The period 1945-1965 in biology is particularly noted for establishing the mouse as an experimental model686. The era was also defined for its research into the effects of radiation (see Chapter 3). Other research interests were catered for however; in Britain, for example, the Marshall School of Reproductive Physiology in Cambridge and Waddington’s Institute of Animal Genetics in Edinburgh were also well-funded at the time. By the late 1960s, mammalian physiology, genetics, and embryology had been linked with clear applications – Graham refers to this as “a powerful cocktail”687. In Britain, this would lead to the mouse being adopted as the animal model for developmental biology; in the USA, Drosophila was the preferred model, due to the better understanding of its genetics at the time688.
2.1 Martin J Evans
Sir Martin J Evans (1941-) is probably most well known as the winner of the 2007 Nobel Prize in Physiology or Medicine (alongside Mario R Capecchi and Oliver Smithies)689. Evans, Capecchi and Smithies were awarded the Nobel Prize “for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells”690. Much of Evans’ life has been well documented, particularly since being awarded the Nobel Prize. Evans’ recollections of his childhood and early university career suggest his constant interest in biology, and some fortunate instances which led him to meet and/or work with influential scientists.
684 Wertz, 2002 p 143.
685 ibid.
686 For example, see Myelnikov, 2015.
687 Graham, 2000 p 51.
688 ibid.
689 Nobel Foundation, 2007.
690 ibid.
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Evans was born on 1st January 1941 near Gloucestershire, and soon moved to Hertfordshire, where Evans recalls his ‘first experiment’: mixing sand cement with water, because he could not understand how the mixture could become solid. After suffering from a burst appendix on what should have been his first day of school, Evans remembers being treated with one of the first antimicrobial drugs (M&B 693), without which he may not have survived; this said, Evans then went on to suffer from several other childhood infections, such as mumps691. Evans suggested that recollecting on the time spent in bed at home as a child, reading, playing with his chemistry set and electric experiment set, he was ‘naturally’ a scientist692. After electing to study Chemistry, Zoology and Botany at sixth form, Evans won a scholarship to Christ’s College, Cambridge, where he found the Natural Sciences options tempting, giving him an opportunity to elect courses he enjoyed; this included biochemistry (taught by noted plant biologists David Coombe (1927-1999), Malcolm Dixon (1899-1985) and Don Northcote (1921-2004)693) and molecular genetics. In the academic year 1962-63, Evans recalled a series of lectures by Jacques Monod (1910-1976)694 and Sidney Brenner (1927-)695 about mRNA; from this point Evans claimed he was resolved to work in either developmental biology or plant biochemistry696. Evans never had the opportunity to sit his final exams however, becoming ill with glandular fever697. Although disappointed at the lack of opportunity to embark on a postgraduate research career at the time, Evans considerd himself fortunate to have been employed as a research assistant with Elizabeth Deuchar at University College London (where he was also able to complete his PhD).
Evans described the atmosphere as somewhat relaxed, stating that Deuchar encouraged but did not direct, allowing Evans the freedom to experiment, innovate, develop techniques and learn a wide range of skills, whilst working on Xenopus development.
691 ibid.
692 ibid.
693 Evans, 2001.
694 Monod was to win the Nobel Prize in 1965 for work on genetic control of virus synthesis.
695 Brenner was another future Nobel Prize winner (2002), and one of the first to see Watson and Crick’s 1953 model of DNA.
696 Evans, 2001.
697 Nobel Foundation, 2007; Evans, 2001.
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During this time, Evans was attempting to isolate developmentally controlled mRNA698. Evans was able to use blastula and gastrula ectoderm, providing an early insight into embryo biology. Evans observed that there were two limiting factors for his research: lack of foreseeable genetics, and difficulty in obtaining enough material for research699. This latter Evans discussed with colleague Robin Weiss (1940-), who suggested using mouse teratocarcinomas, following the 1966 publications by Stevens and Pierce (see Chapter 3). Evans noted that Stevens had presented these rapidly dividing cells which could divide asymmetrically, whilst Pierce had demonstrated their clonality. Stevens sent Evans stocks of mice from JAX, and Evans was taught the tissue culture techniques required by Weiss and Pavel Vesely (visiting from Prague)700.
