Original Article 139 Human Embr yonic Stem Cell Stability Lisa M. Hoffman and Melissa K. Carpenter * Krembil Centre for Stem Cell Biology, Robarts Research Institute, London, Ontario *Correspondence and reprint requests to: Melissa K. Carpenter , Krembil Centre for Stem Cell Biology, Robarts Research Institute, 100 Perth Dr., London, Ontario N6A 5K8, Canada. E-mail: mcarpenter@robarts.ca Abstract Human embryonic stem cells (hESCs) are derived from human preimplantation embryos, and exhibit the defining characteristics of immortality and pluripotency. Indeed, these cell populations can be maintained for several years in continuous culture, and undergo hundreds of population doublings (see refs. 1,2). hESCs are thus likely candidates for source of cells for cell replacement therapies. Although hESC lines appear stable in their expression of cytokine markers, expression of telomerase, ability to differentiate, and maintenance of a stable karyotype, several other aspects of stability have not yet been addressed, including mitochondrial sequencing, methylation patterns, and fine resolution cytogenetic analysis. Because of the potential utility of hESCs, it will be of utmost importance to evaluate the stability of these aspects of ESC biology. Stem Cell Reviews Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1550-8943/05/1:139–144/$30.00 of telomerase, suggesting that a shortening of telomeric length may act as a “mitotic clock” to initiate replicative senescence (9,12–19). Mitochondrial mutations have also been shown to accumulate in culture and to alter stability of cells and their integration after transplantation. Recent studies demon- strate that as mutations in mitochondrial DNA accumulate during the aging process and perhaps in carcinogenesis, there is a progressive decline in mitochondrial func- tion (20–23). Like alterations in the mito- chondrial sequence, epigenetic changes have similarly been found to be associated with cancer and other abnormalities in cell differentiation (24–29). It is clear that epi- genetic changes regulate important aspects of differentiation and as such, alterations in the epigenetic state of the ESCs are likely to have serious consequences. In vitro fertilization clinics, for instance, report an increased incidence of Beckwith- Wiedemann syndrome related to aberrant genomic imprinting (30). These findings indicate that long-term culture of cells can result in significant changes in their characteristics. Somatic Cells: Changes Over Extended Culture Periods Human somatic cells typically exhibit a limited ability to proliferate in vitro; indeed, following prolonged culture periods, somatic cells progressively cease cell divi- sion, undergo a number of characteristic phenotypic and biochemical changes, and enter a permanent growth-arrested state referred to as replicative senescence (3–6). As shown in Fig. 1, on prolonged replication, somatic cells exhibit changes in telomere length, imprinting patterns, methylation status, and also accumulate mutations. Chromosomal analyses in human fibrob- lasts, for instance, demonstrate the occur- rence of abnormalities with increasing passage number ( 7) and donor age ( 8) . Considerable evidence suggests that telo- merase and telomere length may play an important role in preventing the indefinite proliferation of cells in culture. More specif- ically, it has been observed that with increas- ing passage number, somatic cells exhibit shortened telomeres, with senescent cells exhibiting shorter telomeres than primary cells (9–11). Both primary and senescent cells have also been shown to express low levels