routinely colonizing the germline. Moreover, when combined with host conceptuses whose development was compromised by tetraploidy, they could sometimes form offspring in which no host-derived cells were discernible. Thus, these cells, which exhibited all the desirable characteristics of EC cells and few of their shortcomings, came to be called embryonic stem ES cells. Once it had been shown that ES cells could retain their ability to colonize the germline after in vitro transfec-tion and selection, their future was assured. Surprisingly, however, despite the wealth of studies demonstrating their capacity for differentiation in vitro, particularly in the mouse, the idea of harnessing ES cells for therapeu-tic purposes took a long time to take root. Thus, although Robert Edwards explicitly argued the case that human ES cells might be used thus more than 25 years ago, it is only within the past decade that this notion has gained momen-tum, encouraged particularly by derivation of the first cell lines from human blastocysts.
TERMINOLOGY
There is some confusion in the literature about terminol-ogy in discussing the range of different types of cells that ES cells are able to form, an attribute that, in embryologi-cal parlance, is termed their potency. Some refer to these cells as being totipotent in recognition of the fact that, at least in the mouse, they have been shown to be able to give rise to all types of fetal cells and, under certain con-ditions, entire offspring. However, this is inappropriate on two counts. First, totipotency is reserved by embryologists for cells that retain the capacity to form an entire concep-tus, and thus give rise to a new individual, unaided. Apart from the fertilized egg, the only cells that have so far been shown to be able to do this are blastomeres from early cleavage stages. Second, murine ES cells seem unable to form all the different types of cell of which the concep-tus is composed. Following injection into blastocysts, they normally give rise only to cell types that are products of the epiblast or fetal precursor cell lineage. While they can also form derivatives of the primitive endoderm lineage which, for some obscure reason they do much more read-ily in vitro than in vivo, they have never been convincingly shown to contribute to the trophectodermal lineage. Hence, a widely adopted convention is to describe ES cells as pluripotent stem cells, to distinguish them from stem cells like those of the hematopoietic system which have a nar-rower, but nevertheless impressive, range of differentiative potential. A further source of confusion is the surprisingly common practice of referring to cells, particularly putative ES cells from mammals other than the mouse, as totipo-tent because their nuclei have been shown to be able to support development to term when used for reproductive cloning.
Another facet of terminology relates to the definition of an ES cell, which again is not employed in a consist-ent manner. One view, to which the author subscribes, is that use of this term should be restricted to pluripotent cells derived from pre- or peri-implantation conceptuses that can form functional gametes, as well as the full range of somatic cells of offspring. While there are consider-able differences between strains of mice in the facility with which morphologically undifferentiated cell lines can be obtained from their early conceptuses, compe-tence to colonize the germline as well as somatic tissues seems nevertheless to be common to lines from all strains that have yielded them. This is true, for example, even for the non-obese diabetic NOD strain whose lines have so far been found to grow too poorly to enable their genetic modification.
CHAPTER | 1 Pluripotential Stem Cells from Vertebrate Embryos
ES-LIKE CELLS IN OTHER SPECIES
As shown in Table 1-1, cell lines that can be maintained for variable periods in vitro in a morphologically undif-ferentiated state have been obtained from morulae or blas-tocysts of a variety of species of mammals in addition to the mouse. They have also been obtained from the stage X blastoderms in the chick, and from blastulae in several different species of teleost fish. The criteria that have been employed to support claims that such lines are counterparts of murine ES cells are quite varied and, not infrequently, far from unequivocal. They range from maintenance of an undifferentiated morphology during propagation or expres-sion of at least some ES cell markers, through differentia-tion into a variety of cell types in vitro, to production of histologically diverse teratomas or chimerism in vivo.
What such ES-like ESL cells lines have in common with murine ES cells, in addition to a morphologically undif-ferentiated appearance and expression of various genes asso-ciated with pluripotency, is a high nuclearcytoplasmic ratio. Among the complications in assessing cell lines in different species is variability in morphology of the growing colonies. While colonies of ESL cells in the hamster and rabbit are very similar to those of murine ES cells, those of most other mammals are not. This is particularly true in the human whose undifferentiated ESL cell colonies closely resemble those formed by human EC cells of testicular origin, as also are those of ESL cells from other primates. In the marmo-set, rhesus monkey, and human, ESL cells not only form relatively flattened colonies, but also exhibit a number of dif-ferences from mouse ES cells in the markers they express. Since they closely resemble human EC cells in all these respects, the differences were assumed until recently to relate to species rather than cell type.
In two studies in the sheep, colonies are reported to look like those formed by murine ES cells initially, but then to adopt a more epithelial-like appearance rapidly thereafter. This change in morphology bears an intrigu-ing similarity to the transition in conditioned medium of murine ES to so-called epiblast-like EPL cells which is accompanied by loss of their ability to colonize the blas-tocyst. Given that this transition is said to be completely reversible, whether a comparable one is occurring sponta-neously in sheep clearly warrants further investigation.
