Funding opportunities

Sources of Genetic Instability in Human Embryonic Stem Cells.

Funding Type: 
SEED Grant
Grant Number: 
Principle Investigator: 
Funds requested: 
$357 978
Funding Recommendations: 
Recommended if funds allow
Grant approved: 
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
SYNOPSIS: The general goal of this proposal is to compare the efficiency of various DNA repair processes in human ES cell lines to the efficiency of those same processes in human embryonic and somatic fibroblasts. The applicants will use transfection of exogenous DNA templates into these cell lines to specifically examine nucleotide and base excision repair, mismatch repair and double-stranded DNA break repair. In addition they will determine the rates of spontaneous and induced mutations in a marker gene stably propagated on an EBV-derived episome. SIGNIFICANCE AND INNOVATION: This research rates highly for significance. There is anecdotal evidence that human ES cells grown in vitro display genetic instability, primarily in the form of acquired aneuploidy, and alterations in DNA methylation patterns have also been observed. Since the integrity of various DNA repair pathways in these cells has not been thoroughly investigated, the proposed research has the potential to yield important insights into the mechanisms influencing the genetic stability of hESCs. The genetic stability of these lines is critical to developing strategies to produce the most effective and consistent therapeutic products. While the methodology proposed to assess DNA repair mechanisms is well-established, the innovation comes from the application of these techniques to the study of human ES cultures. STRENGTHS: There is very little known about the integrity and efficiency of DNA repair processes in ES cells. The experiments proposed here will, therefore, provide valuable new information. The applicants have considerable experience in ES cell culture and have made important contributions to the study of DNA repair and mutagenesis. The overall approach to study the genetic instability of human ESC cultures has high feasibility and is well-designed. The investigator has pilot data demonstrating the feasibility of each of the assay systems proposed for use in the study. WEAKNESSES: Despite the paucity of work in this important area, the applicants do not make a compelling case that insight into DNA repair processes in ES cells is a pressing issue that will have any immediate or even long-term practical benefit. Acquired aneuploidy may be a problem, but evidence for this is anecdotal rather than quantitative, and it remains to be seen whether it is more severe in ES cells than in any other cell line grown for many passages in vitro. Moreover, it is not evident that aneuploidy would be directly related to any of the repair processes that will be studied under this application. A key technical concern is that the assays for DNA repair primarily involve transfection of large amounts of exogenous DNA. This raises concerns about whether the efficiency of repair measured on these abundant, exogenous templates quantitatively relates to how effectively the cell processes damage to its own genome. There were two other rather minor weaknesses. First, two of the assays may not be sufficiently sensitive to detect defects in DNA repair if the cultures are not homogeneous in their defects. The investigator may want to determine the sensitivity (using spiking of known defective cells into WT cultures) for the Dual luciferase assay to detect DNA repair capacity using host cell reactivation and the double-strand break assays. These other two assays seem to be more sensitive and quantitative and more likely to be able to detect heterogeneity in the culture. Second, why compare the human ESC lines to what appear to be unrelated somatic cultures (unrelated in terms of not coming from the same donor)? In the absence of being able to obtain somatic cells from the same donor, it seems more reasonable that the investigator differentiate the human ES cells and compare the differentiated cells to the undifferentiated ES cells. DISCUSSION: Very little is known about DNA repair in hESC, and this experienced investigator proposes a feasible and well-designed approach to investigating these processes. The innovation of this approach is the application of the proposed techniques to address this important question. The major concern is that there is only anecdotal, rather non-compelling evidence for genetic instability in hESC

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