Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

Mechanisms to maintain the self-renewal and genetic stability of human embryonic stem cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00148
ICOC Funds Committed: 
$2 570 000
Disease Focus: 
Cancer
Genetic Disorder
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESCs) are capable of unlimited self-renewal, a process to reproduce self, and retain the ability to differentiate into all cell types in the body. Therefore, hESCs hold great promise for human cell and tissue replacement therapy. Because DNA damage occurs during normal cellular proliferation and can cause DNA mutations leading to genetic instability, it is critical to elucidate the mechanisms that maintain genetic stability during self-renewal. This is the overall goal of this proposal. Based on our recent findings, I propose to investigate two major mechanisms that might be important to maintain genetic stability in hESCs. First, I propose to elucidate pathways that promote efficient DNA repair in hESCs. Second, based on our recent findings, I hypothesize that another primary mechanism to maintain genetic stability in self-renewing hESCs is to eliminate DNA-damaged hESCs by inducing their differentiation. Therefore, I propose to identify the pathways that regulate the self-renewing capability of hESCs in the presence and absence of DNA damage. In summary, the proposed research will contribute significantly to our understanding of the pathways important to maintain self-renewal and genetic stability in hESCs. This information will provide the foundation to improve the culturing condition of hESCs to promote efficient self-renewal with minimum genetic instability, a prerequisite for the development of hESCs into human therapeutics. One major objective of the proposed research is to improve the genetic manipulation technologies in hESCs, including transgenic and gene targeting technologies. While mouse models are valuable tools to study the mechanisms of the pathogenesis in human diseases, many differences between mouse and human cells can lead to distinct phenotypes as well as the common phenomenon that certain therapeutic interventions work well in mouse models but poorly in humans. Therefore, it is of high priority to create disease-specific hESCs as powerful genetic tools to study the mechanism of the pathogenesis in human diseases. In addition, the unlimited supply of primary cells derived from the disease-specific hESCs will become valuable reagents for drug discovery. There are two ways to generate the disease-specific hESCs. One approach is through nuclear transfer that has been proven extremely difficult in human context and so far unsuccessful. The other is to employ the transgenic and gene targeting techniques to create disease-specific hESCs. Therefore, the proposed research will significantly improve our capability to generate disease-specific hESCs. After experimenting with various existing hESC lines, we found that only the non-federally-approved hESC lines developed recently at Harvard University is most suitable for genetic manipulation technologies. Since the research involving the HUES lines can not be supported by federal government, CIRM is in a unique position to support this proposed research.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) are capable of unlimited self-renewal, a process to reproduce self, and retain the ability to differentiate into all cell types in the body. Therefore, hESCs hold great promise for human cell and tissue replacement therapy. The major goal of the human stem cell research supported by proposition 71 is to improve and even realize the therapeutic potential of hESCs. DNA damage occurs during normal cellular proliferation of hESCs and can cause genetic mutations that will be passaged to derivatives. Any cells with genetic mutations are not suitable for therapeutic purpose since they can cause cancers in the recipient. Therefore, to achieve the therapeutic potential of hESCs, it is critical to elucidate the mechanisms that prevent genetic mutations during the self-renewal of hESCs. This is the overall goal of this proposal. Successful completion of the proposed research will help to optimize the culturing conditions that promotes efficient self-renewal with minimum genetic instability. One high-priority area of hESC research is to create disease-specific hESCs, which can be used as powerful genetic tools to study the mechanism of the pathogenesis in human diseases. In addition, the unlimited supply of primary cells derived from the disease-specific hESCs will become valuable reagents for drug discovery. There are two ways to generate the disease-specific hESCs. One approach is through nuclear transfer that has been proven extremely difficult in human context and so far unsuccessful. The other is to develop the transgenic and gene targeting techniques to create disease-specific hESCs. One major objective of my proposed research is to improve the genetic manipulation technologies in hESCs, including transgenic and gene targeting technologies. The successful completion of the proposed research will significantly improve our capability to generate disease-specific hESCs. In addition, the disease-specific hESCs (ATM-/- and p53-/- hESCs) generated in the course of the proposed studies are valuable tools to study the basis of neuronal degeneration in Ataxia-telangiectsia and development of human epithelial tumors as a result of p53-deficiency. Both of these phenotypes are not observed in mouse models. In summary, the proposed research will benefit California citizens by contributing to the eventual realization of the therapeutic potential of hESCs.
Progress Report: 
  • The goal of this proposal is to investigate the mechanisms that maintain the genomic stability of human ES cells (hESCs). We are focusing on the tumor suppression pathways ATM and p53, which are well established guardians of the genome in differentiated cells. In addition, we are investigating the pathways that govern the self-renewal of hESCs, which might be coordinated with DNA damage responses to maintain the genomic stability in hESCs. During the reporting period, we made significant progress towards our goals. First, we developed high efficiency homologous recombination technology to successfully disrupted ATM and p53 in hESCs. Analysis of the mutant ES cells indicate the roles of ATM and p53 in maintaining genomic stability in hESCs. Second, we identified pathways that are important for the self-renewal of hESCs. Third, we employed the knock-in tech
  • The goal of this proposal is to investigate the mechanisms that maintain the genomic stability of human ES cells (hESCs). We are focusing on the tumor suppression pathways ATM and p53, which are well established guardians of the genome in differentiated cells. In addition, we are investigating the pathways that govern the self-renewal of hESCs, which might be coordinated with DNA damage responses to maintain the genomic stability in hESCs. During the reporting period, we made significant progress towards our goals. First, we developed a bacterial artificial chromosome based gene targeting technology that allows high efficiency homologous recombination in hESCs, and published the first homozygous knockout mutant hESCs in the world (Aims 1 and 3). This achievement, which was described in a publication in the top stem cell journal Cell Stem Cell, has attracted worldwide attention and will help to open up the entire field of hESCs (Song et al., 2010, Cell Stem Cell 6, 180-189). We employed the same technology to generate homozygous phosphorylation site knock-in mutant hESCs to study the mechanism underlying ATM activation in hESCs (Aim 3). Second, we identified a novel Pin1-Nanog pathway that is critical for the self-renewal of hESCs (Aim 2). Using small molecule compounds that inhibit this pathway, we were able to suppress the potential of ES cells to form teratomas. This finding, which is published in the Proceeding National Academy of Science, provides a druggable target to address the teratomas risk associated with the human ES cell based therapy (Moretto-Zita et al., 2010, PNAS, Epub 7/9). Third, to identify ES cell-specific DNA repair pathways, we have identified several ES cell-specific interaction between proteins and DNA breaks (Aim 3).
  • We have made several significant progresses during the past year. We found the important roles of p53 in the differentiation of hESCs. We also identified that Nanog is a major coordinator of the self-renewal and proliferation of ES cells. We found that ATM is important to maintain the genetic stability of cells differentiated from hESCs. In addition, we identified an important phosphorylation event in activating ATM in hESCs. Finally, we identified a novel pathway to activate DNA damage in ES cells.

