Nature Biotechnology 24, 325 - 326 (2006)
doi:10.1038/nbt0306-325
The selfish stem cellPeter W AndrewsPeter W. Andrews is at the Center for Stem Cell Biology and Department of Biomedical Science, The University of Sheffield, Sheffield S10 2TN, UK. P.W.Andrews@sheffield.ac.uk Two new studies describe approaches for optimizing the culture of human embryonic stem cells.One challenge for biologists seeking to develop applications of human embryonic stem (ES) cells is to discover culture conditions that minimize the appearance of genetically variant cells; another is to recognize and eliminate variant cells when they do arise. Two reports in this issue provide significant insights into both of these problems. In one, Pyle et al.1 show that neurotrophins working through the tropomyosin-related kinase (TRK) receptors promote survival of human ES cells. They suggest that neurotrophin-mediated survival may be a key factor in optimizing culture conditions for human ES cells, and that some chromosomal changes may enhance neurotrophin signaling, thereby providing a selective advantage in suboptimal culture conditions. In the second paper, Herszfeld et al.2 have identified for the first time a surface antigen, CD30, that appears to mark karyotypically abnormal human ES cells. Interestingly, expression of CD30 renders cells less sensitive to apoptosis and so also provides a survival advantage.
When human ES cell lines were first derived, they were seen to possess normal diploid karyotypes3. However, all living things are subject to continual selection for genetic variants best adapted to their environments. This truism applies as much to cells in tissue culture as to Darwin's finches in the Galapagos. Thus the recent excitement about the potential of stem cells for regenerative medicine has been tempered by the recognition that these cells may, indeed, acquire karyotypic changes during prolonged culture4,
5. It seems most likely that such changes offer selective advantages by promoting the maintenance of the stem cell state, perhaps by limiting apoptosis or differentiation. These observations have led to a vigorous discussion as some groups argue that they see aberrations less frequently than do others. Whether the different experiences of different laboratories are explained by the different ES lines used, which have diverse starting genotypes, or whether they are due to different subculturing techniques, or culture conditions, or simply 'luck,' remains to be resolved.
Nevertheless, a striking feature of the reported karyotypic changes is that they commonly involve acquisition of extra copies of the same chromosomes, chromosomes 17 and 12. Moreover, extra copies of these chromosomes, or more precisely the long arm of chromosome 17 (17q) and the short arm of chromosome 12 (12p), are almost always found in embryonal carcinoma (EC) cells6,
7, the pluripotent stem cells of teratocarcinomas and the malignant counterparts of ES cells. This commonality of genetic change in ES cells in culture and EC cells in tumors suggests a common cause. An obvious possibility is the selection for variants that affect the molecular decision processes by which a pluripotent stem cell chooses between the alternative fates of death, differentiation or self-renewal; even a small increase in the probability of self-renewal over death or differentiation would provide a strong selective advantage (Fig. 1a). Such variant 'selfish' ES cells may indeed behave more like EC cells in teratocarcinomas2,
8.
 | | Figure 1. Generating the 'selfish' stem cell. | | |  | (a) When they divide, stem cells have a choice between three possible fates: death through apoptosis, differentiation, or the generation of a new stem cell, a process we call 'self-renewal.' Pyle et al. have identified neurotophin signaling as a significant part of the decision machinery whereby human ES cells choose between these fates: specifically, neurotrophins appear to block the apoptotic fate. (b) Elements of the decision process are likely to be key targets for mutation and selection pressure to generate variant ES cells that develop over time in culture. Mutations that promote self-renewal at the expense of death or differentiation will provide the variant stem cells with a selective advantage, and so promote maintenance and expansion of the stem cell population. Such variants are likely to tend towards the transformed phenotype of tumor stem cells. Indeed, it can be envisaged that ES cells in culture are part of a continuous spectrum of adaptation from a 'normal' state of cells within the inner cell mass of an embryo, through cultured ES cells, to the state exemplified by malignant pluripotent stem cells from teratocarcinomas, the EC cells. Herszfeld et al. have shown that the appearance of CD30, which plays a role in suppressing apoptosis, seems to mark a stage in this progressive process of adaptation.
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Currently, we only have fragmentary knowledge of the mechanisms that promote survival and self-renewal of human ES cells. Like murine ES cells, they express the transcription factors Oct4, Sox2 and Nanog. But they also differ from murine ES cells. Leukemia inhibitory factor is unable to support human ES cell growth in the absence of feeders9, and it is reported that high concentrations of fibroblast growth factor 2 and also transforming growth factor  , activin and nodal, but not the bone morphogenetic proteins, promote human ES cell self-renewal10,
11.
The results reported by Pyle et al. and Herszfeld et al. provide important new information. First, Pyle et al. found that TRKB and TRKC receptors are expressed by human ES cells and, most strikingly, addition to the culture of their ligands, the neurotrophins brain-derived neurotrophic factor, neurotrophin (NT)3 or NT4, raises the notoriously low cloning efficiency of these cells some 36-fold (Fig. 1a). Certainly, in our hands, we have only seen such cloning efficiencies in human ES cells that had acquired an abnormal karyotype. Yet in the reported study, the cells remained diploid, and fully pluripotent. In further experiments, Pyle et al. showed that the neurotrophins promote ES cell survival and suppress apoptosis through the PI-3-kinase pathway. At least some of the beneficial effects of the mouse embryo fibroblasts commonly used to support human ES cell growth may be due to their ability to produce neurotrophins.
In their study, Herszfeld et al. monitored the expression of CD30, a member of the tumor necrosis factor receptor superfamily (Fig. 1b). They found that diploid human ES cells do not express CD30, whereas EC cells and several karyotypically abnormal ES cell lines derived from initially diploid lines showed faster population growth rates and all expressed cell surface CD30. These cells also expressed a splice-variant mRNA encoding a truncated form of the CD30 protein consisting of a constitutively active, cytoplasmic domain. This expression of CD30 correlated with an increased resistance to apoptosis, a function that was also demonstrated by transfecting human ES cells with a vector encoding constitutive expression of the cytoplasmic form of CD30; expression of this transgene was associated with a decreased level of apoptosis.
Both papers invoke the importance of controlling survival of ES cells in culture as a factor in optimizing culture conditions for human ES cells. Clearly, neurotrophins are important candidate factors to add to new formulations for defined media for human ES cell culture. Moreover, in any eventual clinical application of ES cell–based regenerative medicine, persisting ES cells that are inadvertently transferred to a patient might be killed using pharmacological inhibitors of the PI3-K pathway. Likewise, the results of Herszfeld et al. suggest that monitoring another pathway involving CD30 and NF B can provide a tool for identifying variant cells in human ES cultures and a means of eliminating them.
Although these two reports now add new important pieces to the jigsaw puzzle of human ES cell growth control, we are still a long way from seeing the whole picture. Pyle et al. noted that the neurotrophin NT3 and the neurotrophin receptor p75NGFR are encoded by chromosomes 12 and 17 respectively, so that amplification of either chromosome could enhance neurotrophin signaling and provide a selective advantage. However, p75NGFR expression was not detected in ES cells. In the case of the expression of CD30, Herszfeld et al. found a correlation with the appearance of an abnormal karyotype, whether or not the abnormalities affected chromosome 1, which encodes CD30. Although the neurotrophin and CD30 pathways might be ways in which biologists, or the ES cells themselves, can control ES cell survival, it seems likely that other pathways and other genes will also be found to play important roles. Nevertheless, not only do these papers provide insights into the mechanisms driving selection of variant human ES cells, but they also suggest approaches to optimizing culture conditions to reduce their selective advantage, and a means to eliminate variant cells when they appear.
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