In this chapter Sir Martin Evans, who was awarded the Nobel Prize for Physiology or Medicine in 2007, describes the identification of stem cells, initially from certain cultured tumour cells (teratocarcinoma cells) and latterly from early mammalian embryos.
The differentiated cells of the adult vertebrate arise from a single fertilized egg and as development proceeds the commitment to differentiation becomes irreversible.
During early development a few cells give rise to all the differentiated tissues; these original cells are thus pluripotential.
In malignant tumours small populations of self-renewing cells may arise; these divide to generate rapidly differentiating cells and others, like themselves, which remain undifferentiated.
The original experimental observation that teratocarcinoma cells spontaneously differentiate into benign cell types, embryos, or primordial germ cells questioned the idea that the cellular differentiation was a spontaneous reversion from malignancy: perhaps stem cells within teratocarcinomas were essentially normal?
Discovery of embryonic stem cells made it possible to generate chimeras in the context of a carrier mouse embryo; the demonstration that these could contribute to the germline provided a route to genetic manipulation (transgenesis) from cells in culture to the intact adult mammal. This has been of critical importance for the generation of experimental animals that serve as authentic models of human inherited disease—with numerous technical refinements inducible and conditional models can be produced, almost at will, for study. It is moreover possible to disrupt any locus in the mouse (and now other mammalian) genome(s) in order to investigate the function of particular genes in the living animal.
Teratocarcinomas also occur in humans and their study has stimulated the developing concept of regenerative medicine—a concept further enriched by the isolation of human embryonic stem cells with differentiation properties similar to those of murine origin. In addition, knowledge about the factors that maintain pluripotency and suppress differentiation has allowed reprogramming of differentiated cells, such as skin fibroblasts, back into the embryonic stem-cell state. These scientific discoveries provide opportunities for using pluripotential cells in regenerative processes; those that are self-derived, would evade immune rejection.
In this chapter I relate the history of isolation of mouse embryonic stem cells and their use as a vehicle for mammalian experimental genetic manipulation—and I will finish with some remarks on the possible future utility of the equivalent human embryonic stem cells as a vehicle for derivation of populations of tissue-specific precursor cells for cell transplantation, on which one form of regenerative medicine may be based.
Embryonic stem cells
During vertebrate development the entire organism with its panoply of diversely differentiated cells arises as the lineal descendant of the fertilized egg. Extensive studies in experimental embryology have demonstrated that commitment to differentiation becomes functionally essentially irreversible. At the earliest stages of development, when cell numbers are small, it is self-evident that there must be dividing cells, the descendants of which will give rise to a large range of differentiated tissues. Such cells would be termed pluripotential. It is not necessarily the case that there will be a self-renewing population of such cells in the normal embryo. Malignant transformation, on the other hand, can produce populations of self-perpetuating tumour cells and may provide a useful experimental source for cell types otherwise inaccessible.
Many tumours display a cell phenotype which is a caricature of their normal cell of origin. Teratocarcinomas are tumours which in addition to their malignancy and continued growth contain many patches of a wide variety of nearly normal tissues. In 1967, Leroy Stevens discovered and developed a strain of inbred mice which had a high spontaneous incidence of testicular teratocarcinomas. He was soon able to show that these arose by spontaneous overgrowth of primordial germ cells in the fetal testis. Stevens demonstrated that some of these tumours could be serially transplanted in the inbred strain of mouse and he said
Following repeated serial transplantations, these tumours have retained their pleomorphic character. Pluripotent embryonic cells appear to give rise to both rapidly differentiating cells and others which like themselves, remain.
This is a definition of an embryonic stem cell.
In the course of this research, Leroy Stevens showed in 1970 that equivalent tumours could be formed by ectopic transplantation of preimplantation embryos. Dr Barry Pierce, as a human pathologist, was particularly interested in the observation that the differentiated cells appeared to be non-malignant whereas the stem cells provided the progressively growing malignant component of the tumours. Using the mouse model system, Pierce working with Kleinsmith in 1964 was able to transplant single cells and recover a fully differentiating tumour, thus proving that these tumours were indeed formed from a progressively growing population of pluripotential stem cells.
All these studies were carried out on tumours and cells derived from them, but increasingly there was evidence suggesting that the stem cells from these tumours were essentially behaving in a normal fashion. These cells, termed embryonal carcinoma cells, could be maintained in tissue culture and could be shown to differentiate in vitro in the tissue culture dish, in vivo in a tumour and in vivo in the context of a normal embryo to form a chimeric mouse. Moreover, by 1975 Gail Martin and I had shown that the differentiation observed in vitro followed a normal embryonic path. In discussion of this in 1981 I wrote
Normal cells do not form tumours and conversely tumour cells are not normal. This concept lies at the heart of much of the study of tumour cell biology. Malignant teratocarcinoma stem cells spontaneously differentiate into benign cell types and normal embryos, or primordial germ cells, are able to initiate teratocarcinoma formation at a relatively high frequency. Is it reasonable to regard this process as a malignant transformation, and cellular differentiation as a spontaneous reversion from malignancy? One alternative … [is that] the teratocarcinoma stem cell is essentially a cell showing a completely normal embryonic phenotype.