Role of the tumor suppressor gene, p16INK4a, in regulating stem cell phenotypes in embryonic stem cells and human epithelial cells.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00444
ICOC Funds Committed: 
$639 150
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The roles of stem cells are to generate the organs of the body during development and to stand ready to repair those organs through repopulation after injury. In some cases these properties are not correctly regulated and cells with stem cell properties expand in number. Recent work is demonstrating that the genes that control stem cell properties are sometimes the same genes that are mutated in cancer. This means that a cell can simultaneously acquire stem cell properties and cancer properties. In order to effectively use stem cells for therapeutic purposes we need to understand the link between these two programs and devise ways to access one program without turning on the other. In other words, we would like to expand stem cell populations without them turning into cancer. Recent work in our laboratory has found that the reduction of a specific tumor suppressor gene, p16, not only removes an important barrier to cancer but also confers stem cell properties within the cell. Cells that have reduced p16 activity can turn on a program that increases and reduces expression of specific genes that control differentiation. In this proposal we will test whether the continued reduction of this tumor suppressor gene creates human embryonic stem cells (hESC) that are unable to differentiate. We hypothesize that the lack of p16 represses multi-lineage potential by activating an epigenetic program and silencing genes that drive differentiation. To test this hypothesis we will first determine if lack of p16 activity is necessary for hESCs to develop into different cell types. Second, we will determine if continued lack of p16 activity is sufficient to inhibit differentiation of hESCs. Finally, we will determine if transient lack of p16 activity is sufficient for a non-stem cell to exhibit properties of a stem cell after propagation in a stem cell niche. Since these types of events are potentially reversible, targeting such events may become clinically useful. These new observations identify novel opportunities. They provide potential markers for determining if someone is susceptible to cancer, as well as, providing potential targets for prevention and therapy. We hypothesize that these properties are critically relevant to the formation of cancer and will provide insights into the role of epigenetic modifications in disease processes and stem cell characteristics.
Statement of Benefit to California: 
Stem cells hold great potential to help us in repairing injured body parts or replacing damaged organs. In order to realize this potential the rules that control stem cell behavior need to be understood. Recent work is demonstrating that the genes that control stem cell properties are sometimes the same genes that are mutated in cancer. In the proposed study we hypothesize that we may learn about a fundamental switch that not only controls stem cells but also controls the formation of a cancer cell. In understanding how this switch works we may be able to identify biomarkers that indicate when a normal looking cell will become a cancer cell or identify a drug that will allow us to stop the potential cancer cell from increasing in number. Since cancer is a common disease in California, any insights we can gain to battle this disease will benefit the citizens of our State. There is also another side to the insights that may arise from the work in this proposal. Currently we believe the roles of stem cells are to generate the organs of the body during development and to stand ready to repair those organs through repopulation after injury. We do not know how to encourage a stem cell to repair, for example, some heart tissue rather than some bone tissue. If we could understand the code that directs the stem cells to differentiate in the proper fashion into one tissue or another, we could use these cells for clinical benefit. The pathways we are studying in this proposal tell the stem cells which genes to silence and which to activate. This is the program that allows the one original cells of your body (the embryo) to diversify into the multitude of specialized cells that work together to make a functioning person (eye cells, skin cells, nerve cells, etc.). In order to effectively use stem cells for therapeutic purposes we need to understand how they code their decisions and whether they can be changed after they have been set. These insights would allow us to aid in maintaining the health of the citizens of California. Finally, if we do gain insight into the code that regulates the differences between cancer cells and stem cells, this information would be the basis of a new area of biotechnology. The generation of knowledge in this area would help in the development of companies, the recruitment of bright young minds and in the fiscal health of our State
Progress Report: 
  • Stem cells hold great potential to help us in repairing injured body parts or replacing damaged organs. In order to realize this potential the rules that control stem cell behavior need to be understood. Our laboratory has found that repression of the tumor suppressor p16 in human mammary epithelial cells (HMECs) endows them with specific properties that are only found in classical stem cells and tumor cells. Indeed, repression of p16INK4a in HMECs enables them to grow in culture for a long time, something that HMECs expressing p16INK4a cannot achieve. Importantly, we have previously shown that repression of p16INK4a is accompanied by the acquisition of pre-malignant features.
  • Thanks to the support of this CIRM grant, we have now established that a sub-population of these cells display stem cell properties. This means that these cells can self-renew but also differentiate in different breast cell types. Unexpectedly, these cells can also give rise to non-breast cells, such as brain cells, when grown in the appropriate cell culture conditions, making this unique cell model a powerful tool for cancer AND regenerative medicine research. Knowing that these cells can generate cells of different tissue types, we can now dissect the rules that dictate those different cell fates. We are also testing whether these exciting findings obtained in cell culture dishes (in vitro) can be confirmed in a mouse model (in vivo). In other words, can these cells generate a functional mammary gland? Other studies, beyond the scope of this application could also test whether these cells could rescue spinal injury.
  • So why do we bother using breast cells to generate brain cells (or other types of cells)? The answer is that we believe that the sub-population of cells we have identified in breast likely exists as a stem cell pool in any tissue (with some tissue-specific variations of course). If this hypothesis is confirmed, these cells could turn out to represent a major advancement in regenerative medicine. Another major advantage of these naturally occurring stem cells, compared to the widely used embryonic stem cell lines, is that they are directly isolated from fresh breast tissue without introducing artifacts that may result from establishment in long-term cell culture systems. Their properties are an accurate reflection of a fully functional stem cell pool actually existing physiologically in our body.
  • Understanding how stem cells code their decisions and whether cell fate can be changed after it has been set is key to the effective use of stem cells for therapeutic purposes. Gaining such insights will greatly improve our ability to manage wound repair and organ replacement. This should also help us characterize fundamental switches that control stem cells as well as control the formation of cancer cells since some of the genes that control stem cell properties are mutated in cancer. A mechanistic understanding of how these switches work may help us prevent adverse events that may result from the use of stem cells during regenerative medicine. Thus, we hope to contribute in improving the health of the citizens of California.
  • An important feature of adult stem cells is the ability to bypass negative growth signals and participate in wound healing. Based on this premise, we identified a small subpopulation of human breast epithelial cells that is capable of bypassing negative growth signals. We identified a differential expression of genes that allowed for the rapid isolation of this novel somatic cell population from fresh disease-free human breast tissue. Importantly, this cell population is characterized by the over-expression of Bmi-1, a protein that plays an essential role in the self-renewal of stem cells and represses the cell cycle inhibitor, p16. This population of cells is therefore poised to express pluripotency markers at a level similar to that measured in human embryonic stem cells. It has the ability to self-renew and can express phenotypes of any of the three mammary lineages in vitro using cell culture differentiation assays. Importantly, these cells are also functional in vivo as observed after implantation in mice. Indeed, these human cells can differentiate into functional mammary outgrowths of human origin in the host mouse as we could document secretion of human milk in mice transplanted with these human somatic cells. We are currently investigating whether these cells can also differentiate into other lineages (tissue types) when cultured in the appropriate conditions. Our preliminary studies support that these cells will hold great promise in regenerative medicine and cell replacement therapy and may help overcome some of the important ethical and technical roadblocks related to the use of human embryonic stem cells.