In no species has production of chimeras with ESL cells rivalled that obtained with murine ES cells. Where it has been attempted, both the rates and levels of chimerism are typically much lower than those found with murine ES cells. An apparent exception is one report for the pig, in which 72% of offspring were judged to be chimeric. However, this figure is presented in an overview of work that remains unpublished, and no details are provided regarding the number of times the donor cells had been passaged before being injected into blastocysts. In a subsequent study in this species using ES-like cells that had been through 11 passages, one chimera was recorded among 34 offspring. However, as the authors of this latter study point out, rates of chimerism of only 10?12% have been obtained follow-ing direct transfer of ICM cells in the pig. Hence, techni-cal limitations may have contributed to the low success with ES-like cells in this species.
The only species listed in Table 1-1 in which coloniza-tion of the germline has been demonstrated are the chicken and the zebra fish, but in both cases this was with cells that had been passaged only 1?3 times before being injected into host embryos. Stem cells from early chick embryos that have been passaged for longer can give strong somatic chimerism, but have not yet been shown to be able to yield gametes. Consequently, in conformity with the terminol-ogy discussed earlier, morphologically undifferentiated cell lines in all species listed in Table 1-1 except the mouse should be assigned the status of ES-like ESL cells rather than ES cells.
Generally, the strategy for attempting to derive ES cell lines in other species has been initially to follow more or less closely the conditions that proved successful in the mouse, namely the use of enriched medium in conjunction with growth-inactivated feeder cells and either leukemia inhibitory factor LIF or a related cytokine. Various modi-fications that have been introduced subsequently include same-species rather than murine feeder cells and, in a number of species including the human, dispensing with LIF. Optimal conditions for deriving cell lines may differ from those for maintaining them. Thus, in one study in the pig use of same-species feeder cells was found to be neces-sary to obtain cell lines, although murine STO cells were adequate for securing their propagation thereafter. Feeder-free conditions were found to work best in the case of both the medaka and the gilthead sea bream. Moreover, the clon-ing efficiency of human ESL lines was improved in serum-free culture conditions.
Unexpectedly, despite being closely related to the mouse, the rat has proved particularly refractory to deriva-tion of ES cell lines see Table 1-1. So far, the only cell lines that have proved to be sustainable in longer term in this species seem to lack all properties of mouse ES cells apart from colony morphology. Indeed, except for the 129 strain of mouse, establishing cells lines that can be propa-gated in vitro in a morphologically undifferentiated state seems almost more difficult in rodents than in most of the other vertebrates in which it has been attempted.
Overall, one is struck by species variability in the growth factors, status of conceptus or embryo, and other require-ments for obtaining pluripotential cell lines in species other than the mouse. So far, one can discern no clear recipe for success. Of course, obtaining cells that retain the capac-ity to colonize the germline following long-term culture is essential only for the purpose of genetically-modifying animals in a controlled manner. Having cells that fall short of this but are nevertheless able to differentiate into a range of distinct types of cells in vitro may suffice for many other purposes, including regenerative medicine.
Recent Findings on Mouse Epiblast Cells
Recent findings in the mouse which may help to explain the differing experiences in other species emerged from attempts to derive ES cells from the epiblast of early post-implantation conceptuses. Stem cells exhibiting pluripo-tency could be obtained thus, but these clearly differed from true ES cells from pre-implantation conceptuses in conditions for their derivation and maintenance, their colony morphology, and also in how their differentiation was induced. Most interestingly, they not only resembled human ESL cells in these respects, but also almost entirely lacked the ability to yield chimerism following introduction
PART | I An Introduction to Stem Cell Biology
into pre-implantation conceptuses. This raises the intrigu-ing possibility that the mouse is peculiar in being per-missive for derivation stem cells at an earlier stage in the epiblast lineage than other species. These novel pluripo-tential mouse cell lines have been termed “epiblast stem cells” or EpiSc.
EMBRYONIC GERM CELLS
The pre-implantation conceptus is not the only source of pluripotential stem cells in the mouse. Sustainable cultures of undifferentiated cells that resemble ES cells strikingly in their colony morphology have also been obtained from primordial germ cells and very early gonocytes in this spe-cies. These cells, termed embryonic germ EG cells, have also been shown to be capable of yielding high rates of both somatic and germline chimerism following injection into blastocysts.
