Essentials of Stem Cell Biology

Robert Lanza,John Gearhart,Brigid Hogan,Douglas Melton,Roger Pedersen,E. Donnall Thomas,James A. Thomson,Ian Wilmut

• Éditeur: Academic Press
• Année: 2009
• ISBN:
  9780080884974
• Édition: 2
• Sujet: Biowissenschaften
• Sous-sujet: Biologie

First developed as an accessible abridgement of the successful Handbook of Stem Cells, Essentials of Stem Cell Biology serves the needs of the evolving population of scientists, researchers, practitioners and students that are embracing the latest advances in stem cells. Representing the combined effort of seven editors and more than 200 scholars and scientists whose pioneering work has defined our understanding of stem cells, this book combines the prerequisites for a general understanding of adult and embryonic stem cells with a presentation by the world's experts of the latest research information about specific organ systems. From basic biology/mechanisms, early development, ectoderm, mesoderm, endoderm, methods to application of stem cells to specific human diseases, regulation and ethics, and patient perspectives, no topic in the field of stem cells is left uncovered.

• Selected for inclusion in Doody's Core Titles 2013, an essential collection development tool for health sciences libraries
• Contributions by Nobel Laureates and leading international investigators
• Includes two entirely new chapters devoted exclusively to induced pluripotent stem (iPS) cells written by the scientists who made the breakthrough
• Edited by a world-renowned author and researcher to present a complete story of stem cells in research, in application, and as the subject of political debate
• Presented in full color with glossary, highlighted terms, and bibliographic entries replacing references

Part I

Introduction to Stem Cells

Chapter 1 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 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.

Terminology

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 modification.

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.

Table 1-1 Vertebrates Other Than Mouse From Which ES-like Cells Have Been Obtained

Species	Basis of Validation*
Rat	M&M & CP
Golden hamster	IVD
Rabbit	M&M, IVD & CP
Mink	M&M, IVD & T (with wide range of cell types in one study)
Dog	IVD
Cat	IVD
Pig	M&M, IVD & CP
Sheep	M&M & **
Cow	M&M, IVD & CP
Horse	IVD
Marmoset	IVD
Rhesus monkey	T
Human	T
Chicken	IVD & CP (including germline with low passage cells)
Medaka	IVD & CP
Zebra fish	IVD (limited) & CP (with short-term cultured cells)
Gilthead sea bream	IVD & (CP with short-term cultured cells)
^* M & M 5 morphology and ES cell markers; IVD 5 differentiation in vitro; T 5 teratoma production in vivo; CP 5 chimaera production by morula aggregation or blastocyst injection.
^** Exhibited an ES-like morphology initially but rapidly acquired a more epithelial one thereafter

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...