Sources of Genetic Instability in Human Embryonic Stem Cells.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00428
ICOC Funds Committed: 
$357 978
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The constant exposure of cells to endogenous and exogenous agents that inflict DNA damage requires active repair processes to eliminate potentially mutagenic events in stem cells leading to cancer. The same agents menace early human embryos with DNA damage that can ultimately lead to mutations, cancer, and birth defects. In vitro, human embryonic stem cells (HESCs) spontaneously undergo events leading to genetic instability and mutations. All these three types of genetic problems can have similar links to malfunctions in DNA repair systems, but little information now exists for HESCs. Therefore, the first step in understanding the causes of HESC genetic instability is to understand which DNA repair systems are defective. We will investigate the basis for this phenomenon in HESCs by evaluating their capacity to either repair DNA or form mutations. First, we will culture two HESC lines and compare HESC repair and mutation formation to that of control cells. We will use a new technique which simplifies the production and use of the feeder cells that support the growth of the HESCs. We will also test the genetic stability of HESCs grown on conventional feeder cells, as well as those grown in feeder free culture. We will use three types of DNA repair assays to monitor the genetic stability of the two HESC lines grown in these different ways. In the first of these assays, DNA molecules with different randomly-induced damage are transferred into HESCs, and DNA repair is followed by the re-establishment of the activity of a reporter protein that is coded for in the damaged DNA. A second assay will introduce specific DNA damage at a unique site in DNA that is transferred to HESCs and repair is determined using a polymerase chain reaction-based technique. Since aneuploidy is also known to be caused by double-strand DNA breaks, we will use two other assays to evaluate capacity of HESCs to repair that type of damage. These experiments will indicate if DNA repair pathways that eliminate DNA damage are dysfunctional and cause genetic instability. The final endpoint for these preliminary experiments is the formation of mutations. To study this, we have modified an assay system so that it will function in normal human cells to monitor mutations which arise spontaneously or those which are induced by various agents. In summary, these investigations will provide the basis for understanding genetic instability in HESCs that can direct cells to tumorgenic outcomes. The employment of HESCs clinically will require such knowledge. Moreover, these results will also yield information on susceptibility to mutations of cells early in development. The practical and basic science aspects of this seed grant proposal should lead to a complete proposal in the near future.
Statement of Benefit to California: 
Human embryonic stem cells (HESCs) hold the potential to cure or alleviate many chronic illnesses, including cancer, but an immense gap exists between the achievement of the goals of stem cell based medicine and the current state of the art. Several stages of development including the following are required: (1) Routine, standardized, simple protocols for the indefinite growth of HESC in the normal, undifferentiated state, in completely defined medium.(2) Control over differentiation of the cells in (1) to all adult cell types of interest. (3) Control over the maintenance of the differentiated state of derivatives of (1), in sufficient complexity to recreate normal functional histology. (4) Techniques, therapies, and protocols that allow immune tolerance of regenerated tissue, without rendering the human recipient immunodefficient. Researchers are still struggling with steps 1 and 2. Although claims of feeder cell and animal product-free, long-term, undifferentiated HESC culture have been made, this is not the current state of the art in laboratories. These claims may be fortuitous or true for only a few HESC lines. The public anticipates a quick success of human stem cell technology and application to human disease, but the promise of stem cell therapy requires basic scientific work that is critical, but may not make headlines. Imprudent claims of miraculous cures could dim public enthusiasm. Few if any data exist regarding DNA repair systems or mutation frequencies of HESCs. We propose to investigate mechanisms underlying genetic instability in cultured HESCs. This instability limits HESC research and therapeutic applications. The data generated by this research according to Proposition 71 will be of lasting value to the People of the State of California for the following reasons: (1) This proposal focuses on a serious, basic difficulty with respect to the growth of undifferentiated HESCs that is a barrier to their human therapeutic use.(2) In the future, if the focus of the stem cell field shifts to the as yet unavailable somatic nuclear transfer (SNT) methods, this proposed research, will provide a basis for the comparison of HESCs and SNT cell lines. (3) All humans begin as embryonic stem cells, therefore data generated by the proposed research will impact maternal health, well baby programs, early childhood development/learning, etc, because mutations are involved in birth defects as well as cancer. Therefore, understanding the causes of mutations in HESCs could assist in avoidance or reduction in birth defects that would aid both the families and the government of California.(5) All of the work described in this proposal will be conducted by individuals in California and most probably will result in the hiring of a graduate of a California institution of higher education, thus reducing unemployment and helping educate a new generation of California researchers in HESC use.
Progress Report: 
  • Human embryonic stem cells (hESCs) originate directly from human embryos, whereas induced pluripotent stem cells (iPSCs) originate from body (somatic) cells that are re-programmed by producing or introducing proteins that control the process making specific RNAs. Together, both these pluripotent cell types are referred to as human pluripotent stem cells (hPSCs). Several reports have observed that in hESCs grown for long times, their genetic material, DNA, is unstable. The stable maintenance of DNA is performed by groups of proteins functioning in different systems globally known as DNA repair pathways. Since the development of aneuploidy is closely linked to cancer and to deficiencies in DNA repair, we have studied the propensity of hPSCs to repair their DNA efficiently by 4 major known DNA repair pathways. In addition, we are also investigating if specific damage to DNA in either hPSCs or somatic cells is processed differently and could lead to deleterious mutations.
  • One major goal of the CIRM SEED grant mission is to bring new researchers into the hPSC field. The results we obtained during the funding period indicate that we have succeeded in that objective, since initially our laboratory had little experience with hESC culture. However, through courses and establishing critical collaborations with other hESC laboratories, we developed expertise in hPSC culture techniques. Most conditions for hPSCs growth require cells (feeder cells) that serve as a matrix and provide some factors needed for the pluripotent cells to divide. In accomplishing this aim, we perfected a method to generate reproducible feeder cells that significantly reduces the time and cost of feeder cell maintenance, and also developed a non-enzymatic and non-mechanical way to expand hPSCs. We now have experience with at least 5 hPSC lines and have methods to introduce foreign DNAs into hESCs and iPSCs to monitor DNA repair in hPSCs.
  • In Aim II of our grant, we used our accumulated knowledge of hPSCs and DNA repair to investigate 4 DNA repair mechanisms in hPSCs and in somatic cells. Depending on the DNA damage, there is often a preferred DNA repair pathway that cells use to alleviate potential harm. We initiated our investigation by treating hPSCs using different DNA damaging agents, including ultraviolet light and gamma radiation. However, we found that hPSCs exposed to these agents rapidly died compared to treatments that allowed somatic cells to continue growing. Therefore, we developed methods to study DNA repair in hPSCs without directly treating the cells with external agents. We treated closed, circular DNA (plasmids) with damaging agents separately, outside the hPSCs and then introduced them into the hPSCs. The plasmid DNA has a sequence that codes for a protein that is produced only when the damage is repaired. The length of time for repair both in hPSCs and in somatic cells was followed by determining the protein production. We have shown superior DNA repair ability and elevated protection against DNA damage in hPSCs compared to somatic cells for ultraviolet light and oxidative damage, two common sources of damage in cells. A major pathway for joining double-strand DNA breaks in mammalian cells, non-homologous end-joining (NHEJ) repair (error prone), is greater in H9 cells than in iPSCs. Another way to repair double-strand DNA breaks that uses similar (i.e., homologous) sequences is lower in iPSCs compared to hESCs and somatic cells. Further study of these repair pathways is warranted, since several methods can be used to form iPSCs. Therefore, the genomic stability for iPSCs could depend on the method used for their generation.
  • DNA repair analysis is critical to understanding how hPSCs protect against damage, but if left unrepaired, cells can turn damage into mutations when the damage is copied by enzymes (DNA polymerases) before repair occurs. Therefore, to monitor the mutations that ultimately lead to cancer or alter hPSC biology, we are using a plasmid that is damaged outside the cells and will make copies in hPSCs and somatic cells. That plasmid is introduced into cells and then the copies are recovered. The number of mutations found in the plasmid DNA indicates the likelihood of observing mutations in hPSCs compared to mutations in somatic cells. Together, these results will yield data on the stability of hPSCs and also a basis to monitor cells for stability which could serve as an indicator of safety for clinical use.

