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Project
Chromosomal abnormalities in human preimplantation embryos and embryonic stem cells: causes, mechanisms and consequences for IVF and regenerative medicine (FWOAL535)
Human preimplantation embryos are known to carry chromosomal aneuploidies in as much as 40 to 70%, depending on the source of the study (Fauser, 2008). Most of these aneuploidies have been found in embryos that underwent blastomere biopsy, followed by FISH for up to 9 chromosomes, during an IVF cycle with preimplantation genetic screening (PGS). PGS has been applied in IVF patients with a bad prognosis to improve IVF results. Although these aneuploidies are probably the most important factor influencing the potential of the embryo to implant in the uterus, the extent to which aneuploidies play a role is not clear. Firstly, the high number of aneuploidies would predict an even lower rate of implantation after embryo transfer in IVF, and secondly the presence of mosaicism in preimplantation embryos has led researchers to suspect that some of these aneuploidies and mosaicism may self-correct during early development of the embryo. These factors most likely explain why PGS has failed to show an effect on IVF pregnancy rates as shown by our own group (Staessen et al., 2004) and others (Twisk et al., 2006). However, not much is known at the cellular level on how these abnormalities arise, how they self-correct, and what their ultimate influence on embryonic development is.
Human embryonic stem cells (hESC) are derived from preimplantation embryos, and hold a great promise in the field of regenerative medicine because of their potential to develop into any cell type of the adult individual. Intensive research over the past 10 years has yielded numerous protocols for the differentiation of hESC into adult cell types (f.i. insulin-producing pancreatic cells, Kroon et al., 2008). Currently, one of the important issues is the genomic stability of hESC during culture. We (Spits et al., 2008) and others (Baker et al., 2007) have shown that hESC are prone to chromosomal abnormalities, including aneuploidies, fragile site expression and small duplications with a hotspot at 20q21.11. This genomic instability strongly reminds of the behaviour of cancer cells, and is of great concern for the use of hESC for therapeutic purposes or as reliable research models. Nevertheless, little is know about the mechanisms and the influence of the culture conditions on the origin of these abnormalities.
We hypothesize that human embryos and stem cells have a cell cycle that differs from somatic cells in their checkpoints, and that this may be responsible for the proneness of these cells to chromosomal abnormalities.
The eukaryotic cell cycle contains the following four checkpoints: the start checkpoint in late G1, the S phase checkpoint, the G2 to M checkpoint and the spindle attachment checkpoint (SAC) at the metaphase-to-anaphase transition.
The first checkpoint will decide on the commitment of the cell to start the chromosome replication, and will arrest the cells in G1 if there is DNA damage (but also by contact inhibition, growth factor withdrawal and replicative senescence). The intra-S checkpoint checks for DNA damage and unfinished DNA replication (f.i. stalled replication forks). The role of the G2 to M checkpoint is mainly to check for DNA damage, through a pathway similar to the G1 checkpoint. Finally, the spindle checkpoint controls that all the centromeres are correctly attached to the mitotic spindle.
The different checkpoints are under the regulation of large protein complexes and cascades for activation, induction and controlled degradation. In most cells, arrest due to prolonged checkpoint activation will lead to apoptosis. Nevertheless, embryonic stem cells (ESC) of different species have been reported to be able to escape apoptosis by overriding the checkpoint (checkpoint adaptation, Syljuasen, 2007) or to have checkpoint uncoupling or an inactive checkpoint. G1 checkpoint uncoupling has been found in Rhesus monkey (Fluckiger, 2006), mouse (Aladjem et al., 1998) and human ES (Qin et al., 2007). The G2 checkpoint has not yet been studied in detail in stem cells, although the results of Chuykin et al. (2008) on mouse ESC suggest an active DNA damage detection system at this stage. The SAC has been shown to be active in mESC, but the cells proved able to escape the arrest and avoid apoptosis (Mantel et al., 2007). All the checkpoints appear to become fully active and are able to induce apoptosis upon differentiation of the cells.
On the other hand, the cell cycle control in human preimplantation embryos is largely unknown. The high incidence of aneuploidy in human embryos may lead to think that they may lack the SAC or are able to undergo checkpoint adaptation.
hESC are derived from the inner cell mass of preimplantation embryos, and have often been suggested as a good model for the study of early human development. We hypothesize that the three cell cycle checkpoints are inactive during human preimplantation development, possibly only during the cleavage stages, or even only before the activation of the embryonic genome which in the human occurs during the four-cell stage (Caufmann et al., 2006). The SAC would be the first checkpoint to be fully functional (possibly at late cleavage-stage or blastocyst stage, which could explain the ability of self-correction) and the other two checkpoints would be activated later in development, the further the cells differentiate, in analogy to hESC, where at least one of the remaining checkpoints (the G1) is fully functional in differentiated hESCs, but not in the undifferentiated state.
