EMBRYONIC STEM CELL
5. Embryonic stem cells
5.1 Experimental embryology of late nineteenth century Europe
In the late 1880s, Wilhelm Roux began ‘pricking experiments’ using two-cell frog (Rana esculenta) embryos. Using a fine, hot needle, Roux would puncture one of the cells (with the aim of killing it, and rendering it no longer capable of contributing to development), whilst leaving the second cell to develop normally.
Roux observed that the usual result of the experiment was that half an embryo would develop from the cell left intact. Therefore, Roux argued, the material for development of one half of the embryo was contained in one of the cells at the two-cell stage204.
A few years later, Hans Driesch (1867-1941) experimented with early sea urchin (Echinus microtuberculatus205) embryos, separating cells by shaking them in sea water. This experiment was important, since it demonstrated that Roux’s findings were not as a result of any effect from the pricked cell206 (i.e. the death of one cell at the two-cell stage). Instead of observing half-embryos as Roux had, Driesch’s sea urchins were notably smaller, but nevertheless formed fully developed larvae. The same occurred after separating cells at the four-cell stage, and, occasionally, the eight-cell stage (also refered to as “¼ blastomeres”207)208.
“The isolated half-cells did in fact cleave as if they were still connected with their sisters, and formed half-cleavage stages resembling half of a hollow ball. However, this then closed to a small whole ball, and I obtained on occasion, quite contrary to my expectations, a dwarf pluteus”209.
Driesch also considered whether the same results would be found using amphibian embryos, however claimed that he was not skilful enough to make this experiment a success210.
Such new observations required new experimental protocols to examine the changing ideas about developmental biology (see above). Swiss anatomist Wilhelm
204 Spemann, 1938 p 19-20; Baltzer, 1967.
205 Baltzer provides the alternative nomenclature Psammechinus microtuberculatus (1967, p 107).
206 Spemann, 1938 p 21.
207 Baltzer, 1967 p 109.
208 Driesch, 1893.
209 Driesch, 1951 p 74.
210 McKinnell, 1978 p 8.
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His (1831-1904) demanded that developmental biology needed to describe the structure and function of the fertilised egg as it developed, and that these descriptions should be derived from mechanical explanations and direct causal relationships, allowing each step to be understood as a consequence of the proceeding one211. For emerging developmental biology, it was considered important to track the fates of the early embryonic cells, still believed to relive their phylogenies. August Rauber (1841-1917) also took His’ approach, and set to work carefully observing cleavage events in order to better understand the importance of geometrics in morphogenesis.
Weismann was part of another investigative strand of His’ developmental biology, concerning himself with the carriers and passage of hereditary material.
5.2 American cell lineage studies
Development of evolutionary theory after the publication of Darwin’s Origin (1859) meant that the development of individual organisms could no longer be adequately explained by referring back to conformity of species type – these ‘types’
no longer existed (if species were changing all of the time). The emerging ideas about evolution suggested that individuals could inherit from distant as well as recent ancestors212; this was captured in Haeckel’s biogenetic law, however it was attracting criticism. Gegenbaur’s ideas (basically rejection of Haeckel’s ‘phylogenies beget ontogenies’ beliefs) were particularly influential in American embryology of the era, especially influencing a new school of study: cell lineage. This was the study of exactly what happened to each cell of the developing fertilised egg over the course of the first few cell divisions of the early embryo213. In particular, Mainenschein has highlighted that there were six members of this school (in addition to students or the occasional visitor); in her account, the school began and ended with these researchers.
The six researchers included EB Wilson (see above), CO Whitman, Edwin Grant Conklin (1863-1952), Aaron L Treadwell (1866-1947), AD Mead (1869-?), and Frank Rattray Lillie (1870-1947)214. For Wilson, Whitman, Conklin, Treadwell, Mead, and Lillie, examination of the earliest stages of development would shed light on fundamental biological processes, taking a more modern approach to interpreting
211 His, 1874 p 2
212 Maienschein, 1978.
213 Maienschein, 1978; Guralnick, 2002.
214 Maienschein, 1978.
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Haeckel’s biogenetic law. They accepted that ontogeny and phylogeny were likely related; however, they saw that although phylogenies of adult forms may have similarities to ancestral ontogeny, there was no causal relationship215. The section below will briefly highlight the contributions of Conklin, Lillie, and Wilson, since Conklin’s and Wilson’s interpretations of results were so different to the extent of almost opposing each other, whereas Lillie’s, although more like Conklin’s, were more moderate216.