Using human embryonic stem cells to treat radiation-induced stem cell loss: Benefits vs cancer risk

Funding Type: 
SEED Grant
Grant Number: 
RS1-00413
ICOC Funds Committed: 
$625 617
Disease Focus: 
Cancer
Neurological Disorders
Skeletal Muscle
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
A variety of stem cells exist in humans throughout life and maintain their ability to divide and change into multiple cell types. Different types of adult derived stem cells occur throughout the body, and reside within specific tissues that serve as a reserve pool of cells that can replenish other cells lost due to aging, disease, trauma, chemotherapy or exposure to ionizing radiation. When conditions occur that lead to the depletion of these adult derived stem cells the recovery of normal tissue is impaired and a variety of complications result. For example, we have demonstrated that when neural stem cells are depleted after whole brain irradiation a subsequent deficit in cognition occurs, and that when muscle stem cells are depleted after leg irradiation an accelerated loss of muscle mass occurs. While an increase in stem cell numbers after depletion has been shown to lead to some functional recovery in the irradiated tissue, such recovery is usually very prolonged and generally suboptimal.Ionizing radiation is a physical agent that is effective at reducing the number of adult stem cells in nearly all tissues. Normally people are not exposed to doses of radiation that are cause for concern, however, many people are subjected to significant radiation exposures during the course of clinical radiotherapy. While radiotherapy is a front line treatment for many types of cancer, there are often unavoidable side effects associated with the irradiation of normal tissue that can be linked to the depletion of critical stem cell pools. In addition, many of these side effects pose particular threats to pediatric patients undergoing radiotherapy, since children contain more stem cells and suffer higher absolute losses of these cells after irradiation.Based on the foregoing, we will explore the potential utility and risks associated with using human embryonic stem cells (hESC) in the treatment of certain adverse effects associated with radiation-induced stem cell depletion. Our experiments will directly address whether hESCs can be used to replenish specific populations of stem cells in the brain and muscle depleted after irradiation in efforts to prevent subsequent declines in cognition and muscle mass respectively. In addition to using hESC to hasten the functional recovery of tissue after irradiation, we will also test whether implantation of such unique cells holds unforeseen risks for the development of cancer. Evidence suggests that certain types of stem cells may be prone to cancer, and since little is known regarding this issue with respect to hESC, we feel this critical issue must be addressed. Thus, we will investigate whether hESC implanted into animals develop into tumors over time. The studies proposed here comprise a first step in determining how useful hESCs will be in the treatment of humans exposed to ionizing radiation, as well as many other diseases where adult stem cell depletion might be a concern.
Statement of Benefit to California: 
Radiotherapy is a front line treatment used in California for many types of cancer, including brain, breast, prostate, bone and other cancer types presenting surgical complications. Treatment of these cancers through the use of radiation is however, often associated with side effects caused by the depletion of critical stem cell pools contained within non-cancerous normal tissue. While radiotherapy is clearly beneficial overall, many of these side effects have no viable treatment options. If we can demonstrate that human embryonic stem cells (hESC) hold promise as a safe therapeutic agent for the treatment of radiation-induced stem cell depletion, then cancer patients may have a new treatment for countering many of the debilitating side effects associated with radiotherapy. Once developed this new technology could position California to attract cancer patients throughout the United States, and the state would clearly benefit from the increased economic activity associated with a rise in patient numbers.
Progress Report: 
  • We have undertaken an extensive series of studies to delineate the radiation response of human embryonic stem cells (hESCs) and human neural stem cells (hNSCs) both in vitro and in vivo. These studies are important because radiotherapy is a frontline treatment for primary and secondary (metastatic) brain tumors. While radiotherapy is quite beneficial, it is limited by the tolerance of normal tissue to radiation injury. At clinically relevant exposures, patients often develop variable degrees of cognitive dysfunction that manifest as impaired learning and memory, and that have pronounced adverse effects on quality of life. Thus, our studies have been designed to address this serious complication of cranial irradiation.
  • We have now found that transplanted human embryonic stem cells (hESCs) can rescue radiation-induced cognitive impairment in athymic rats, providing the first evidence that such cells can ameliorate radiation-induced normal-tissue damage in the brain. Four months following head-only irradiation and hESC transplantation, the stem cells were found to have migrated toward specific regions of the brain known to support the development of new brain cells throughout life. Cells migrating toward these specialized neural regions were also found to develop into new brain cells. Cognitive analyses of these animals revealed that the rats who had received stem cells performed better in a standard test of brain function which measures the rats’ reactions to novelty. The data suggests that transplanted hESCs can rescue radiation-induced deficits in learning and memory. Additional work is underway to determine whether the rats’ improved cognitive function was due to the functional integration of transplanted stem cells or whether these cells supported and helped repair the rats’ existing brain cells.
  • The application of stem cell therapies to reduce radiation-induced normal tissue damage is still in its infancy. Our finding that transplanted hESCs can rescue radiation-induced cognitive impairment is significant in this regard, and provides evidence that similar types of approaches hold promise for ameliorating normal-tissue damage throughout other target tissues after irradiation.
  • A comprehensive series of studies was undertaken to determine if/how stem cell transplantation could ameliorate the adverse effects of cranial irradiation, both at the cellular and cognitive levels. These studies are important since radiotherapy to the head remains the only tenable option for the control of primary and metastatic brain tumors. Unfortunately, a devastating side-effect of this treatment involves cognitive decline in ~50% of those patients surviving ≥ 18 months. Pediatric patients treated for brain tumors can lose up to 3 IQ points per year, making the use of irradiation particularly problematic for this patient class. Thus, the purpose of these studies was to determine whether cranial transplantation of stem cells could afford some relief from the cognitive declines typical in patients afflicted with brain tumors, and subjected to cranial radiotherapy. Human embryonic (hESCs) and neural (hNSCs) stem cells were implanted into the brain of rats following head only irradiation. At 1 and 4 months later, rats were tested for cognitive performance using a series of specialized tests designed to determine the extent of radiation injury and the extent that transplanted cells ameliorated any radiation-induced cognitive deficits. These cognitive tasks take advantage of the innate tendency of rats to explore novelty. Successful performance of this task has been shown to rely on intact spatial memory function, a brain function known to be adversely impacted by irradiation. Our data shows that irradiation elicits significant deficits in learning and spatial task recognition 1 and 4-months following irradiation. We have now demonstrated conclusively, and for the first time, that irradiated animals receiving targeted transplantation of hESCs or hNSCs 2-days after, show significant recovery of these radiation induced cognitive decrements. In sum, our data shows the capability of 2 stem cell types (hESC and hNSC) to improve radiation-induced cognitive dysfunction at 1 and 4 months post-grafting, and demonstrates that stem cell based therapies can be used to effectively to reduce a serious complication of cranial irradiation.

