NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 2 FEBRUARY 2007 207 Adaptation to culture of human embryonic stem cells and oncogenesis in vivo Duncan E C Baker 1,4 , Neil J Harrison 2,4 , Edna Maltby 1 , Kath Smith 1 , Harry D Moore 2 , Pamela J Shaw 3 , Paul R Heath 3 , Hazel Holden 3 & Peter W Andrews 2 The application of human embryonic stem cells (HESCs) to provide differentiated cells for regenerative medicine will require the continuous maintenance of the undifferentiated stem cells for long periods in culture. However, chromosomal stability during extended passaging cannot be guaranteed, as recent cytogenetic studies of HESCs have shown karyotypic aberrations. The observed karyotypic aberrations probably reflect the progressive adaptation of self-renewing cells to their culture conditions. Genetic change that increases the capacity of cells to proliferate has obvious parallels with malignant transformation, and we propose that the changes observed in HESCs in culture reflect tumorigenic events that occur in vivo, particularly in testicular germ cell tumors. Further supporting a link between culture adaptation and malignancy, we have observed the formation of a chromosomal homogeneous staining region in one HESC line, a genetic feature almost a hallmark of cancer cells. Identifying the genes critical for culture adaptation may thus reveal key players for both stem cell maintenance in vitro and germ cell tumorigenesis in vivo. The inner cell mass (ICM) of the mammalian embryo can give rise to all somatic cell types. HESC lines are derived from the ICM, and these cells seem to maintain in vitro the same pluripotent properties as ICM cells in vivo. However, these two systems are not equivalent, since the moment that HESCs are transferred from the embryo to the culture dish they are subject to selective pressures from their new environment. Indeed, a recent report comparing epigenetic control in murine ICM and ES cells noted differences in histone methylation and acetylation 1 , perhaps indicating a change in cell regulation in culture. The facility for indefinite self-renewal is a key feature for HESCs, yet this is not a property of ICM cells in vivo and must be a characteristic selected dur- ing initial outgrowth in culture. As such, the establishment of an HESC line must involve some form of adaptive process, most likely epigenetic in origin as, at least in the mouse, established ES cells can revert to an ICM-like state when replaced in a blastocyst 2 . On initial derivation HESCs have shown diploid karyotypes, which may remain stable for extended periods 3 . However, several reports indicate that they may acquire chromosomal abnormalities during pro- longed culture (Table 1). For dividing cells, the occurrence of genetic aberrations is not an unusual phenomenon; yet the nonrandom survival of such variants and their ability to overtake the normal diploid cells in a culture implies that the chromosome abnormalities observed in HESCs can impart a growth advantage to those cells in which they arise. As such, we consider cells that are karyotypically abnormal and show an increased growth rate as ‘culture adapted’, and the process through which this occurs to be ‘adaptation’. Such adaptation may also result in enhanced cloning efficiencies after plating single cells 4 ; a reduced ten- dency for apoptosis in adapted HESCs has also been reported 5 . Another expectation is that culture-adapted cells may show a reduced capacity for differentiation, but this is difficult to assess quantitatively and has not yet been studied systematically, although the retention of undifferentiated stem cells in a xenograft tumor of a culture-adapted HESC line has been reported 6 . The culture adaptations discussed below are all based on the observation of chromosomal changes, though adaptation could also involve other genetic and epigenetic changes that are not observed by standard cytogenetic analyses 4,7 . Here we discuss the karyotypic changes observed in culture adaptation, the parallels between adaptation and germ cell tumorigenesis and the implications of this process in the main- tenance and therapeutic use of HESCs. Genetic change and germ cell transformation For many years genetic change has been known to be associated with neoplasia 8 . In tumor development, genetic abnormalities, which can affect apoptotic pathways, differentiation control or cell cycle, arise in precancerous cells, resulting in an uncontrolled increase in growth. Such a process may be considered similar to adaptation in HESCs, in which a mutation can provide a growth advantage that allows the abnormal cell to dominate a culture over a series of passages. Thus the functions of the same genes might be subject to similar change in both situations, though, since the growth conditions of a cancer cell in vivo and a stem cell in vitro are not the same, not all of the progressive changes are likely to be congruent. The concept that HESCs adaptation and malignancy are related has most direct relevance to testicular germ cell tumors (TGCTs). These tumors develop from pluripotent germ cells 9 , by way of intermediate carcinoma in situ (CIS) cells 10 , and are the most common malignancy in young men 11 . TGCTs consist of two histologically distinct groups: seminomas and nonseminomas. Seminomas are generally uniform 1 Sheffield Regional Cytogenetics Service, Sheffield Children’s Trust, Western Bank, Sheffield S10 2TH, UK. 2 Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK. 3 Academic Neurology Unit, University of Sheffield, School of Medicine and Biomedical Science, Beech Hill Road, Sheffield S10 2RX, UK. 4 These authors contributed equally to this work. Correspondence should be addressed to P.W.A. (p.w.andrews@sheffield.ac.uk). Published online 7 February 2007; doi:10.1038/nbt1285 PERSPECTIVE © 2007 Nature Publishing Group http://www.nature.com/naturebiotechnology