2.2 Gail R Martin
Gail Martin (née Zuckman) was born in New York, USA, in 1944, and went to the University of Wisconsin before beginning her research career at the University of California at Berkeley. She obtained her PhD in 1971. Martin then moved to England with her English husband Steven Martin (also a biologist), after he had been offered a job there. Martin herself found work in Martin Evans’ biochemistry group at University College London (UCL), where she credited Robin Weiss with introducing her to teratocarcinomas701. Martin was not the only American at the time to visit the UK and Europe after completing a PhD; the National Institutes of Health (NIH) and March of Dimes provided funding for fellowships. In fact Graham estimated that such funding doubled the research in mammalian developmental biology in the UK between 1960 and 1980702. Martin returned to the USA in 1976 to work at the University of California at San Francisco (UCSF), where she has remained until the present. Martin is currently Emeritus Professor in the Department of Anatomy, working particularly on development and organogenesis in mice and chickens. Martin was also President of the Society for Developmental Biology in 2006-2007 after winning the Edwin Grant Conklin Medal from the Society in 2002.
698 Nobel Foundation, 2007.
699 ibid.
700 ibid.
701 Robin Weiss was working in a shared laboratory alongside Steven Martin at the Imperial Cancer Research Fund’s Laboratories in London in 1971. It was Robin Weiss (whose primary interest was retroviral biology) who introduced Gail Martin and Martin Evans.
702 Graham, 2000.
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In 2007, Martin received the Pearl Meister Greengard prize (along with Beatrice Mintz and Elizabeth Robertson), celebrating women in science. She is also a member of the American Academy of Art and Sciences and the National Academy of Sciences (cellular and developmental biology), and in 2015 was elected to be a Foreign Member of the Royal Society.
Gail Martin began her research career investigating collagen, in particular its use in laboratory tissue culture and its cross-linking properties703. It was likely to be this experience in cell culture and associated techniques which made Martin a promising postdoctoral addition to Evans’ laboratory at UCL.
2.3 The SIKR Cell Line
Publications from Evans’ laboratory suggest that the group were spending almost all of their time and resources on mouse work, including their ongoing studies into teratocarcinomas. However, if one is to believe Graham’s interpretation of the era, this was relatively unheard of. Graham instead proposes that few groups invested in mammalian developmental biology full-time, instead preferring to top-up funding for their mouse work with other areas of research in the 1970s704. It is possible that Evans, who was only beginning to establish his own small group in the early 1970s, could garner enough funding for his laboratory to concentrate solely on murine cell biology.
In 1972, Evans published “The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratoma cells” in the Journal of Embryology and Experimental Morphology. In this paper, Evans described a clonal culture of cells isolated from a teratoma of strain 129 mice, obtained from Stevens. Evans made it clear that tissue culture work using mouse teratoma cells was already well underway, citing the work by Stevens and Little (1954), Stevens (1964, 1967, 1968 and 1970) and Kleinsmith and Pierce (1964). In 1970, two further groups had demonstrated that teratomas had a single cell origin (Kahan and Ephrussi, 1970; Rosenthal, Wishnow and Sato, 1970705). Evans differentiated his work from his predecessors by stating that his paper demonstrated the ‘isolation of pluripotent stem cells from a solid
703 For example, The nature of the collagen synthesized by cultured human fibroblasts and the review Recent progress in collagen research, both published in 1971.
704 Graham, 2000.
705 These papers had described homozygous teratocarcinoma lines, whereas Gail Martin and Martin Evans described a heterozygous cell line.
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teratocarcinoma derived from the implantation of an early embryo into the adult testis’706. This then is continuing a trend highlighted previously – the comparison of cancerous growth and normal development (i.e. that teratomas arise from initially non-pathological cells), and studying their parallels. The paper described the nature of “SIKR” cells707 (see Figure 6), which were capable of producing teratomas in mice when reintroduced (demonstrating differentiation into ten tissue types).
Figure 6: Isolation, culture and tumourigenicity testing of Evans’ SIKR cell line.
Constructed using information in Evans, 1972.
In culture, two sub-clones were isolated: C-type (tumourigenic, pluripotent and comparable to SIKR cells in differentiation range) and E-type (non-tumourigenic and with lower differentiation capacity). C cells grew as monolayers on E cells; C cells were unaffected by colony density, whereas E cells would not proliferate once the culture had reached a certain density. The C cells were considered to be more primitive than E cells; E cells alone were not tumourigenic, but could become so
706 Evans, 1972 p 164.
707 “SIKR” appears to be a shortened version of the longer name given to the cell line, OTT 5568S/1/KR; OTT 5568 was a transplantable tumour obtained from Stevens in May 1969.
The tumours were maintained by continuous transplantation, with the slowest growing tumours selected for Evans’ project. These were named OTT 5568S. One particular clone of OTT 5568S, OTT 5568S/1/KR, grew well in vitro, and demonstrated good differentiation in vivo. This was selected for use by Evans, and the name shortened to SIKR. See Evans, 1972.