Screening for Oncogenic Epigenetic Alterations in Human ES Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00408
ICOC Funds Committed: 
$685 000
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Embryonic stem cell-based therapies hold great promise for the treatment of many human diseases. These therapeutic strategies involve the culture and manipulation of embryonic stem cells grown outside the human body. Culture conditions outside the human body can encourage the development of changes to the cells that facilitate rapid and sustained cell growth. Some of these changes can resemble abnormal changes that occur in cancer cells. These include “epigenetic” changes, which are changes in the structure of the packaging of the DNA, as opposed to “genetic” changes, which are changes in the DNA sequence. Cancer cells frequently have abnormalities in one type of epigenetic change, called “DNA methylation”. We have found that cultured embryonic stem cells may be particularly prone to develop the type of DNA methylation abnormalities seen in cancer cells. A single rogue cell with DNA methylation abnormalities predisposing the cell to malignancy can jeopardize the life of the recipient of stem cell therapy. We have developed highly sensitive and accurate technology to detect DNA methylation abnormalities in a single cell hidden among 10,000 normal cells. In this seed grant, we propose to screen DNA methylation abnormalities at a large number of genes in different embryonic stem cells and compare their DNA methylation profiles to normal and cancer cells. This will allows us to identify the dangerous DNA methylation abnormalities most likely to occur in cultured embryonic stem cells. We will then develop highly sensitive assays to detect these DNA methylation abnormalities, using our technology. We will then use these assays to determine ES cell culture conditions and differentiation protocols most likely to cause these DNA methylation abnormalities to arise in cultured ES cells. The long-term benefits of this project include 1) an increased understanding of the epigenetics of human embryonic stem cells, 2) insight into culture conditions to avoid the occurrence of epigenetic abnormalities, and 3) a technology to monitor for epigenetic abnormalities in ES cells intended for introduction into stem cell therapy patients.
Statement of Benefit to California: 
The successful implementation of human embryonic stem cell therapy will require rigorous quality control measures to assure the safety of these therapies. Cells cultured outside the human body are known to be at risk of developing abnormalities similar to those found in cancer cells. Since a single rogue cell hidden among thousands of normal cells could cause cancer in an embryonic stem cell therapy recipient, it will be essential to have highly sensitive and accurate assays to detect these abnormalities in cultured embryonic stem cells before they are introduced into the patient. The goal of this proposal is to develop such sensitive and accurate assays. The citizens of the State of California will benefit from the availability of such assay technology to help assure the safety of human embryonic stem cell therapies.
Progress Report: 
  • Embryonic stem cell-based therapies hold great promise for the treatment of many human diseases. These therapeutic strategies involve the culture and manipulation of embryonic stem cells grown outside the human body. Culture conditions outside the human body can encourage the development of changes to the cells that facilitate rapid and sustained cell growth. Some of these changes can resemble abnormal changes that occur in cancer cells. These include epigenetic changes, which are changes in the structure of the packaging of the DNA, as opposed to genetic changes, which are changes in the DNA sequence. Cancer cells frequently have abnormalities in one type of epigenetic change, DNA methylation. In this grant, we screened for DNA methylation abnormalities at a large number of genes in different embryonic stem cells and compare their DNA methylation profiles to normal and cancer cells. This allowed us to identify potentially dangerous DNA methylation abnormalities, which occur in cultured embryonic stem cells. In the first year of this seed grant, we have developed a custom microarray to screen for DNA methylation changes at predisposed genes. In addition, we have analyzed DNA methylation in embryonic stem cells at more than 14,000 genes on a generic platform. This has allowed us to identify hundreds of genes that are abnormally methylated in various types of human cancers, and that show some evidence of this alteration in ES cells.
  • In the last phase of our study, we have screened the DNA methylation level of 1,536 genes in 142 different human embryonic stem cell pairs. Each member of the pair differed in the length of time it was in culture. Thus, our sample set was comprised of 284 paired specimens, one derived from an early passage and one derived from a late passage.
  • Our results indicate that the levels of DNA methylation varied considerably at a significant portion of the screened genes, some of which gained and some of which lost DNA methylation. These results indicate that DNA methylation in human embryonic stem cells seems to be susceptible to change over, at least in the genes examined in this study. Overall, our results suggest that the monitoring of DNA methylation changes in human embryonic stem cells may have to be incorporated as a routine protocol in stem cell manipulation.
  • During the past 12 months we have made significant progress on the data analysis of 141 paired (early passage-late passage) human embryonic stem cell lines (HESCs). The data in question was generated using a custom Illumina GoldenGate array of known Polycomb targets in HESCs, as described by Lee et al 2006. Briefly, we profiled the DNA methylation status of 1,536 loci on 282 specimens. This profiling was used to determine whether DNA methylation changes in HESCs arise as a result of time in culture at the examined loci. This determination was made by comparing the DNA methylation status of a sample of an early passage line with a late passage sample of the same line.
  • Interestingly, we found that DNA methylation in Polycomb target genes is highly affected by time in culture in a cell line-specific manner. That is, in some cell lines few DNA methylation changes were observed, while in the majority of them a large number of loci showed either an increase or decrease in DNA methylation. Via collaboration with the University of Sheffield, we were able to determine that DNA methylation instability seems to be independent of genetic instability. Furthermore, genetic instability seems to be a function of passage time in culture.

Functions of RB family proteins in human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00298
ICOC Funds Committed: 
$520 777
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Nearly one out of every two Californians born today will develop cancer at some point in their lives, and it is likely that one in five persons will die of the disease. We propose to study the mechanisms of action of the RB gene, which is mutated in a broad range of human cancers, including pediatric cancers of the eye and the bone, and adult tumors such as lung, breast, prostate and liver cancers. RB normally acts as a tumor suppressor. When RB is mutated, cells lose the ability to sense when to cycle or not and they divide too much, thereby initiating cancer. Because RB is mutated in so many human cancers, therapies that could re-introduce RB function in cancer cells would benefit a great number of cancer patients. A key question is to determine in which cell type loss of RB function is most detrimental. Knowing the answer to this question would help to diagnose cancer early and target specific cells within tumors, making treatment more effective. Recent evidence suggests that loss of RB may initiate cancer in stem cells . Because human embryonic stem cells (hESCs) give rise to any other stem cells, we will study the role of RB in hESCs. The results of these experiments will thus be applicable to a broad range of human cancers. Specifically, we will use novel tools that will allow us to precisely alter RB levels in hESCs. We will then study the consequences of these manipulations for the proliferation of these cells; lower levels of RB may promote proliferation, while higher levels of RB may slow proliferation and push these embryonic stem cells to become more mature. We will then investigate the molecular mechanisms underlying these observations, beginning with the cellular pathway leading to retinal development because of RB’s involvement in retinal cancer. Because RB is usually deleted in cancer cells, there is no simple way to re-express RB function in these cells. However, two genes related to RB, p107 and p130, are rarely deleted in cancers and can compensate for loss of RB in mouse cells. Therefore, we will also study the role of p107 and p130 in hESCs, to determine if the functions of these two genes also overlap with RB function in these human cells and their progeny. If this is the case, knowing how to control the expression of p107 and p130 in hESCs may result in the development of a novel therapeutic strategy against human cancers associated with loss of RB. A better knowledge of the cells from which cancer arises and of the molecular mechanisms by which cancer initiates will lead in the future to the development of novel means to diagnose cancer earlier, thereby increasing the chances of a successful therapy. In addition, because of the central role of RB family members in multiple cellular functions, these experiments in hESCs may provide novel insight into the basic biology of these stem cells, which will eventually allow us to manipulate these cells more efficiently to treat a broad range of human diseases.