Two centres became crucial for cell lineage study in the US: the University of Chicago, and the Marine Biological Laboratory (MBL) at Woods Hole, Massachusetts (where Conklin was the first director, from 1888). In 1894, at an MBL Friday Evening Lecture, Wilson noted that “no-one believes that ontogeny is actually a true and complete record of phylogeny”217. Wilson and Gegenbaur appeared to have similar views: that homology could only be accurately assessed using comparative anatomy and morphology218. (The search for homologies also became a key concept for cell lineagists, who used this approach to help understand body plans and variation, for example219.) The aim of the cell lineage researchers was to first, learn the extent to which an individual embryo is a product of its distant ancestors, and secondly, to learn how the individual embryo is affected by external pressures (perhaps resulting in adaptation and change). This fit well with the proposed Darwinian theory of natural selection; it should also shed light on whether changes due to selective pressures occurred in the past, or whether they were still occurring220. Maienschein claimed that although not the priority of cell lineage studies, it was the clarification provided on the relationship between ontogeny and phylogeny that enabled later study of development221. Gross goes as far as to say that the cell lineage studies were successful as they could be carried out “largely free of preoccupation with phylogeny”, enabling the researchers to demonstrate the somewhat superficial nature of Haeckel’s style of embryology; without such shackles, a new way of studying the developing form of the embryo could emerge222.
215 ibid p 134.
216 Guralnick, 2002.
217 Wilson, 1894 p 102.
218 Laubichler and Maienschein, 2003.
219 Guralnick, 2002.
220 Maienschein, 1978 p 135.
221 ibid p 157.
222 Gross, 1985.
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Wilson’s contemporary and, Maienachein claimed, “inspirational leader” of the cell lineage group, Conklin, concluded that the germ layer was not the best place to start looking for homologies223. Instead, Conklin preferred to study the cleavage patterns of of blastomeres. This would also help Conklin learn more about the various factors which he believed had an influence on evolution, including growth, differentiation, variation, metabolism, and inheritance, for example224. From his studies on blastomeres, Conklin could examine any phylogenic significance of early cleavages without the many extra factors at play that affected cleavage in later development. Conklin saw development as an expression of internal or intrinsic factors alone, believing from his results that the cleavage and cell divisions were all precisely inherited functions (i.e. unaffected by any external influence). For Conklin then, understanding early ontogeny was the key to understanding evolution225. Conklin therefore rejected Haeckel’s biogenetic law. Conklin’s approach was historical; he explained that whilst cleavages were morphogenetic, this did not fit with recapitulation theory226. Laublichler and Maienschein highlight the difficulties with such studies however; although the practicalities of such experiments were difficult enough, there were also issues with disseminating results. It was expensive to publish the results of such observational studies, since so many figures and plates were required to adequately describe the processes occurring227.
Lillie’s cell lineage work began around a decade after Conklin’s, after attending an 1892 session at the MBL, then working with Whitman there. Lillie completed his PhD under Whitman in 1894. Lillie worked with Unio (a fresh-water mussel), receiving acclaim for providing impressive insight in his 1895 paper The embryology of Unionadae229. Lillie believed that the cleavage options of the fertilised egg were limited by information inherited from the parents, and the orientation of its cytoplasm. External pressures would not affect any adaptation at this stage, however could come into play on the organism as a whole; like Conklin then, Lillie believed that ontogeny was influenced only by internal factors.
223 Maienschein, 1978 p 134.
224 ibid p 146.
225 ibid p 147, 149.
226 Guralnick, 2002 p 549.
227 Laubichler and Maienschein, 2003.
229 Maienschein, 1978 p 151.
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Wilson began his cell lineage work in the 1870s, whilst still studying under William Keith Brooks at Johns Hopkins University. Wilson’s early papers, published in the late 1870s and early 1880s, trace the early developmental stages of various invertebrae230. Wilson interpreted his observations as showing that each cell of the 4-cell embryo developed into a different part of the body. He also learned that cleavage for cell division occurred in a specific way, so that new cells always developed at a slight angle, resulting in what became known as ‘spiral cleavage’231. As his research continued, he found that he became less and less convinced by Haeckel’s biogenetic law, having rejected most of it by the early 1890s. Guralnick claimed that Wilson’s Nereis (a polychate worm) work from 1892 in particular demonstrated that a different approach from Haeckel’s version of recapitulation was needed, as Wilson launched a
“vitriolic” attack on Haeckelian methods in his MBL lecture of 1894232. Instead, Wilson believed that ontogeny was actually a series of organogenies – each organ appeared to develop from a single cell (a blastomere). To achieve this, the egg divides depending on the role the new cell will later have, so the morphology and location or pattern of cleavage and division was regulated to achieve this. This regulation may have been affected by both internal and external (such as environmental) factors. The impact of external factors meant that although early embryos may have the same cleavage events and cell divisions, and the resulting new cells all be arranged in the same way, they may eventually have different morphologies. Each cell was influenced by its inherited factors, whilst the whole embryo was also influenced by its immediate environment233, a belief that most cell lineagists would agree with234. This appeared to set Wilson’s next goal: to elucidate how external factors could affect internal functions, resulting in differences to adult homologies; yet again, cell lineage studies were useful here, but were not the only way to learn more about embryology235. Eventually, by the beginning of the twentieth century, Wilson had also accepted cell homology, and summed up the work of cell lineagists by noting the similarities revealed by studies on molluscs and
230 Maienschein, 1978.
231 Guralnick, 2002 p 541.
232 ibid p 544; Wilson, 1894 p 104.
