Funding opportunities

Genetic Instability of Pluripotent Cells and the Impact on Differentiation Capacity

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01342
Funds requested: 
$1 072 562
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
This proposal addresses the genetic stability of human pluripotent stem cells (hPSCs), a fundamental aspect of stem cell biology that has important long-term implications on the clinical usefulness of these cells. Due to their abilities to proliferate indefinitely and to produce a wide variety of cell types, hPSCs have incredible potential as tools for cell therapy, biomolecule production, and drug development. However, in order for these cells to be useful, they must stably maintain useful characteristics (such as the ability to differentiate into a desired cell type) and not acquire dangerous properties (such as the ability to form malignant tumors). Since genetic changes can lead to changes in cell behavior, and since cancer is associated with genetic instability, we would generally prefer to use genetically normal cells. Our laboratory has a longstanding interest in this area, as shown by our Preliminary Results, which present the results of a pilot study on 40 samples, including 20 hESC samples from 14 hESC lines. We used a microarray technique to identify areas of the genome that were preferentially duplicated in human embryonic stem cells (hESCs) compared to differentiated cells and tissues. In this work, we found that genomic duplications were significantly more common in hESC cultures than in the other samples. Many of these genetic abnormalities were too small to be detected by standard cytogenetic methods, such as karyotyping. The most striking finding was that 5 out of 14 hESC lines contained a duplication of the pluripotence-associated gene, NANOG. These early results suggested to us that much more work in this area is warranted. In this project, we will use a similar microarray method to map genetic aberrations in an expanded set of cell lines. This set of cells will be large enough to allow the identification of genetic aberrations that are significantly more likely to be found in pluripotent stem cells compared to other cell types. We will also determine whether the same genomic changes are found in hESCS and iPSCs. We will also test the ability of next-generation sequencing to probe for aberrations in that are not detectable by the microarray method. We will determine whether there are any differences in the frequency and type of genetic aberrations that occur at different stages of hPSC derivation and culture (short-term, medium-term, and long-term). Finally, we will determine the effect of common genetic aberrations on the abilities of hPSCs to proliferate, differentiate, and form tumors. In summary, we propose to use advanced genomic and cellular methods to determine which genetic aberrations are commonly found in hPSCs, to understand the factors that influence how frequently these aberrations arise, and to assess the effects of common aberrations on important properties, such as differentiation and tumor formation.
Statement of Benefit to California: 
The size and diversity of California's population presents a challenge to scientists and clinicians who hope to contribute to the future of medical care in this state. Fortunately, California has a tradition of being a leader in terms of medical and scientific research, technology development, and bringing new products to patients and consumers. Approximately 20,000 Californians await organ transplants, and more than a million have progressive degenerative diseases such as Alzheimer disease, Parkinson’s disease, neuromuscular diseases such as amyotrophic lateral sclerosis (ALS) and muscular dystrophy, chronic liver disease, and diabetes. The possibility of applying cell replacement therapy to these problems could dramatically improve the length and quality of life for those who suffer from incurable diseases and life threatening injuries. Human pluripotent stem cells can differentiate to many different cell types in the body, and thus hold promise as the source of cells for these therapies. The research community has the responsibility to make human embryonic stem cells as safe and effective as possible for cell therapy, by ensuring that they retain normal, noncancerous qualities. California scientists have made tremendous progress toward clinical applications of pluripotent stem cells by developing new ways to derive these cells and to differentiate them into cell types that can be used to replace damaged tissues. However, we must also understand the genetic stability of human pluripotent stem cells in order to ensure that the cells used for cell therapies do not form tumors in patients. In this project, we will identify the types and frequencies of genetic anomalies present in human pluripotent stem cells, the factors that increase and decrease the genetic stability of these cells, and which genetic anomalies are potentially harmful. In carrying out this research, we will be contributing to California's economy. The vast majority of the supplies we will be using for this project will be sourced from California companies. In addition, we will be hiring new personnel and providing technical training. Since [REDACTED] collaborates closely with [REDACTED], and [REDACTED] laboratory will be hosting interns from the CIRM Bridges Program, interns can choose to participate in this project as part of their training.