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following spontaneous transformation in vitro. C and E cells were also pluripotent708. The ratio of C and E cells in culture could also be manipulated by seeding the cells at different densities. When mentioning previous reports of teratocarcinoma cell line establishment, Martin and Evans suggested that:
“These reports primarily have been concerned with demonstrating that these in vitro teratocarcinoma lines are pluripotent. This evidence was obtained by reinjecting the cells of these in vitro lines into mice and making a histological examination of the tumors formed. There has, however, as yet been no detailed description of the characteristics in vitro of these cell lines.”709
Martin and Evans also suggested that in vitro cell lines would be useful as a research tool, as large numbers of these cells could be cultured in controlled environments. This would allow more detailed study, and an understanding of the
“biological and biochemical characteristics of pluripotency” to be established710. What Martin and Evans had done was to produce a heterogenous teratocarcinoma cell line, unlike homogeneous lines described by Kahan and Ephrussi (1970) and Jakob et al. (1973)711. Importantly, Martin and Evans concluded that the C cells were ‘the stem cell line of teratocarcinomata’; this was supported by evidence including morphological examination and molecular biology techniques (in this case, experiments testing alkaline phosphatase levels)712. The cells were described as having large clear nuclei with prominent nucleoli, and a minimal, dark cytoplasm. In culture, these cells formed small, tight colonies. In bacteriological dishes (where the cells had no opportunity to adhere to the plastic base of the plate), embryoid bodies developed, noted as morphologically similar to early post-implantation embryos713. Martin and Evans also concluded that C cells gave rise to E cells and that this process
708 Martin and Evans, 1974.
709 ibid p 163.
710 ibid p 163.
711 BW Kahan was at the Department of Zoology, University of Wisconsin and Boris Ephrussi, a Russian geneticist was at Laboratoire de Génétique physiologique, Gif-sur-Yvette, France, in 1970. The Jakob et al. group were based in France; H Jakob was at the Institut Pasteur, Paris.
712 Martin and Evans (1974) suggest that these homogeneous cultures consisted of only C cells. In 1973, a group working across the UK, USA and France published a paper specifically describing alkaline phosphatase activity in murine teratomas (Bernstine et al., 1973). This publication demonstrated that a correlation between alkaline phosphatase activity and embryonal carcinoma cells (the stem cells of teratomas) had been established.
713 Martin and Evans, 1974; Martin and Evans 1975a; Martin and Evans 1975b; Martin, 1975.
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was not reversible; this was identified as a possible route to studying cell determination (although this was difficult in vivo with E cells as they were not malignant).
To follow this, Martin and Evans published another paper less than twelve months later, describing the nature of SIKR subclones714. Where C cells were subcloned, the cultures were homogeneous, consisting of only embryonal carcinoma cells. When injected into strain 129 mice, these cells gave rise to teratocarcinomas with differentiated tissues present, such as nervous tissue, cartilage and epithelium.
With appropriate cell culture techniques, the C cell colonies could be induced to differentiate in vitro. The first stage of this was the development of aggregates of cells which produced an endodermal outer layer – these formations were found to be identical to the embryoid bodies observed in the ascites fluid of mice with intraperitoneal teratocarcinomas715 (and also similar to developmental events which had been shown to occur during normal murine embryogenesis716). If allowed to continue growing for several weeks in vitro, a variety of cell types could be observed, including keratinising epithelium, cartilage, endodermal cysts, neural cells, muscle, fibroblasts and pigmented cells. Martin and Evans had identified that the early differentiation processes of teratocarcinoma cells that occurred in vitro were identical to those which occurred in vivo717. In addition, this process was shown to be highly organised, and paralleled early development of the mouse embryo; in their conclusions however, Martin and Evans did not suggest that this would be a useful tool for studying the early stages of embryogenesis. Three months later, Martin published a review describing how teratocarcinomas might be useful for studying embryogenesis and neoplasia. This detailed paper described teratomas, embryoid bodies, embryogenesis, embryonal carcinoma cells, and the development of derived cell lines. Here then, Martin seems to be highlighting herself the parallels between normal and pathological development718. Martin also described techniques used to learn more about these cell lines, including morphology, karyotyping, the biochemical
714 These subclones were produced by culturing colonies from a single cell of the original SIKR cell line.
715 As described by Teresky et al., 1974.
716 This was described byTarkowski and Wroblewska in 1967; this is the reference Martin and Evans refer to in their description of the process.
717 This is further investigated and published in a separate paper: Martin and Evans, 1975b.
718 Since Martin appears aware of the significance of the parallels in the 1970s, this observation may have driven her focus towards development, as seen in her later work.