The above findings have prompted those struggling to derive ES cell lines in other species to explore primordial germ cells as an alternative for achieving controlled genetic modification of the germ line. As shown in Table 1-2, EG-like EGL cells have been obtained in several mammals as well as the chick, but as with ES-like cells, their ability to participate in chimera-formation has, with one exception, only been demonstrated at low passage. Moreover, while donor cells have been detected in the gonad of a chimera obtained from low passage EG-like cells in the pig, no case of germline colonization has been reported except with cells from chick genital ridges that were cultured for only five days. Even here, the proportion of offspring of donor type was very low.
CHAPTER | 1 Pluripotential Stem Cells from Vertebrate Embryos
t is, however, noteworthy that even in the mouse rates of malformation and perinatal mortality appear to be higher in EG than in ES cell chimeras. This may relate to erasure of imprinting in the germ line which seems to have begun by the time primordial germ cells have colonized the genital ridges or, for certain genes, even earlier. It is perhaps because of such concerns that the potential of EG cells for transgenesis in strains of mice that have failed to yield ES cells has not been fully explored. Interestingly, unlike in the mouse, EGL cell lines derived from genital ridges and the associated mesentary of 5?11 week human fetuses seem not to have embarked on erasure of imprint-ing. Obviously, it is important to confirm that this is the case before contemplating use of such cells as grafts for repairing tissue damage in humans.
FUTURE CHALLENGES
The value of ES and ESL cells as resources for both basic and applied research is now acknowledged almost univer-sally. Present barriers to exploitation of their full potential in both areas are considered below, together with possi-ble ways of addressing these. Fundamental to progress is gaining a better understanding of the nature and the basic biology of these cells.
BIOLOGY OF ES AND ES-LIKE CELLS
Germline Competence
Although murine ES cells have been used very extensively for modifying the genome, there are still a number of problems that limit their usefulness in this respect. Among these is loss of competence to colonize the germline, a common and frustrating problem whose basis remains elusive. It is not attributable simply to the occurrence of sufficient chromosomal change to disrupt gametogenesis, because it can occur in lines and clones that are found to be karyotypically normal. At present, it is not known whether it is due to failure of the cells to be included in the pool of primordial germ cells, or their inability to undergo appropriate differentiation thereafter, possibly as a conse-quence of perturbation of the establishment of genomic imprinting or its erasure. Even within cloned ES lines, cells have been found to be heterogeneous with respect to expression of imprinted genes. Given that many ES cell lines are likely to have originated polyclonally from sev-eral epiblast founder cells, there is the further possibility that they might, ab initio, consist of a mixture of germline-competent and non-competent sub-populations. Recent studies on involvement of BMP signaling in the induction of primordial germ cells have been interpreted as evidence against a specific germ cell lineage in mammals. Particular significance has been attached to experiments in which distal epiblast, which does not usually produce primor-dial germ cells, was found to do so when grafted to the proximal site from whence these cells normally originate. However, because of the extraordinary degree of cell mix-ing that occurs in the epiblast before gastrulation, descend-ants of all epiblast founder cells are likely to be present throughout the tissue by the time of primordial germ cell induction. Hence, yet to be excluded is the possibility that competence for induction is lineage-dependent and thereby segregates only to some epiblast founders cells. Because ES cell lines are typically produced by pooling all colonies derived from a single blastocyst, they might therefore orig-inate from of a mixture of germline-competent and non-competent epiblast founder cells.
Male ES cell lines have almost invariably been used in gene targeting studies, even though this complicates work on X-linked genes whose inactivation may lead to cell-autonomous early lethality or compromised viability in the hemizygous state. Here, female XX lines would, in prin-ciple, offer a simpler alternative, except they are gener-ally held to suffer partial deletion or complete loss of one X-chromosome after relatively few passages. It is, however, not clear how secure this conclusion is, because few refer-ences to their use have appeared in literature since the early reports in which consistent loss of all or part of one X was first documented. More recently, one of only two female lines tested was found to be germline-competent, but the entirely donor-derived litters were unusually small, raising the possibility not entertained by the authors that the line in question was XO. Interestingly, human ESL cell lines seem not to show a similar propensity for X-chromosome loss and, indeed, nor do murine EpiSc.
Origin and Properties of ES and ES-like Cells
It is evident from the earlier overview that there is con-siderable diversity even among eutherian mammals in the characteristics of cells from early conceptuses that can be perpetuated in vitro in a morphologically undifferen-tiated state. The reason for this is far from clear, particu-larly since the great majority of such cell lines have been derived at a corresponding stage, namely the pre-implanta-tion blastocyst, often using inner cell mass tissue isolated from there. In the mouse, in contrast to their EC counter-parts, ES cells have not been obtained from post-implanta-tion stages, arguing that there is a rather narrow window during which their derivation is possible. What this relates to in developmental terms remains obscure, although the finding that ES cells can shift reversibly to a condition that shows altered colony morphology and gene expression, in conjunction with loss of ability to generate chimeras fol-lowing blastocyst injection, offers a possible approach for addressing this problem. Whether the late blastocyst stage sets the limit for obtaining ESL cell lines has not yet been addressed critically in other mammals. However, the pos-sibility that ESL cells in other mammals correspond to murine EpiSc clearly warrants further consideration.