In the same year I also published a discussion of why, if these cells are indeed normal embryo cells, were we unable to derive them directly into tissue culture but it only through a tumour formed by ectopic transplantation of the embryo.
I surmised that there might be three explanations relating to number, timing, and rapidity of differentiation:
1 The number of pluripotential cells in the embryo at any one time might be very low; sufficient in vivo but insufficient in vitro where there is greater cell mortality.
2 There might be a short time window—in vivo this is extended by growth of the embryo up to this point or regression of some of the cells of a later embryo following damage of transplantation.
3 Embryonal carcinoma cells which differentiate readily are more difficult to maintain in tissue culture than those which are more culture adapted and differentiate less well; ‘… the genuine embryonic cell counterpart may differentiate and lose its pluripotency and rapid growth characteristics all too readily under culture conditions …’
In retrospect we now know that it is possible to isolate these cells from mouse embryos between 1 and 4.5 days of normal development, so the second premise is unfounded, but the other two—cloning efficiency in vitro and use of culture conditions most conducive to maintenance of the undifferentiated state—are vital.
By that time I had, by using cloning efficiency assays, optimized all aspects of embryonal carcinoma cell culture; the media, serum, substrate, and feeder cells included. It was then that in 1981, in a collaboration with Matt Kaufman, we attempted to grow cells from implantationally delayed blastocysts and successfully isolated embryonic stem cell cultures. These diapause embryos have the advantage of a slight increase in cell number compared with the 3.5 day blastocyst. This technique was immediately effective in allowing pluripotential cell colonies to grow out directly from the inner cell mass. The use of delayed blastocysts was unnecessary but certainly improves efficiency of embryonic stem cell isolation. Much more is now known about the factors maintaining pluripotency and suppressing differentiation. Moreover, as shown by Takahashi and Yamanaka in 2006, by using conditions conducive to embryonic stem cell derivation and growth it has now proved possible to reprogram other differentiated cell types back to the embryonic stem cell state.
Because of all the antecedent work with culture of mouse embryonal carcinoma cells the expected properties for directly isolated mouse pluripotential stem cells were well understood and characterized. These were all rapidly verified, including—importantly—the ability to make chimeras in the context of a carrier mouse embryo. Andrew Bradley and other colleagues showed that these chimeras made with freshly isolated embryonic stem cell lines chimaerized not only the soma but also the germ line of the resulting mice, hence giving a route to genetic manipulation from culture to creature.
During normal early development there may be no more than a couple of dozen embryonic stem cells and they are constrained in the normal time course and present for only a short time. In contrast, many millions of embryonic stem cells may be maintained in tissue culture indefinitely and all of them retaining their pluripotentiality. This means that clones bearing a rare genetic change may be identified and used to pass that change into the mouse germ line and hence the breeding mouse genome. These cells are therefore a vector to an experimental mammalian genetics.
There are essentially two approaches: random mutagenesis and selection/screening, or targeted mutagenesis. Embryonic stem cells in culture are amenable to most forms of mutagenesis and it is therefore the screen that is pivotal. In the late 1980s, when we were first able to use embryonic stem cells to transfer mutation from culture to creature, sequence information for the mouse genome was very limited and many loci unidentified. When mutation is being induced randomly into as yet unidentified loci it is useful to ensure that these are marked and for this reason methods of insertion mutagenesis have tended to be favoured. With Andrew Bradley, I found that retroviral vectors were often the vehicle of choice because of their efficient transfection and clean integration leading to readily identifiable mutation.
With the notable exception of hypoxanthine-guanine phosphoribosyl transferase, informative mutations could not be readily selected in vitro and so screening of the resultant progeny after intercrossing was required—a lengthy process which did, however, result in the identification of some interesting loci. One of the more interesting was 413d/Nodal, found by Elizabeth Robertson and colleagues, which appeared as an organizational early embryonic lethal later identified by Zhou and colleagues as a factor resembling transforming growth factor beta (TGFβ). This was identified by the early death of homozygous mutant embryos.
The random mutagenesis approach was greatly strengthened by the introduction of the idea of gene trapping by transfection of embryonic stem cells using vectors with reporters which would be transcribed only when integrated into a suitable site in the genome. This allowed a screen not only of mutagenic effect (most usually in the homozygote) but of developmental and tissue-specific expression both in vitro and usefully in the immediate chimaera and the heterozygote offspring. Integration using a retroviral vector has the apparent disadvantage that the long terminal repeat sequences necessarily surround the trapping construct. But by using a splice acceptor as the trapping element and by having it in reverse orientation, thus avoiding interference with the transcription of retroviral vector itself, Friedrich and Soriano brought the advantages of retroviral transfection to gene trapping in the early 1990s. Intelligently constructed mutagenic reporters of gene function such as these have been used for extensive screens.