233 Wilson, 1892; Maienschein, 1978.
234 Guralnick, 2002.
235 Maienschein, 1978.
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annelids, and other such creatures as all basically following the same general plan of development236.
The cell lineage studies of the late nineteenth and early twentieth centuries resulted, claimed Gross, in the modern approach to studying embryonic development.
Gross argued that embryology had been viewed as an especially useful discipline through which evolution could be studied; by the end of the nineteenth century however, embryology had begun to be seen as a discipline in its own right (see also Chapter 2)237. The question of inheritance versus adaptation was important in the latter half of the nineteenth century, and cell lineage studies were able to begin investigating this, eventually showing that both internal and external factors were influential238. In the 1890s, cell lineage work was mainly based on observation. This is a different methodological approach to the emerging experimental embryology of researchers such as Roux in Europe (see Chapter 2). Whilst observation was of course initially useful, the experimental embryologists argued that observation alone could not provide explanation for developmental phenomenon (Grualnick has argued however that Wilson’s Nereis work supported mechanistic explanations of cleavage patterns239). The experimental embryologists, making use of similar creatures for their work as the cell lineagists, demonstrated that the early cleavage events could be disrupted without affecting the embryo’s capacity to continue normal development240; observation alone could not have achieved such understanding. Arguably, cell lineage studies had helped to elevate the field of embryology from its study under the paradigm of Haeckel’s biogenetic law. The study of the embryo and embryogenesis could potentially reveal much more about multicellular life than the study of recapitulation theory241. Potentially, this may have also been a significant factor in the end of cell lineage studies in the early twentieth century; as highlighted by Guralnick, published accounts of new cell lineage studies had stopped by 1907242. Guralnick offered a potential explanation for the decline of cell lineage studies: that researchers stopped carrying them out because the general patterns of cleavage
236 Guralnick, 2002 p 559.
237 Gross, 1985 p 76.
238 Guralnick, 2002.
239 ibid p 537.
240 Gross, 1985 p 71.
241 ibid p 76.
242 Those papers published after 1900, also focused more on comparative homologies than causation. Guralnick, 2002.
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observed were relatively similar, whilst simultaneously, specific cell divisions of embryos showed too much variation243. Although significant amounts of data had been produced by this time, no-one really knew how to process it all into a theory of embryonic development. It was only through experimental embryology, particularly as it developed in the early twentieth century, that biologists such as Hans Spemann (1869-1941) became capable of assimilating and evaluating the observations produced regarding cell lineage studies, utilising the data alongside experimental results. In 1915 for instance, Spemann noted the significant influence of the homology theme of cell lineage work, claiming that it was useful to link the causal-analytical and historical approaches of previous embryological studies244.
6. Conclusions
There was significant growth in cell biology studies through nineteenth-century Germany, for which several factors were responsible. Firstly, there were significant funds available through the state for university appointments and research.
Secondly, the academics employed, and trained in new techniques of systematic observation and experiment, also had access to dyes and microscopes (for example, Paul Ehrlich’s [1853-1915] influence on the use of dyes in microscopy in the late nineteenth century249). Organicism drove the search for the cell, and then its structure and function. In Germany in particular, this was aligned with idealistic views of the state; Haeckel, for example, compared cells to good citizens of the Kulturstaat, which could only flourish by the division of labour. Different political views would be manifested in different theories on ‘the cell state’. Eventually, social concepts such as
‘colonies’, ‘migration’, and ‘culture’ would remain in the cytology that developed250. Henri Dutrochet’s idea of the vital phenomena that allowed all life to flourish, and the translation of his thoughts to experimentation, took the first steps towards modernising cell physiology. So great were Dutrochet’s microscopic skills, that he was one of the first to observe a somatic cell. Experimentation was also taken up by
243 These variations, Guralnick argues, are not addressed in the cell lineage papers published.
In part, this may have been because there was no easy way (at the time) of analysing the mass of data produced on such variation in any meaningful or quantitative manner. Guralnick, 2002 p 537; 561.
