Human embryonic stem cells (hESCs) hold great promise for treating many human diseases because of their ability to become any type of cell in the human body. In order to choose the optimal hESCs for therapeutic use in various diseases, it is critical to characterize fully the safety and potential of different stem cell lines. Chromosomes are large packages of DNA within a cell, and changes to a cell’s chromosomes can affect its function. Large scale chromosome changes include: the loss and gain of whole chromosomes, termed aneuploidy; partial loss of chromosomes, deletion; and movement of large chromosome segments between chromosomes, translocation. As chromosomal changes can dramatically alter the properties of many cell types, it is important to assess and compare the prevalence and consequences of chromosomal changes in hESC lines. Our proposed research will use a technique that fluorescently labels each chromosome pair within a cell with a unique color, to allow us to identify aneuploidy, deletions and translocations within stem cells. We will further observe whether extended culture time in the laboratory can influence the characteristics of chromosomes within stem cells. Once we know what changes are occurring at the chromosome level within a stem cell population, it is crucial to determine how these changes affect their capacity to become different cell types, including normal and/or tumor cells. The second part of our proposal will compare the ability of stem cell populations with or without chromosome changes, to survive, divide and become neurons. The data gained from this proposal will provide key information for future stem cell research regarding the therapeutic potential and safety of hESCs.
Statement of Benefit to California:
The California Institute of Regenerative Medicine will be funding many research projects designed to use human embryonic stem cells for treating many human diseases, including Parkinson’s and Alzheimer’s diseases, diabetes and cancer. Clearly this research will benefit the lives of hundreds of thousands of Californians who are affected by these diseases, if it leads to safe and effective treatments. However, in order to spend California’s limited research dollars on the most promising research, it is vital to know which stem cell lines have the best therapeutic potential. Our proposal addresses this key issue through assessing genetic changes at the chromosome level that can influence the function and survival of different stem cell populations. Strong California-based expertise in the hESC field will attract biotechnology industries and top academic researchers to the area, bringing more jobs and money into the state of California. San Diego is currently a hotbed for stem cell research with multiple research institutions that have dedicated stem cell research programs, including The Scripps Research Institute and The Burnham Research Institute. The collaborative nature of our research proposal will foster high quality stem cell research in this region that is based on a clear understanding of the basic genomic characteristics of the cells being studied.
SYNOPSIS: The application proposes to evaluate the extent of aneuploidy and other chromosomal aberrations in human ES cell lines by spectral and DAPI karyotyping. The applicants will evaluate at least 100 metaphases from many different ES cell lines, both NIH-approved and non-NIH-approved. They will evaluate whether different growth conditions affect the extent of karyotypic abnormalities. In Aim 2 the applicants will correlate karyotype abnormalities with global patterns of gene expression, as well as differentiation potential. SIGNIFICANCE AND INNOVATION: The karyotypic stability of human ES cell lines has not previously been analyzed systematically. While the experimental approach is not innovative, the proposed study to map chromosomal mosaicism using a more sensitive method of spectral karyotyping and correlating the mosaicism with phenotypic effects in terms of gene expression and differentiation has the potential to yield very important data for the field of ES cell biology. To date, traditional karyotype studies have indicated a high degree of genetic stability, but if these studies contradict those previous results, it will be very important to track down the biological implications of those data - which is exactly what this investigator plans to do. In addition, the proposed studies will determine the utility of using spectral karyotyping in addition to current methodologies to assess the genetic stability and uniformity of different ES cell lines. STRENGTHS: The applicants are highly qualified to perform the proposed experiments. They have already demonstrated the utility of this technique to identify chromosomal abnormalities in other cell types where others did not (see their ref #7). Spectral karyotyping provides a level of resolution not offered by the more conventional karyotyping techniques that are standardly used. Therefore, there is a possibility that the investigators might uncover a degree of genetic instability not previously appreciated. In addition to their experience in using SKY, they are also experienced in culturing murine embryonic stem cells, and in collaboration with Dr. Jeanne Loring will have access to expertise in the culture and differentiation of human embryonic stem cells. The experiments proposed under specific Aim1 also seem highly feasible given the experience of the investigator in performing these analyses. The additional analysis under two different culture conditions and comparing three different passages (low, pass 10 and 25) also seem to be feasible. Using the information gained from Aim 1, the investigators will perform studies to correlate various anueploid genotypes to alterations in gene expression using microarray, or to altered ability to proliferate or differentiate. WEAKNESSES: Aim 1 may be useful to the extent that it could possibly raise the alarm that ES cells can have genetic changes that are invisible to more standard methods of karyotyping. Nevertheless, karyotype abnormalities are stochastic and will therefore differ among labs for a given cell line. Thus, the extent of these changes would still have to be determined by each lab working with a particular ES cell line. In any event, it seems likely any cell line that will be used therapeutically would be subject to at least this level of scrutiny and reviewers questioned the need for doing such an extensive, lab-specific survey at this point. The second part of this aim is to look at how culture conditions will affect karyotype instability. Unfortunately only two conditions will be tested, among the many possibilities. A key variable to look at would be the impact of low oxygen culture conditions (2%) on suppressing unwanted genetic instability. While the experiments in Aim 2 are feasible, the lack of an hypothesis makes this aim very weak. It seems obvious that changes in karyotype will affect gene expression, and the rationale for correlating global gene expression changes with random variations in karyotype is unclear. Moreover, differences among cell lines will probably mask any gene expression changes that arise as primary or secondary effects of chromosome abnormalities. Similarly, it seems very unlikely that useful biological information will emerge by comparing the differentiation potential of ES cell lines with random differences in their karyotypes. DISCUSSION: This proposal is interesting in the context of validation of this methodology for detection of chromosomal mosaicism. The Chun lab was the first to find a fair amount of aneuploidy in human brain cells, thus it may be somewhat interesting to raise the alarm that this could lead to diversity. The major criticism of the proposal is the overall hypothesis for Aim 2. Descriptive information provided by transcriptional profiling is unlikely to be very informative in the proposed chromosomal mosaicism studies. Although reviewers noted that this kind of study was not generally useful since it should be done for any given line at a given passage per lab, they felt that the first part of Aim 1 should be supported with high enthusiasm. Alerting the field to the extent of hidden genetic instability in their ES cells is very worthwhile. An application more focused on this aspect of the work would receive much stronger consideration if future funding opportunities arise.