Review Summary: 
The goal of this proposal is to investigate the genetic instability of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) in culture and the effects of genetic aberrations on the differentiation potential and tumorigenicity of these cells. In Aim 1, the applicant proposes to analyze a large number of hESC and iPSC lines using high resolution single-nucleotide polymorphism genotyping followed by copy number variation (CNV) analysis, and compare genetic aberrations in these lines to those found in other cultured cells, such as adult stem cells, primary cell lines and in tissue samples. In Aim 2, the applicant proposes to determine when genetic aberrations in pluripotent cells are likely to occur: during embryogenesis or hESC derivation, during iPSC generation, or during long-term culture of these cells. This will be accomplished by comparing early passage hESC lines to their parental DNA, as well as by comparing multiple hESC lines derived from several embryos donated by the same couple and multiple iPSC lines derived from the same fibroblast cell line. Finally, in Aim 3, the applicant proposes to examine the effects of genetic aberrations on the potential of hESCs and iPSCs to form teratomas and to undergo differentiation, both undirected differentiation to embryoid bodies (EBs) and directed differentiation to oligodendrocyte precursor cells. The reviewers agreed that this proposal is focused on an important problem that has largely been ignored, but they were concerned that the resulting data would be more qualitative than quantitative, limiting its impact. Given the variety of genetic aberrations that may be detected, it is not clear that even the large number of cell lines proposed will lead to statistically significant results and allow correlation of individual aberrations with differentiation potential or tumorigenicity. Reviewers did not find the project to be particularly innovative, noting that the technologies proposed are standard in the field. They also commented that the proposal does not focus on a potential mechanism and does not present a testable hypothesis as to why hESCs and iPSCs might be more genetically unstable than other cell types. Reviewers agreed that the proposal is feasible and praised the substantial preliminary data. However, they raised a number of issues with the research design. Reviewers appreciated the large collection of cell lines and tissues available to the applicant for the studies in Aim1, but would have liked more information about how many samples come from early vs. late passages, or are available from multiple passages. With regard to Aim 2, reviewers noted that studies of the genomic stability of "normal" cells during culture are lacking, as are functional analyses of such cells before and after mutations. They felt that it is crucial to understand the rate that cells usually acquire mutations in culture, the nature of these mutations and their effects, so that a good set of negative controls can be established to compare to the genetic changes that occur during the derivation and long-term culture of hESCs and iPSCs. They also noted that the identification of a higher frequency of genomic changes in hESC may not be due to increased genetic instability but rather may result from selection for improved growth in culture. Reviewers further cautioned that current algorithms may not detect CNVs accurately. The vast increase in CNV detection the applicant cites using one method when compared to another may be due to false positives, rather than improved sensitivity, and it was recommended to validate interesting changes using another method, such as quantitative PCR. With regard to Aim 3, reviewers were concerned that the strategy of comparing cells that have acquired genetic aberrations to others of the same line that have not fails to take epigenetic differences into account, and a more direct functional test that involves the targeted introduction of the observed genetic change would be more informative. Reviewers also commented that pitfalls and alternative approaches could have been described in more detail. Reviewers generally agreed that the applicant is qualified to perform the work proposed. They differed in their opinions about the track record of the applicant, who is a very junior investigator. For a junior investigator, the applicant was considered quite accomplished. However, reviewers were unclear about the investigator's independence, given that the work would be performed in another investigator's laboratory, but were reassured by a strong letter of support from this investigator. Reviewers praised the caliber of the collaborators but noted their low percent efforts. One reviewer suggested the addition of a genomic computational analysis expert to the research team, particularly one sufficiently informed of the limitations of existing CNV analysis programs. In summary, reviewers acknowledged the significance of the scientific problem addressed by this proposal and praised its preliminary data, but they questioned the potential impact and raised a number of significant concerns about the research plan.
Conflicts: 

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