Statement of Benefit to California: 
Despite significant decreases in the incidence and mortality rates of cancers in California over the past decade, nearly one out of every two Californians born today will still develop cancer at some point in their lives, and it is likely that one in five persons will die of the disease. Overall, in 2007, more than 50,000 people will die of cancer in California. These statistics underscore the need for the development of novel approaches to detect and treat human cancers. Stem cells hold the promise of treatments and cures for human diseases that affect millions of people. In particular, recent models suggest that cancer may arise from mutant stem cells whose progeny form the bulk of the tumor. Thus, in the future, one anti-cancer strategy may be to replace mutant stem cells in patients with normal stem cells. Another approach may be to repair the defects in these mutant stem cells. However, these approaches will only be possible when the mechanisms controlling the proliferation of these stem cells and their capacity to produce their functional progeny are better understood under normal and pathological conditions. We propose to study the mode of action of a key cancer gene, the RB gene, in human embryonic stem cells (hESCs). RB is inactivated in a broad range of human tumors, including adult lung, brain, breast, and prostate cancers, as well as pediatric eye and bone tumors. Thus, RB is a major target for the development of therapeutic strategies that may benefit a wide range of cancer patients. However, the mechanisms by which RB mutation triggers cancer are still poorly understood, hampering the development of such anti-cancer strategies. We believe that by studying RB function in hESCs, we will gain novel insights into both the mechanisms of action of RB and the biology of these stem cells. Exploring the effects of altering RB levels in hESCs will increase our knowledge of RB's mode of action and will eventually provide new ways to treat human cancers. In addition, these experiments may identify novel means of manipulating hESCs to control the fate of these cells when transplanted into patients. Because hESCs have the capacity to form any type of cell in the human body, these experiments will be relevant to the numerous cancer types associated with loss of RB function and may be ultimately translated into novel anti-cancer strategies. In addition, the results of these experiments may lead to novel avenues of research and may lay the groundwork for the development of therapies against diseases occurring in organs in which RB plays a central role, such as the eyes and the bones. Thus, the proposed research may benefit a broad range of patients, from young children to senior citizens, in California and elsewhere.
Progress Report: 
  • Human embryonic stem cells (hESCs) hold promise for treating a broad range of human diseases. However, at the time when we submitted this proposal, there was a striking paucity of published studies on how the fate of hESCs is controlled. For instance, we know that hESCs can form tumors upon transplantation, but the mechanisms governing cell division in these cells were still largely unknown. Given the central role of the retinoblastoma (RB) family of genes at the interface between proliferation and differentiation, our goal was to study the function of RB and its family members p107 and p130 in human embryonic stem cells (hESCs). In the last two years, we have examined the consequences of altering the function of RB, p107, and p130 for the proliferation, self-renewal, and differentiation potential of hESCs.
  • We have found that overexpression of RB results in cell cycle arrest in hESC populations, indicating that the RB pathway can be functionally activated in these cells. We have also found that loss of RB function does not result in significant changes in the biology of hESCs. In contrast, inactivation of several RB family members at the same time leads to self-renewal, proliferation, and differentiation defects.
  • Together, these studies indicate that the level of activity of the RB family is critical in hESCs: too much or too little RB family function results in loss of proliferative potential.
  • Our future goal is to precisely manipulate the levels of RB family genes to determine if we can identify conditions to manipulate the fate of hESCs, reducing their ability to proliferate (suppressing cancer) while allowing them to differentiate into specific lineages.

hESC as tools to investigate the neural crest origin of Ewing's sarcoma

Funding Type: 
SEED Grant
Grant Number: 
RS1-00249
ICOC Funds Committed: 
$675 001
Disease Focus: 
Solid Tumor
Cancer
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESC) hold great promise as sources of tissue for regenerative medicine and therapeutics. In addition, their utility as tools to study the origins and biology of human disease must not be underestimated. hESC give rise to tissue-specific adult stem cells and, ultimately, to all mature tissues in the body. As such, disruptions to normal stem cell function can have catastrophic consequences and result in life-threatening or debilitating disease. Understanding how such diseases arise will afford novel insights into how we can better prevent and treat them. Laboratory based studies with hESC therefore stand to make extraordinary contributions to human health. Human tumors, and in particular the cancers that affect children, often look like tissues that have not developed normally and whose growth has gone unchecked. In fact, recent studies have shown that, in many cases, tumors arise because genetic mutations in the DNA of normal stem cells lead to disordered development, resulting in formation of malignant rather than normal tissues. For example, leukemia can arise when a mutation occurs in a normal blood stem cell, thus inducing formation of cancerous rather than normal blood. Analogous situations exist in other human tissues and their respective tumors. However, because of the relative rarity of normal stem cells in other parts of the body and our inability to effectively isolate them, very little is yet known about how these stem cells go awry and create cancer. hESC, therefore, represent an invaluable resource for the generation of tissue-specific stem cells and for studies of the genesis of human, and in particular, pediatric cancer. Several different human cancers are believed to arise either directly or indirectly from stem cells called neural crest stem cells (NCSC). NCSC exist in small numbers throughout the body and contribute to the formation of multiple different tissues including the peripheral nervous system and the pigment cells of our skin. It is our central hypothesis that NCSC-derived tumors arise because genetic mutations in NCSC lead to disordered tissue development and the initiation of cancer. Ewing’s sarcoma family tumors (ESFT) are highly aggressive tumors that primarily affect children and young adults. ESFT have a specific mutation in their DNA and this mutation leads to the creation of a cancer-causing gene. We believe that expression of this abnormal gene in NCSC disrupts normal stem cell differentiation and development and, ultimately, leads to ESFT formation. In this proposal we will use hESC as tools to prove or disprove this theory. Unfortunately, despite highly toxic and aggressive treatment, the survival rate for patients diagnosed with ESFT remains poor. By creating novel hESC-based models to study the origin and biology of ESFT we aim to gain novel insights into the origin and biology of these tumors that will aid in the development of more effective, less toxic therapies.
Statement of Benefit to California: 
Human embryonic stem cells (hESC) represent a tremendous resource as tools to study numerous human diseases, including cancer. Cancer claims the lives of over 50,000 Californians, including over 300 children, annually. Laboratory based studies using hESC, such as those proposed in this application, stand to make extraordinary and unique contributions to our understanding of the origin and biology of human cancer. These contributions will ultimately aid in the development of novel therapeutic strategies designed to improve survival and quality of life of cancer patients. In this proposal we will exploit the power of hESC to study the cellular origins of sarcomas. Sarcomas arise in the bones and soft tissues and primarily affect children and young adults. Despite intensive therapy, the survival rate of patients diagnosed with sarcoma remains poor. The proposed research will provide much needed insight into sarcoma biology and will enable development of novel sarcoma-targeted therapies. In addition, the hESC-derived models that we establish will be readily adaptable to and available for studies of other human cancers.
Progress Report: 
  • Recent studies have shown that mutations in the DNA of adult stem cells can lead to the formation of cancerous rather than normal tissues. However, with the exception of blood, adult stem cells are rare and not readily accessible for isolation or study. Thus, very little is yet known about how these stem cells are hijacked to cause cancer.
  • Our laboratory is studying how mutations in stem cells give rise to Ewing sarcoma. Ewing sarcoma family tumors (ESFT) are highly aggressive tumors that primarily affect children and young adults. ESFT have a specific mutation in their DNA that leads to the creation of a cancer-causing gene called EWS-FLI1. It is our hypothesis that expression of EWS-FLI1 in adult stem cells generates ESFT. In particular, we are interested in a very rare population of adult stem cells called neural crest stem cells (NCSC) and these cells have been the focus of our CIRM-funded grant.
  • We initially proposed that human embryonic stem cells (hESC) could be used to generate NCSC and that these cells would be invaluable tools with which to study the origin of ESFT. In the first year of the grant we successfully achieved this goal and the work has been published. In the second year of the grant we have studied the consequences of activating the EWS-FLI1 on these cells. Importantly, our work shows that NCSC that express EWS-FLI1 do not differentiate normally. Instead they acquire properties of cancer stem cells. Thus, we propose that ESFT arise from NCSC that acquire a genetic mutation that prevents them from developing normally. These abnormal stem cells then go on to develop into full blown tumors.