The alternative approach is directed mutagenesis. Two scientists who received the Nobel prize with me, Oliver Smithies and Mario Capecchi, both pioneered the method showing that a DNA construct with substantial homology with a chromosomal sequence could recombine into the endogenous sequence at relatively high efficiency when introduced into cells in tissue culture. With the advent of full knowledge of the genome, this method of gene targeting by homologous recombination has become the choice for gene inactivation. Even when a specific point mutagenesis or random point mutagenesis at a particular locus is desired it is more effective to recurrently target a marked locus with mutagenized vector rather than hitting the whole genome.
Homologous recombination works well with embryonic stem cells and is only limited by the need to keep the cells in conditions which retain their full pluripotentiality and germ line chimaerization ability. The design of the targeting vector and screening to find correctly targeted clones becomes the main consideration. In addition to simple mutation, methods have been developed which allow both spatial and temporal control of gene deletion or of function. All these studies are dependent upon the combination of in vitro cell genetic manipulation and selection coupled with true in vivo observation of the physiological consequences in the context of the whole animal. This has been made possible by tissue culture of embryonic stem cells. It is important to note that virtually any desired ‘designer’ change may be made by using the techniques of homologous recombination gene targeting ranging from single point mutation to large chromosomal alterations. All these techniques are applied to mice and this has provided the experimental genetic approach to mammalian genetics illuminating understanding of data emerging from the study of the human genome. Mouse embryonic stem cells have established at least two platforms for research: an in vitro system of cell differentiation equivalent to that in the early embryo and a vectorial system for experimental mammalian genetics in vivo.
Teratocarcinomas carcinomas occur not only in mice but also in humans. Thompson and colleagues showed in 1998 that embryonic stem cells isolated from human embryos have similar differentiation properties to those of mice. Clearly their use for genetical experiment would entirely unethical in addition to being impracticable, but their in vitro differentiation not only allows fundamental studies of human embryonic and cellular development but is also providing a possible platform for derivation of tissue-specific precursor cells in vitro. This concept of their utility to provide cells for tissue repair was clearly stated in the original publication by Jamie Thomson and the idea of both pluripotential and tissue-specific stem cells has been a very powerful stimulant for human regenerative medicine.
The beginnings of regenerative medicine
Regenerative medicine springs from the powerful idea of treatment of cellular insufficiency or tissue damage by replacement or supplementation with appropriately differentiated cells. Treatment with live cells is not a new concept; blood transfusion and bone marrow transplantation use cell suspensions and there are numerous transplantation therapies replacing whole organs. In some cases, such as liver transplantation, a regenerative tissue is introduced. Interestingly, skin grafting provides the example of transfer of organized tissue with the aim of transferring the regenerative skin stem cells and this therapy has advanced in some applications to the use of disaggregated cells either freshly isolated or from passaged tissue culture.
The question arises as to whether such treatments are medical or surgical. This is not just a question of professional demarcation: it affects which type of regulation is going to be applied. Will we see regenerative medicine and medical procedures regulated under rules designed for the safety of medicines, or under rules designed for the safety of tissue and organ transplantation? As presently framed, the two are very different. Regulations for drugs and other pharmacological formulations are framed both to ensure exact reproducibility and on the basis of large-scale clinical trials for efficacy. Regulations for organ and tissue transplantation focus upon the ethical source of the tissue and informed consent for its use and upon ensuring, as far as is practicable, that it does not transfer infection. The actual use is dependent upon professional judgement of its utility for the patient and good practice by the physician. It is much more a patient-specific approach than a specific formulation for use on many patients. Cell preparations, on the other hand, particularly when allogeneic, have been licensed by the United States Food and Drug Administration under conditions of good manufacturing practice and scrutiny not dissimilar to that of a drug. If the promise of widespread patient-specific treatment by autologously derived cells becomes a reality, then the personalized approach of the transplantation-based rules might be more appropriate.
There is another important practical question to be pondered; that is the cost of patient-specific treatment. Clearly, even when perfected and streamlined, preparing a specific precursor cell population autologous for each patient will be very expensive. On the other hand, successful treatment for what would otherwise be a chronic condition could be a one-off cure and the cost of this would have to be contrasted with the costs of long-term treatment and care, which might be only palliative. The current model of pharmaceutical intervention—using, in the main, small foreign molecules (drugs) to perturb and modulate the patient’s physiology—should be contrasted with a future model of long-term regenerative repair by endogenous self-perpetuating natural effectors (cells). The cost-effectiveness of this approach may well eventually favour the cell model.
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