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marker alkaline phosphatase, immunological properties and some genetics, focusing on the T locus719. A section was also devoted to describing the similarities between embryonal carcinoma cells and normal embryonal cells:
“No significant differences have yet been detected among embryonal carcinoma cells found in tumors from different sources…There are at least two possible ways in which embryonal carcinoma cells could arise from their progenitor cell types. First, some malignant change occurs in the progenitor cell types. Second, that embryonal carcinoma cells are normal undifferentiated embryonic cells (or primordial germ cells) which behave abnormally because they are not in their normal environment”720.
Martin commented that this latter hypothesis was first suggested by Cohnheim and Ribbert in the nineteenth century (see Chapter 3). Ivan Damjanov and Davor Solter also discussed this embryonic theory in two papers published in 1974. It was an attractive theory as the genetic and chromosomal stability of embryonal carcinoma cells was demonstrated over many generations both in vitro and in vivo; tumours also developed from early embryos transferred to extra-uterine sites, suggesting that malignancy would occur readily in this situation. Martin then reasoned that if embryonal carcinoma cells were normal pluripotent embryo cells (and if pluripotent embryo cells could be cultured from early embryos), then these cells should have the same characteristics of embryonal carcinoma cells in vitro, and should form teratocarcinomas in vivo. Martin highlighted however that Michael Sherman (at the Roche Institute of Molecular Biology, New Jersey) had so far been the only researcher to produce cell lines from mouse embryos, and these cells appeared
719 Mutations at this locus had been previously shown to affect mouse embryogenesis and the antigen expression of spermatozoa. Artzt, Bennett and Jacob (at Laboratoire de Genetique Cellulaire, Institut Pasteur et College de France, Paris) (1974) reasoned that this could therefore also affect the antigen expression of early embryos and embryonal carcinoma cells.
In addition to this, in a paper published in late 1975, Peter Stern (at the Neuroimmunology Unit at University College London) alongside Martin and Evans, described the investigation of the antigens expressed by teratocarcinoma cells. As the cells differentiated, their surface antigen profile changed. In the conclusions, there were hints that the importance of such work would be eventually realised in developmental biology, if cell determination could be identified by changes in cell surface antigen expression. This suggests that Martin may not have been the only researcher at the time to have recognised the significance of the parallels between pathological and non-pathological development, and the importance of this for a variety of research projects.
720 Martin, 1975 p 240. This could be seen as a development of earlier work by Knox, although Martin does not reference this previous research, so it is unknown whether she was aware of it or not.
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somatic, not pluripotent, and in no way similar to embryonal carcinoma cells721. Evans and Matthew Kaufman (see below) would later develop a method to improve on Sherman’s results.
2.4 Embryonal carcinoma stem cells
Martin’s next set of experiments revealed that there were common proteins in both embryonic carcinoma cells and the embryonic ectoderm (which were not observed in other cell types)722. Furthermore, it appeared unlikely that embryonic carcinoma cells and preimplantation embryonic cells were homologous. This led the group to suggest that whereas embryonic carcinoma cells begin to differentiate by developing an endoderm outer layer, they were more biochemically similar to normal embryonic cells which had already developed this layer – i.e. these embryoid bodies were developing ‘inside-out’. It was therefore suggested that the embryonic carcinoma cells were a better model system for studying the embryonic ectoderm than earlier embryonic development (since it was more easily accessible)723. In addition, later 1970s work studied the expression of a protein in both embryonic carcinoma cell lines and cells isolated from the ICM (inner cell mass) of the blastula at 4 days post-conception. The protein investigated (large external transformation-sensitive protein, or LETS protein) was expressed correlating with differentiation. A comparable result was observed – that teratocarcinoma cells were similar to the embryonic ectoderm724.
This work led Martin to write another, updated, review comparing teratocarcinomas and embryogenesis, again highlighting the importance of the parallels between abnormal development and embryogenesis for researchers in this field.
“There is…some uncertainty about the normal embryonic equivalent of embryonal carcinoma cells, and whether pluripotent embryonal carcinoma cells isolated from different tumors are all derived from the same embryonic cell type. Nevertheless, these tumor cells, particularly those cell lines that synchronously form embryoid
721 Sherman, 1975. Sherman had developed a culture medium which promoted the hatching of mouse blastocysts; the free cells would then adhere to the culture dish. These cells would swiftly differentiate however. In 1978, Mintz, Cronmiller, and Custer also published a paper
721 Sherman, 1975. Sherman had developed a culture medium which promoted the hatching of mouse blastocysts; the free cells would then adhere to the culture dish. These cells would swiftly differentiate however. In 1978, Mintz, Cronmiller, and Custer also published a paper