Genetic Instability of Pluripotent Cells and the Impact on Differentiation Capacity

Funding Type: 
Basic Biology I
Grant Number: 
ICOC Funds Committed: 
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Embryonic Stem Cell
Directly Reprogrammed Cell
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.
Progress Report: 
  • During this year, we have demonstrated that hematopoietic stem cells are originated from the cells that line the inside of blood vessels, named endothelial cells. Budding of hematopoietic stem cells from endothelial cells occurs during a specific and restricted time window during development and progress has been made to elucidate the regulatory genetic networks involved in this process. We have also demonstrated that hemogenic endothelium is derived from one specific embryonic tissue (lateral plate mesoderm). This information will be used to recapitulate similar conditions in vitro and induce the growth of hematopoietic stem cells outside the body from adult endothelial cells.
  • The objective of this proposal was to identify factors that allow blood vessels to generate hematopoietic stem cells early in the embryonic stage. The process of blood generation from vessels is a normal step in development, but it is poorly understood. We predicted that precise information related to the operational factors in the embryo would allow us to reproduce this process in a petri dish and generate hematopoietic stem cells when needed (situations associated with blood transplantation or cancer).
  • In the second year of this proposal, we have made significant progress and identified critical factors that are responsible for the generation of hematopoietic stem cells from the endothelium (inner layer of blood vessels). These experiments were performed in mouse embryos, as it would be impossible do achieve this goal in human samples. The genes identified are not novel, but have not been associated with this capacity previously. To verify our findings we have independently performed additional experiments and validated the information obtained from sequencing the transcripts.
  • In addition, we developed a series of novel tools to test the biological relevance of the genes identified in vivo (using mouse embryos). Specifically, we have tested whether forced expression of these genes could induce the generation of hematopoietic stem cells. Interestingly, we found that a single manipulation was not sufficient, but multiple and specific manipulations resulted in the generation of blood from endothelium. This was a very exciting result as indicated that we are in the right track and identified factors that can reprogram blood vessels to bud blood stem cells. With this information at hand, we moved into human cells (in petri dishes).
  • The first step was to test whether human endothelial cells could offer a supportive niche for the growth of hematopoietic cells. To our surprise, we found that in the absence of any manipulation, endothelial cells could direct differentiation and support the expansion of CD34+ cells (progenitor blood cells) to a very specific blood cell type, named macrophages. These were rather unexpected results that indicated the ability of endothelial cells to offer a niche for a selective group of blood cells. The final question in the proposal was to test whether the modification of endothelial cells with the identified factors could induce the formation of blood from these cells. For this, we have generated specific reagents and are currently performing the final series of experiments.
  • In this grant application we have been able to investigate the mechanisms by which endothelial cells, the cells that line the inner aspects of the entire circulatory system, produce blood cells. This capacity, called “hemogenic” (giving rise to blood) can be extremely advantageous in pathological situations when generation of new blood cells are needed, such as during leukemia or in organ-transplantation. Although the hemogenic capacity of the endothelium is, under normal conditions, restricted development we have been able to “reprogram” this ability in endothelial cells. For this, we first investigated the genes that responsible for this hemogenic activity during development using mouse models and tissue culture cells. Using this strategy we found key transcription factors in hemogenic endothelium not present in other (non-hemogenic) endothelial cells. Subsequently, we validated that these genes were able to convey hemogenic capacity when expressed in non-hemogenic sites. Finally, using human endothelial cells, we have been able to impose expression of these key transcription factors in endothelial cells. Our data indicates that the forced expression of these factors is able to initiate a program that is likely to result in blood cell generation. The progress achieved through this grant place us in a remarkable position to carry out pre-clinical trials to evaluate the potential of this technology.

© 2013 California Institute for Regenerative Medicine