Human embryonic stem cells (hESCs) have important potential in the treatment of human disease. Because they can change into a large number of different cell types, they may be useful in restoring a variety of damaged tissues. One potentially harmful side effect of hESC therapy is cancer due to unregulationed growth of the hESCs introduced in the body. hESCs have the potential to grow almost indefinitely. Therefore if they should become "transformed" into cancer cells while being cultured in the laboratory, they may cause cancer in the individuals into which they are injected. Transformation of normal cells into cancer cells can occur through changes in their DNA, which contains the information telling cells to grow or not to grow. Because multiple changes must occur for cells to begin the unchecked growth of cancer cells, the likelihood of cancer is low. However, some cellular changes can increase the rate at which subsequent changes occur, which greatly increases the probability that a cell will acquire all of the changes necessary to become a cancer cell. This increased rate of changes in DNA is called genomic instability, which is proposed to be an early step in many cancers. One mechanism by which genomic instabiiity can occur is through the loss of the caps that protect the ends of chromosomes that contain the DNA. Loss of these caps, called telomeres, can make the DNA highly unstable. This proposal will study whether the loss of telomeres is a cause of instability in hESCs during their growth in the laboratory. Information on this process will allow steps to be taken to avoid this potential harmful effect during hESC therapy.
Statement of Benefit to California:
Human embryonic stem cells (hESCs) have important potential in the treatment of human disease. Because they can change into a large number of different cell types, they may be useful in restoring a variety of damaged tissues. This study will investigate a potentially harmful side-effect involving genetic changes that may occur during growth of hESCs in the laboratory that could lead to cancer when they are injected into people. Understanding the process involved in generating these genetic changes will allow scientists to avoid them and limit the likelihood of these complications in the clinic.
SYNOPSIS: The general goal of this proposal is to understand various factors that contribute to genomic instability of hESCs growing in vitro. The experiments focus primarily on the effect of spontaneous and double-strand DNA break (DSB)-induced telomere loss. In Aim 1 the applicant proposes to study telomerase and telomere maintenance, the status of cell cycle checkpoints, and chromosome stability under different culture conditions in hESCs. In Aim 2 the applicant will create hESC lines containing a telomeric selectable marker or a sub-telomeric recognition site for the I-SceI endonuclease. In Aim 3 these lines will be used to determine the spontaneous rate of telomere loss in cultured hESCs, the frequency of telomere restoration after telomere loss, and the frequency of chromosome abnormalities (especially those induced by breakage/fusion/bridge cycles) following telomere loss.
SIGNIFICANCE AND INNOVATION: The genetic stability of hESC lines is unclear. Some reports have documented chromosome abnormalities or even highly unstable karyotypes, whereas others found no evidence for this. The difference may lie in culture conditions, but this too is unclear. Mouse ESCs lack the p53 DNA damage checkpoint, but under normal conditions this does not appear to result in a high degree of genetic instability. However extrapolating those observations to humans is difficult. Human telomeres are shorter than those in murine ESCs, and it is not know whether the p53 checkpoint is operative in hESCs. Thus, the extent of chromosome instability in hESCs, and the conditions that affect it remain uncertain.
Chromosome instability in hESCs during propagation in vitro would potentially limit their usefulness for cell transplantation and tissue repair because the chromosome changes could be initiating events in tumorigenesis. Previous work from the applicant’s laboratory has demonstrated DNA breaks near telomeres of murine ES cells lead to chromosome instability, characterized by B/F/B cycles, and ultimately chromosome healing by the acquisition of new telomeric DNA. The frequency of telomere loss in hESCs is not known, nor is it known how hESCs respond to telomere damage. Prior to the use of hESCs in therapies it will be important to determine the extent to which they undergo chromosome instability and to identify specific methods for propagating hESCs that either enhance or diminish these chromosome changes.
The significance of this work is based upon the presumption that chromosomal instability and telomere loss is an important issue for hESCs. The arguments are not strong. ESCs have a robust mechanism for maintaining telomeres and thus chromosome stability. While DSB imparted by ionizing radiation or long term culturing could impair telomere maintenance, there is no strong evidence that this is a practical issue in hESC research or has potential use in hESC for therapeutic purposes.
STRENGTHS: The applicant has a long and productive history studying chromosome instability, in particular instability caused by DNA breaks near chromosome ends. He is also the discoverer of the ALT pathway of telomere maintenance. The technology and concepts that form the basis for this application have previously been developed and tested in murine stem cells. For example, the applicant has previously shown that murine ESCs, which lack a p53-dependent G1 checkpoint, continue to proliferate and show chromosome abnormalities in response to telomere erosion. He has also developed in murine ES cells the technology necessary for simultaneously monitoring the rate of telomere loss, the chromosome instability that results from telomere loss, and the mechanism of acquisition of new telomeres. It would not be surprising if there are significant differences between human and mouse ESCs with respect to telomere maintenance and responses to telomere damage. Therefore extending these studies to human cells is important.
The strength of this proposal is the investigator's experience in telomere biology. Certainly the issues the PI raises are important biological problems that are worthy of study. However, it is not established that this type of research on hESC will make important contributions to our understanding of hESC biology.
WEAKNESSES: The applicant correctly emphasizes that culture conditions may affect the baseline genetic instability in hESCs, and proposes to determine whether specific conditions increase or decrease chromosome aberrations. However, only two culture conditions will be tested, either bulk or mechanical processing by standard methods. The strength of the application would be increased if the applicant were able to suggest specific culture conditions that could be tested which might mitigate the extent of background of spontaneous instability. Otherwise, the immediate practical benefit of these data may be less obvious.
The major weakness is the practical relevance to hESC research. The authors make the best case they can for relevance, but is not very convincing. In addition, many experiments are incremental in significance. For example, it is well known that ESCs contain telomerase. Yet the PI proposes to assay for telomerase in hESCs. While this is good and prudent action in the normal course of achieving a larger aim, it should not be the major goal of the aim. Thus, aim 1 is not substantial. Aim 2 proposes to place a reporter gene near telomeres in hESCs, as has been done in other cell lines. Again this strikes me as more of a technical step rather than a substantial aim.
DISCUSSION: In general there was not much enthusiasm for this proposal because the importance of the work in uncertain. Why should one study telomere instability in hESCs? How important is an understanding of cell cycle checkpoints in hESCs? In addition, the Aims were not very substantial. Aim 1 presents descriptive experiments that yield data worth knowing, but only two culture conditions are being examined. Aim 2 is potentially problematic since it requires homologous recombination, and Aim 3 depends on the success of Aim 2 in order to create telomere loss.
The applicant is a great investigator with a good track record and has a long and productive history studying chromosomal instability including in mouse ESCs. One of the reviewers questioned the emphasis of the proposal given the reviewer's uncertainty about the importance of chromosomal instability in hESCs. Another reviewer noted that the applicant proposes prolonged growth of hESCs over 30-60 generations. This reviewer considers understanding stabilty as important and believes the proposed study will provide really practical knowledge.