244 Spemann, 1915; Guralnick, 2002.
249 For example, see Ehrlich, 1877.
250 Weindling, 1981.
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Raspail, now considered important for the development of the discipline of cytochemistry. Franco-German politics however diminished the reception of Raspail’s work, and the initial impact it had in Europe.
A further example of such political influences on scientific endeavour, that of Purkynĕ’s contemporary at Berlin, Müller, being far more influential, since he was German as opposed to Czech. Although Purkynĕ’s student Valentin was one of the first to publish comparisons between animal and plant cells in the 1830s, his efforts were dismissed by the popular Müller student Schwann, when he published Mikroskopische Untersuchungen in 1839.
Although the details of Schwann’s Cell Theory were repudiated within a relatively short amount of time, the basis of Cell Theory (i.e. that all organisms were made-up of cells) was influential in biology for much longer; the ideal of unifying the disciplines of zoology, botany, anatomy, and physiology, strengthened by German organicism, would influence the type and structure of research in the biological sciences well into the twentieth century.
This had an effect on the developing discipline of embryology. Although there had been an interest in development throughout history, it was the development of the microscope and staining that gave scientists the first opportunities to take a closer look at early mammalian development. Fuelled by debates concerning epigenesis, evolution, and preformation, pre-nineteenth century studies focused on dissection and microscopy. Following the initial boost of information provided by microscopy in the late 1600s, little technological development in microscopy through the 1700s stymied progress. Late eighteenth-century dissections were of some use, but the popular conclusions drawn by Haller distracted some research avenues as other theories were dismissed.
It was not until 1861 that Gegenbaur clearly argued that the egg itself was a cell. I would argue that this is an important point in embryonic stem cell history - without identifying the unfertilised egg as a cell, it would be difficult to consider the first ‘products’ of the fertilised egg as cells too. The first influential observations on fertilised eggs were made in the early nineteenth century. Prévost and Dumas described how ‘furrows’ would appear in the hours following fertilisation in the rabbit egg. Rusconi followed suit, going further than Prévost and Dumas by declaring that the furrowing was not only a surface phenomenon - in fact there was segmentation
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occurring throughout the fertilised egg, resulting in division and subdivision of the egg.
Soon after Rusconi published his observations, Barry published the first of three papers that would describe his studies on egg development and maturity, fertilisation, and early embryonic development in detail. In the second paper (1839), Barry described segmentation like Rusconi, referring to division of vesicles with nuclei. Barry compared the vesicles he observed with globules identified in the nervous system by Valentin. This, I argue, is the first time cells of the newly-forming embryo were compared with the cells observed in an adult.
Barry’s and Rusconi’s observations were confirmed by others working with invertebrates. The theory was strengthened by the publication of Remak’s book on embryology. Remak claimed that the nuclei in cells of the adult were all produced from the first nucleus created at fertilisation. This again shows that the preformation / epigenesis and ovist / spermist debates of previous centuries still required conclusions; Remak’s work seems likely to have been influenced by this, given the conclusions provided.
It is important to consider how the works of Remak, Barry, and others influenced stem cell biology. Since Haeckel initially coined the term Stammzelle, it is prudent to understand how and why, and in what context. Haeckel was influenced early on in life and education by botany and botanists. Haeckel’s time at Würzburg and Berlin where he met Müller, Kölliker, and Gegenbaur (amongst others) was important, since this influenced Haeckel’s interests and career path. Dröscher argued similarly - that Haeckel’s exposure to Cell Theory, Müller, Virchow, Darwin, and Gegenbaur allowed him to conceive his ‘stem tree’ idea, from which influences Haeckel would develop Stammeltern and Stammorganismen. The use of Stamm appears to follow from the works of others in similar disciplines of study. Stammzelle seems to alter its meaning in Haeckel’s works, dependent on context. Stammzellen were derived from Moneren, giving rise to the first unicellular and multicellular
It is important to consider how the works of Remak, Barry, and others influenced stem cell biology. Since Haeckel initially coined the term Stammzelle, it is prudent to understand how and why, and in what context. Haeckel was influenced early on in life and education by botany and botanists. Haeckel’s time at Würzburg and Berlin where he met Müller, Kölliker, and Gegenbaur (amongst others) was important, since this influenced Haeckel’s interests and career path. Dröscher argued similarly - that Haeckel’s exposure to Cell Theory, Müller, Virchow, Darwin, and Gegenbaur allowed him to conceive his ‘stem tree’ idea, from which influences Haeckel would develop Stammeltern and Stammorganismen. The use of Stamm appears to follow from the works of others in similar disciplines of study. Stammzelle seems to alter its meaning in Haeckel’s works, dependent on context. Stammzellen were derived from Moneren, giving rise to the first unicellular and multicellular