  • By creating novel stem cell models to study the origin of ESFT we are gaining new insights into how these tumors arise in children. These insights will ultimately aid in the development of more effective therapies that can be designed to destroy abnormal cancer-causing stem cells whilst sparing normal stem cells.

Derivation and Characterization of Cancer Stem Cells from Human ES Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$642 500
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
oldStatus: 
Closed
Public Abstract: 
Cancer is the leading cause of death for people younger than 85 (1). High cancer mortality rates underscore the need for more sensitive diagnostic techniques as well as therapies that selectively target cells responsible for cancer propagation (1) Compelling studies suggest that human cancer stem cells (CSC) arise from aberrantly self-renewing tissue specific stem or progenitor cells and are responsible for cancer propagation and therapeutic resistance (2-9). Although the majority of current cancer therapies eradicate rapidly dividing cells within the tumor, the rare CSC population may be quiescent and then reactivate resulting in disease progression and relapse (2-9). We recently demonstrated that CSC are involved in progression of chronic phase chronic myelogenous leukemia (CML), a disease that has been the subject of many landmark discoveries in cancer research(19-30), to a more aggressive and therapeutically recalcitrant myeloid blast crisis (BC) phase. These CSC share the same cell surface markers as granulocyte-macrophage progenitors (GMP) but have aberrantly gained the capacity to self-renew as a result of activation of the Wnt/-catenin stem cell self-renewal pathway (4). Because human embryonic stem cells (hESC) have robust self-renewal capacity and can provide a potentially limitless source of tissue specific stem and progenitor cells in vitro, they represent an ideal model system for generating and characterizing human CSC (10-18). Thus, hESC cell research harbors tremendous potential for developing life-saving therapy for patients with cancer by providing a platform to rapidly and rationally test new therapies that specifically target CSC (2-18). To provide a robust model system for screening novel anti-CSC therapies, we propose to generate and characterize CSC from hESC (10-18). We will investigate the role of genes that are essential for initiation of CML such as BCR-ABL and additional mutations such as b-catenin implicated in CSC propagation (19-30). The efficacy of specific Wnt/b-catenin antagonists at inhibiting BCR-ABL+ human ES cell self-renewal, survival and proliferation alone and in combination with potent BCR-ABL antagonists will be assessed in sensitive in vitro and in vivo assays with the ultimate aim of developing highly active anti-CSC therapy that may halt cancer progression and obviate therapeutic resistance (4,31).
Statement of Benefit to California: 
The research outlined in this proposal represents a unique opportunity for collaborations between investigators from disparate disciplines to use human embryonic stem cells to challenge an existing paradigm namely that leukemic blasts are responsible for progression of chronic myelogenous leukemia (CML) rather than leukemic stem cells (LSC). Current clinical diagnostic tests are not sufficiently sensitive to predict timing of progression for all patients with CML nor are they adequate for determining the type of therapeutic intervention required. Moreover, the primary therapy for CML, Abl kinase inhibition, was shown to be cardiotoxic when given long-term at high doses. Furthermore, amplification of BCR-ABL is not the sole event that occurs during CML progression to blast crisis. Identification and inhibition of molecular mutations responsible for the generation of LSC in CML blood and/or marrow may prevent progression to blast crisis (BC) and would represent an innovative, effective form of CML therapy. Modeling of LSC responsible for CML progression in human embryonic stem cells could have a significant impact on our understanding of the pathophysiology of CML, provide novel diagnostic and therapeutic modalities and improve the quality and possibly quantity of life of patients with CML. By using BCR-ABL transduced human embryonic stem cells, we will rigorously evaluate the LSC hypothesis and as a consequence, the additional molecular events required for progression to blast crisis CML. The ultimate aims of this grant are to develop more sensitive methods to predict leukemic progression and to identify novel molecular therapeutic targets through the development of LSC models using human embryonic stem cells. We aim to provide a robust, reproducible system for testing novel anti-LSC compounds alone and in combination in order to expedite the development of novel therapeutic agents for anti-LSC clinical trials at {REDACTED}. Not only may the translational research performed in the context of this grant speed the delivery of innovative anti-LSC therapies for Californians with leukemia, it will help to train California’s future R&D workforce in addition to developing leaders in translational medicine. This grant will provide the personnel working on the project with a clear view of the importance of their research to cancer therapy and a better perspective on future career opportunities in California.
Progress Report: 
  • SEED Grant Research Summary
  • Compelling studies suggest that cancer stem cells (CSC) arise from primitive self-renewing progenitor cells. Although many cancer therapies target rapidly dividing cells, CSC may be quiescent i.e. asleep resulting in therapeutic resistance. Recently, we demonstrated that CSC drive progression of chronic phase (CP) chronic myeloid leukemia (CML), a subject of many landmark cancer research discoveries, to a therapeutically recalcitrant myeloid blast crisis (BC) phase. CML CSC share cell surface markers with granulocyte-macrophage progenitors (GMP) and have amplified expression of the CML fusion gene, BCR-ABL. In addition, they aberrantly gain self-renewal capacity, in part, as a result Wnt/β-catenin activation. Because human embryonic stem cells (hESC) have robust regenerative capacity and can provide a potentially limitless source of tissue specific progenitor cells in vitro, they represent an ideal model system for generating and characterizing human CSC. The main goals of this research were to generate CSC from hESC to provide an experimentally amenable platform to expedite the development of sensitive diagnostics that predict progression and combined modality anti-CSC therapy.
  • To this end, we tested whether BCR-ABL expression in hESC is sufficient to induce changes characteristic of CML stem cells. Unlike mouse ESC, introduction of a novel lentiviral BCR-ABL vector into hESC did not drive myeloid differentiation nor did it induce stromal independence in vitro underscoring key differences between mouse and human hESC and the importance of in vivo models. Notably, Hues16 cells had a higher propensity to differentiate into CD34+ cells than other hESC lines particularly in AGM co-cultures and thus, were used in subsequent in vivo experiments. Moreover, this SEED grant funded Yosuke Minami in Professor Jean Wang’s lab to create a unique CML blast crisis mouse model typified by GMP expansion and resistance to a BCR-ABL inhibitor, imatinib (Minami et al, PNAS 2008;105:17967-72). In addition, a bioluminescent humanized model of blast crisis CML was created based on transplantation of GMP from patient blood into immune deficient mice (RAG2-/-gc-/-). Cells were tagged with firefly luciferase that emits a bioluminescent signal so that leukemic transplantation efficiency could be tracked in vivo (IVIS). As few as 1,000 human blast crisis CML GMP could transplant leukemia in immune deficient mice thereby providing an important model for studying the molecular events that contribute to leukemic transformation (Abrahamsson et al, PNAS 2009;106:3925-9).
  • In the second aim, we hypothesized that BCR-ABL is sufficient for generating CML from self-renewing stem cells. In these studies, Hues16 cells differentiated into CD34+ cells were lentivirally transduced with BCR-ABL leading to sustained BCR-ABL engraftment in 50% of transplanted mice. Chronic phase CD34+ cells derived from CML blood were less efficient at sustaining CML engraftment (7%) suggesting that hESC derived CD34+ cells have higher self-renewal potential and are similar to advanced phase CML progenitors.
