Essentials of Stem Cell Biology
Copyright © 2009 Elsevier Inc.
All right reserved. ISBN: 978-0-08-088497-4
Chapter One Pluripotential Stem Cells from Vertebrate Embryos: Present Perspective and Future Challenges
Richard L. Gardner
Many have contributed to the various developments that brought recognition of the enormous potential of cells of early embryonic origin for genetic modification of organisms, regenerative medicine, and in enabling investigation 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 mainstream pathologists and oncologists. While Stevens developed 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 significant advance in enabling dramatic enrichment of the morphologically 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 teratocarcinomas that contained as rich a variety of differentiated tissues as the parent tumor. Hence, the embryonal carcinoma (EC) cell, as the stem cell of teratocarcinomas has come to be known, was the first self-perpetuating pluripotential 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 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 circumstances was more limited than in vivo, it could nevertheless 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 development 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 injecting the cells into blastocyst stage embryos. The results from each laboratory led to the same rather striking conclusion. EC cells which if injected into an adult, would grow progressively and kill it, were able to participate in entirely normal development following their introduction 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 exceptionally include the germline. The potential significance of this finding was considerable in terms of its implications for possible controlled genetic manipulation of the mammalian 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.
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 infrequently formed tumors, with those that proved to be teratocarcinomas often being evident already at birth. Therefore, it seems likely that regulation of growth of atleast some of the transplanted EC cells failed altogether. Other chimeras developed more specific tumors such as rhabdomyosarcomas 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 differentiation 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 culture militated against retention of a normal genetic constitution 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 immunologically compromised non-syngeneic adult hosts. They differed from EC cells principally in giving much more frequent and widespread somatic chimerism following reintroduction into the preimplantation 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 transfection 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 therapeutic 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 momentum, encouraged particularly by derivation of the first cell lines from human blastocysts.
There is some confusion in the literature about terminology in discussing the range of different types of cells that ES cells are able to form, an attribute that, in embryological 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 conditions, 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 conceptus, 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 conceptus 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 readily 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 narrower, 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 totipotent 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 consistent 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 considerable differences between strains of mice in the facility with which morphologically undifferentiated cell lines can be obtained from their early conceptuses, competence 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
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 undifferentiated state have been obtained from morulae or blastocysts 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 expression of at least some ES cell markers, through differentiation 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 undifferentiated appearance and expression of various genes associated with pluripotency, is a high nuclear/cytoplasmic 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 marmoset, rhesus monkey, and human, ESL cells not only form relatively flattened colonies, but also exhibit a number of differences 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 intriguing 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 blastocyst. Given that this transition is said to be completely reversible, whether a comparable one is occurring spontaneously 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 following direct transfer of ICM cells in the pig. Hence, technical 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 colonization 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 terminology discussed earlier, morphologically undifferentiated cell lines in all species listed in Table 1-1 except the mouse
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 modifications 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 necessary 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 cloning 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 derivation 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 propagated 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 requirements 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 capacity 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 pluripotency 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 into pre-implantation conceptuses. This raises the intriguing possibility that the mouse is peculiar in being permissive for derivation stem cells at an earlier stage in the epiblast lineage than other species. These novel pluripotential mouse cell lines have been termed "epiblast stem cells" or EpiSc.
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