Genetic Instability of Pluripotent Cells and the Impact on Differentiation Capacity
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.
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.