We propose to derive new human embryonic stem (hES) cell lines, and begin an investigation of epigenetic abnormalities in these lines. Epigenetics refers to changes in the behavior of genes that are not caused by changes in the DNA itself (i.e. mutations) but rather by alterations to protein and chemical (methyl) groups bound to the DNA. The overall goals of this project are to develop the capacity to derive hES cell lines, and to perform pilot projects on the natural history of epigenetic marks in hES cells and the body cells derived from them. The project builds on our efforts to establish the capacity to derive hES lines in collaboration with an IVF facility. In keeping with the goals of CIRM, this proposal is intended to serve as the nucleus for a program of ES cell research; we think that it is important that this program have the ability to derive ES cells. It is often assumed that ES cells do not retain epigenetic marks acquired in previous generations. Some evidence indicates that epigenetic marks can arise in the human germline and cause a disease syndrome; they may also be inherited. Epigenetic inheritance requires that an epigenetic mark be retained in cells of the inner cell mass, and thus presumably in ES cells as well. This raises the possibility that individual ES cell lines carry epigenetic marks, either inherited from the parent or acquired in the derivation process, that are capable of affecting the phenotype of their progeny somatic cells. We propose a pilot project to investigate this hypothesis. To prepare for the derivation of hES cell lines, we have established a collaboration with an IVF facility, which provides us with access to extensive expertise in human embryo manipulation. We have recruited a technician with substantial ES cell experience, which we have extended by further training, and are ready to begin derivation of hES cell lines. We propose to derive new hES cell lines from spare embryos by established methods; these will be used in our own studies, and will also be made available to the wider research community. In the hES lines we derive, we will study the occurrence and natural history of epigenetic abnormalities. We will identify epigenetic marks using antibodies, then choose likely candidates for further analysis. We will study several such marks in multiple hES lines. Because there is a new area of study, it is difficult to predict just what we will find; this is entirely appropriate for a pilot study.
Statement of Benefit to California:
Human embryonic stem (hES) cells hold tremendous promise for the alleviation of human suffering, because they are able to form new cells that can be used to replace cells that are defective due to inherited or other types of disease, and because by studying them we may learn how to change the behavior of cells within the human body. If cells derived from hES cells are to be useful, we must develop ways to assess not just their efficacy, but their safety, because cellular therapies may require implanted cells to survive indefinitely in a patient’s body. We are aware that one possible source of defects in ES cells are epigenetic changes, which alter the behavior of genes without changing the DNA of those genes. Epigenetic defects can predispose to diseases such as cancer. We need methods to assess hES cells for such defects and understand their natural history and consequences; we are proposing a pilot project that begins to explore ways of detecting and analyzing them. As part of this effort, we also propose to derive new hES cell lines from spare embryos donated for research. These new hES lines will be made available to other investigators for their projects. Thus this project will benefit California by producing new hES lines, which are badly needed to advance stem cell research generally, and by exploring methods to assess the integrity of existing hES cell lines.
SYNOPSIS: The general goal of this proposal is to study the occurrence of epimutations, in the form of DNA methylation, in human ES cell lines. In aim 1, the applicant will derive new human ES cell lines. In Aim 2 the applicant will use global approaches to determine the pattern of chromosomal DNA methylation, and then bisulfite conversion to precisely map methylation sites at specific loci. INNOVATION AND SIGNIFICANCE: This proposal seeks to test a hypothesis that epimutations may arise during hES cell line derivation or be propagated by hES cell line cultivation. The approach is cutting edge in their use of genome-wide ChIP assays using a methyl-cytosine antibody for immunoprecipitation and genome-wide tiling array as readout. The group has well-documented expertise in DNA methylation analysis using bisulfite treated DNA for specific locus analyses. The plan to perform genome-wide DNA methylation analysis of hES cells is not innovative per se, as many groups have and are pursuing variations on this type of approach. What sets this proposal apart is the testing of a specific hypothesis regarding the origin of epimutations in the germline as compared to de novo epimutations occurring in cell culture during line establishment. STRENGTHS: The strength of the proposal is the excellent track record of the collaborating PIs in their respective fields of origins and biology of epimutations and reproductive biology. The applicant has made important contributions to the study of epigenetic changes in mammalian genomes and now proposes to extend his studies to ES cells. In addition, the team has recruited a skilled technician who has the training to carry out the proposed new line derivation, although this skill is currently untested due to lack of funding for the project. WEAKNESSES: The primary weakness of this proposal is that the experimental design does not seem to have the power to address the hypothesis being tested. In particular, the high cost of the platform chosen for unbiased global methylation analysis precludes reiterative analysis of the same sample to provide the necessary statistical evaluation of the quality of the dataset. This proposal would be significantly strengthened if it aimed to evaluate existing lines for possible sites of epimutation, rather than leap into the bigger question of their origin. It is necessary to demonstrate the assay has the power and sensitivity before trying to tackle this larger and very interesting question. In addition, there was no estimation of the frequency of new epialleles created during gametogenesis, and the likelihood these would be identified within a single or few ES cell lines with the limited experimental plan. The project goals do not appear feasible as proposed. Aim 1 is entirely technical, and moreover the applicant has no prior experience in this technology. There is scant justification for supporting this under this funding mechanism. The majority of Aim 2 uses array technology with which the applicant is unfamiliar, and therefore proposes to subcontract this work to a biotechnology company. The only work under this Aim which will actually be done in the applicants lab is bisulfite conversion DNA sequencing to precisely determine the sites of DNA methylation at loci identified with the micoarrays. These experiments seem premature, as the more biologically important issues would remain to be addressed. Thus, the applicant proposes no experiments to address whether DNA methylation has affected gene expression, nor whether this has any impact on the functional properties of the ES cells. Without this knowledge, further information about the exact nucleotides that are methylated seems unnecessary. Indeed, it has alread been reported that human ES cell lines can have unique patterns of DNA methylation (Bibikova et al., 2006), and it is not evident how this proposal represents a substantial advance over this prior work. DISCUSSION: There was no further discussion following the reviewers' comments.