The aims of this project are:
* Comparatively investigate key elements of the cell cycle checkpoints in human preimplantation embryos and human embryonic stem cells and evaluate hESC as models for early human development.
* Explain the high chromosomal instability of hESCs and improve the culture conditions to minimize chromosomal abnormalities.
* Explain the high aneuploidy rates in human embryos, unravel their mechanism of origin and self-correction and the significance of their presence, and possibly provide a reliable and easy screening tool to replace PGS.
Human embryonic stem cells (hESC) are derived from preimplantation embryos, and hold a great promise in the field of regenerative medicine because of their potential to develop into any cell type of the adult individual. Intensive research over the past 10 years has yielded numerous protocols for the differentiation of hESC into adult cell types (f.i. insulin-producing pancreatic cells, Kroon et al., 2008). Currently, one of the important issues is the genomic stability of hESC during culture. We (Spits et al., 2008) and others (Baker et al., 2007) have shown that hESC are prone to chromosomal abnormalities, including aneuploidies, fragile site expression and small duplications with a hotspot at 20q21.11. This genomic instability strongly reminds of the behaviour of cancer cells, and is of great concern for the use of hESC for therapeutic purposes or as reliable research models. Nevertheless, little is know about the mechanisms and the influence of the culture conditions on the origin of these abnormalities.
We hypothesize that human embryos and stem cells have a cell cycle that differs from somatic cells in their checkpoints, and that this may be responsible for the proneness of these cells to chromosomal abnormalities.
The eukaryotic cell cycle contains the following four checkpoints: the start checkpoint in late G1, the S phase checkpoint, the G2 to M checkpoint and the spindle attachment checkpoint (SAC) at the metaphase-to-anaphase transition.
The first checkpoint will decide on the commitment of the cell to start the chromosome replication, and will arrest the cells in G1 if there is DNA damage (but also by contact inhibition, growth factor withdrawal and replicative senescence). The intra-S checkpoint checks for DNA damage and unfinished DNA replication (f.i. stalled replication forks). The role of the G2 to M checkpoint is mainly to check for DNA damage, through a pathway similar to the G1 checkpoint. Finally, the spindle checkpoint controls that all the centromeres are correctly attached to the mitotic spindle.
The different checkpoints are under the regulation of large protein complexes and cascades for activation, induction and controlled degradation. In most cells, arrest due to prolonged checkpoint activation will lead to apoptosis. Nevertheless, embryonic stem cells (ESC) of different species have been reported to be able to escape apoptosis by overriding the checkpoint (checkpoint adaptation, Syljuasen, 2007) or to have checkpoint uncoupling or an inactive checkpoint. G1 checkpoint uncoupling has been found in Rhesus monkey (Fluckiger, 2006), mouse (Aladjem et al., 1998) and human ES (Qin et al., 2007). The G2 checkpoint has not yet been studied in detail in stem cells, although the results of Chuykin et al. (2008) on mouse ESC suggest an active DNA damage detection system at this stage. The SAC has been shown to be active in mESC, but the cells proved able to escape the arrest and avoid apoptosis (Mantel et al., 2007). All the checkpoints appear to become fully active and are able to induce apoptosis upon differentiation of the cells.
On the other hand, the cell cycle control in human preimplantation embryos is largely unknown. The high incidence of aneuploidy in human embryos may lead to think that they may lack the SAC or are able to undergo checkpoint adaptation.
hESC are derived from the inner cell mass of preimplantation embryos, and have often been suggested as a good model for the study of early human development. We hypothesize that the three cell cycle checkpoints are inactive during human preimplantation development, possibly only during the cleavage stages, or even only before the activation of the embryonic genome which in the human occurs during the four-cell stage (Caufmann et al., 2006). The SAC would be the first checkpoint to be fully functional (possibly at late cleavage-stage or blastocyst stage, which could explain the ability of self-correction) and the other two checkpoints would be activated later in development, the further the cells differentiate, in analogy to hESC, where at least one of the remaining checkpoints (the G1) is fully functional in differentiated hESCs, but not in the undifferentiated state.
The aims of this project are:
* Comparatively investigate key elements of the cell cycle checkpoints in human preimplantation embryos and human embryonic stem cells and evaluate hESC as models for early human development.
* Explain the high chromosomal instability of hESCs and improve the culture conditions to minimize chromosomal abnormalities.
* Explain the high aneuploidy rates in human embryos, unravel their mechanism of origin and self-correction and the significance of their presence, and possibly provide a reliable and easy screening tool to replace PGS.
Date:1 Jan 2010 → 31 Dec 2013
Keywords:reproductive genetics, andrology, clinical genetics, embryology, assisted reproductive technology
Disciplines:Basic sciences, Biological sciences