Human embryonic stem cells (HESC) are pluripotent cell lines derived from the inner cell mass (ICM) of blastocyst stage embryos. These cells possess the capacity of self-renewal and can differentiate into all three embryonic germ layers. These unique characteristics have made HESC not only an attractive potential therapeutic tool for numerous human diseases, but also a research tool for studying early human developmental processes and an interesting cellular model for human disorders, particularly for those for which there is no animal model available (Ben-Nun and Benvenisty, 2006).
HESC are immortal and often kept in culture for long periods of time without this apparently affecting their pluripotent capacity. Nevertheless, these cells probably undergo spontaneous mutations at a rate of 10e-9 per nucleotide, as any other cell type does. It has been reported that HESC can accumulate chromosomal changes and epigenetic modifications (Inzunza et al., 2004, Maitra et al., 2005). Taking into account the great potential applications of HESC, it is important to ensure that these cell lines keep a general genomic stability, or, if this is not the case, to study the extent and consequences of the instability.
Our research centre includes a HESC laboratory, where currently 13 HESC lines have been derived and new lines are regularly added (Mateizel et al., 2006). Among the established lines, we count 6 lines carrying a dynamic mutation in the DMPK, HD, FMR1 (2 lines), ATXN7 and FSHD genes respectively.
This project would involve two main lines of research:
Our first aim is to study the (epi)genetic stability of the HESC. If these cells are to be used in cell therapy, all precautions must be taken to guarantee the safety of the recipient. Moreover, changes in the genome of these cells may influence their gene expression profiles, and consequently influence their capacity to differentiate to different cell lineages.
Our second aim is to contribute to the unravelling of the mechanisms of instability in dynamic mutations, which is of importance for later treatment of these diseases. HESC carrying dynamic mutations are interesting cell models that may replace animal modelling. It is thus important to fully characterize the cell lines carrying dynamic mutations before any further experiments can be performed.
The main objectives of this project are to study the (epi)genetic stability of HESC at several levels and to evaluate the consequences of possible abnormalities:
A. The cells will be characterised for their chromosomal content and for small genomic modifications, and possible abnormalities will be correlated with gene expression patterns.
B. The role of aberrant DNA methylation will be investigated in genes with abnormal gene expression profiles.
C. The behaviour of dynamic mutations will be investigated in gametes and preimplantation embryos (as in vivo model) as well as in HESC lines (as in vitro model).