Just as ES cell lines have been obtained from pre-blastocyst stages in the mouse, so have ESL cell lines from other mammals. However, in the case of the mouse, ES cell lines derived from early cleavage stages and moru-lae have not been found to differ in properties or devel-opmental potential from those obtained from blastocysts, implying that all originate at the same very early stage in differentiation of the epiblast lineage. Thus claims that lines isolated from morulae have an advantage over those from blastocysts in being able to produce trophoblast have not been substantiated. However, species- as opposed to stage-related differences in the ability of cell lines to produce trophoblast tissue have been encountered. Early claims that mouse ES cells can form trophoblastic giant cells are almost certainly attributable to short-term persistence of contaminating polar trophectoderm tissue. Thus, produc-tion of such cells seems to be limited to the early passage of ES lines derived from entire blastocysts. It has never been observed with lines established from microsurgically isolated epiblasts. While the situation is not clear in many other species, in primates differentiation of trophoblast has been observed routinely in ESL cell lines estab-lished from immunologically-isolated inner cell masses. Moreover, differentiation of human cell lines to the stage of syncytiotrophoblast-formation has been induced efficiently by exposing them to BMP4.
Pluripotency
A seminal characteristic of ES or ESL cells is their pluripo-tency. The most critical test of this, which is not practica-ble in some species, particularly the human, is the ability to form the entire complement of cells of normal offspring. This assay, which was originally developed in the mouse, entails introducing clusters of ES cells into conceptuses whose development has been compromised by mak-ing them tetraploid, either by suppressing cytokinesis or through fusing sister blastomeres electrically at the two-cell stage. ES cells are then either aggregated with the tetraploid cleavage stages or injected into tetraploid blastocysts. Some of the resulting offspring contain no discernible host cells. It seems most likely that host epiblast cells are present ini-tially and play an essential role in “entraining” the donor ES cells before being outcompeted, since groups of ES cells on their own cannot substitute for the epiblast or inner cell mass author’s unpublished observations. Selection against tetraploid cells is already evident by the late blasto-cyst stage in chimeras made between diploid and tetraploid morulae. Aggregating ESL cells between pairs of tetraploid morulae has been tried in cattle, but resulted in their con-tributing only very modestly to fetuses and neonates.
PART | I An Introduction to Stem Cell Biology
The second most critical test is whether the cells yield widespread, if not ubiquitous, chimerism in offspring following introduction into the early conceptus, either by injection into standard blastocysts or aggregation with morulae. The third is the formation of teratomas in ectopic grafts to histocompatible or immunosuppressed adult hosts, since it is clear from earlier experience with murine and human EC cells that a wider range of differentiation can be obtained in these circumstances than in vitro. For such an assay to be incisive it is necessary to use clonal cell lines and thus ensure that the diversity of differentiation obtained originates from one type of stem cell rather than a medley of cells with more limited developmental potential. While teratoma formation has been demonstrated with clonal ESL in the human, this is not true for corresponding cell lines in other species. A note of caution regarding the use of teratomas for assessing pluripotency comes from work-ers who found that hepatocyte differentiation depended not only on site of inoculation of mouse ES cells, but also the status of the host. Thus, positive results were obtained with spleen rather than hind-limb grafts, and only when using immmunologically-compromised nude rather than syn-geneic mice as hosts.
Conditions of Culture
ES and ESL cells are usually propagated in complex culture conditions that are poorly defined, by virtue of including both growth-inactivated feeder cells and serum. This com-plicates the task of determining the growth factor and other requirements necessary for their maintenance, as well as for inducing them to form specific types of differentiated cells. While differentiation of murine ES cells in a chemically-defined medium has been achieved, their maintenance under such conditions has not. Murine ES cells can be both derived and maintained independently of feeder cells providing a cytokine that signals via the gp 130 receptor is present in the medium. However, whether the relatively high incidence of early aneuploidy recorded in the two stud-ies in which LIF was used throughout in place of feeders is significant or coincidental is not clear. It is important to resolve this in order to learn whether feeder cells serve any other function than acting as a source of LIF or a related cytokine. Production of extracellular matrix is one possibil-ity. However, species and cell-type variability is also a fac-tor here since LIF is not required for maintaining human ESL lines, whose cloning efficiency is actually improved by omission of serum, although feeder cells are. The norm has been to use murine feeder cells both for obtaining and perpetuating ESL cell lines in other mammals, including the human. Recently, however, there has been a move to use feeders of human origin for human ESL cells. This is a nota-ble development since it would obviously not be acceptable to employ xenogeneic cells for growing human ESL cell
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