  • Thirdly, we hypothesized that BCR-ABL was necessary but not sufficient for progression to blast crisis. Introduction of lentiviral activated beta-catenin or shRNA to GSK3beta, together with BCR-ABL did not enhance BCR-ABL engraftment compared with BCR-ABL transduction of hESC alone. These studies suggested that hESC may already have sufficient self-renewal capacity to sustain the malignant CML clone and are molecularly comparable to advanced CML progenitors that behave like CSC. In addition, through extensive cDNA sequencing of human blast crisis CML progenitors, we found that 57% of samples harbored a misspliced form of GSK3beta that promoted tumor production and could serve as a novel prognostic marker in CML clinical trials (Abrahamsson et al, PNAS 2009;106:3925-9).
  • In the final aim, we hypothesized that CML CSC are not eliminated by BCR-ABL inhibitors alone and that combined modality therapy will be required. In collaborative research involving in vitro analysis of imatinib resistant CML progenitors and more recently in a humanized mouse model of blast crisis CML, we found that dasatinib, a potent BCR-ABL inhibitor, is necessary but not sufficient for CSC eradication. Discovery of a GSK3beta deregulation, a negative regulator of both beta-catenin and sonic hedgehog (Shh) pathways (Zhang et al, Nature 2009), led us to disover that Shh combined with BCR-ABL inhibition abrogated CSC driven tumor formation (manuscript in preparation) providing the impetus for an upcoming Pfizer sponsored Shh inhibitor clinical trial for refractory hematologic malignancies.

The APOBEC3 Gene Family as Guardians of Genome Stability in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
ICOC Funds Committed: 
$777 467
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The successful use of human embryonic stem cells (hESCs) as novel regenerative therapies for a spectrum of currently incurable diseases critically depends upon the safety of such cell transfers. hESCs contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of their genetic material. While many of these retroelements have been permanently silenced during evolution by crippling mutations, many remain active and capable of moving to new chromosomal locations potentially producing disease-causing mutations or cancer. More mature differentiated cells control retroelement movement (retrotransposition) by methylating the DNA comprising these elements. Strikingly, such DNA methylation is largely absent in hESCs because these cells must be able to develop into a wide spectrum of different tissues and organs. Thus, in order to protect the integrity of their genomes, hESCs must deploy an additional defense to limit retroelement retrotransposition. Recent studies of HIV and other exogenous retroviruses have identified the APOBEC3 family of genes (A3A-A3H) as powerful anti-retroviral factors. These APOBEC3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also used by retroelements for their successful retrotransposition. We hypothesize that one or more of the APOBECs function as guardians of genome integrity in hESCs. We propose to compare and contrast which APOBEC3s are expressed in one federally approved and nine nonapproved hESC lines and to assess the natural level of retroelement RNA expression occurring in each of these lines. Next we will test whether the knockdown of expression of these APOBEC3s in the hESCS lines by RNA interference leads to a higher frequency of retrolement retrotransposition. Finally, if higher levels of retrotransposition are detected, we will examine whether these cells display an impaired ability to differentiate into specific tissue types corresponding to the three germ cell layers (ectoderm, mesoderm, and endoderm) and whether increased retrotransposition is associated with a higher frequency of malignant transformation within the hESC cultures. These studies promise to provide important new insights into how genomic stability in is maintained in hESCs and could lead to the identification of specific GMP culture conditions that minimize the chances of such unwanted retrotransposition events in cells destined for infusion into patients. These studies are directly responsive to the CIRM request for application. If funded, these studies would allow the entry of my laboratory with extensive APOBEC experience, into the exciting field of stem cell biology.
Statement of Benefit to California: 
Harnessing the exciting potential of embryonic stem cells as therapies for a wide range of diseases like diabetes, Alzheimer’s disease, myocardial infarction among others first requires ensuring that the infusion of these cells into patients can be performed safely. Of note, human embryonic stem cells contain up to 3 million “jumping genes” or mobile genetic retroelements that can potentially move from location to another in the genome. Great harm could occur if the movement of these retroelements in human embryonic stem cells results in the mutation of key genes or the inactivation of tumor suppressor genes, the latter could facilitate the development of cancer in recipients of these cells. The safety of stem cell therapy thus depends on the rigorous maintenance of genomic integrity and stability within the embryonic stem cell during its manipulation. Strikingly, the major cellular defense against the movement of the retroelements to new genetic locations, DNA methylation, is greatly reduced in human embryonic stem cells. A general state of hypomethylation is likely required to permit these pluripotent cells to differentiate into multiple cell types. With DNA methylation no longer able to constrain the activity of these retroelements, we believe a second natural defense springs into action to protect these stem cells. We proposeto identify and characterize this defensive network. These studies could lead to new approaches for maintaining or even enhancing this defense when embryotic stem cells are manipulated in culture, thereby helping to ensure the safety of embryonic stem cells destined for therapeutic transfer. Thus, the results of these studies will have both scientific and practical value. As such, we believe these studies will benefit the citizens of California certainly at a societal level and potentially at a personal level.
Progress Report: 
  • Human embryonic stem cells contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of human genome. While many of these retroelements have been silenced during evolution by crippling mutations, many remain active and capable of jumping to new chromosomal locations potentially producing disease-causing mutations or cancer. In tissues, mobility of these elements is suppressed by DNA methylation, which inactivates expression of the retroelement RNAs. In sharp contrast, embryonic stem cells exhibit very dynamic changes in DNA methylation, where the methylation patterns are gained and lost at high rates. During periods of low DNA methylation, retroelement RNA expression likely increases. Accordingly, hESCs must deploy other defensive strategies in order to maintain genomic integrity. Recent studies have identified the APOBEC3 family of genes (A3A-A3H) as powerful antiviral factors. These A3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also employed by retroelements for their successful retrotransposition. We hypothesized that one or more of the APOBECs function as guardians of genome integrity in hESCs. In the last two years we have found that six out of the seven human A3 genes located in a tandem array on chromosome 22 are expressed in hESCs. A3A, which in prior studies was suggested to exert the greatest anti-retroelement effects, surprisingly is not expressed in hESCs. Further, we find that the A3 proteins decrease when pluripotent cells differentiate into somatic cells suggesting an important function of these A3 proteins in pluripotent hESCs. We established a LINE1 retrotransposition assay in hESCs that allows us to visualize genetic jumping of this class of “marked” retroelements via flow cytometry. Using this assay we have found that LINE1 elements effectively jump in hESCs. To test our central hypothesis, namely that A3 proteins guard the genome in hESCs, we have established experimental conditions for RNAi knock-down of all expressed A3 genes. By combining the knock-down and the retrotransposition assay we demonstrated that the knock-down of one member of the A3 protein family leads to a 3.5-fold increase in LINE1 retrotranspositon. This finding highlights a protective role for the A3 family of cytidine deaminases that helps safeguard the genome integrity of hESCs.

Genetic Enhancement of the Immune Response to Melanoma via hESC-derived T cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00203
ICOC Funds Committed: 
$642 501
Disease Focus: 
Melanoma
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The overall goal of the proposed studies is to utilize human gene therapy approach using human embryonic stem cells to direct our body’s defenses to specifically attack melanoma tumor cells. Current technologies try to accomplish this by genetically manipulating certain circulating T lymphocytes, such that they will target tumor cells. T lymphocytes are the major cell type of our body’s immune system. However it is likely that this type of approach will not result in the presence of stable, lifelong genetically modified T cells. In contrast, a potentially more long-lasting approach would be to genetically modify human embryonic stem cells with the same therapeutic gene. Stem cells have the ability to form any type of blood cell, including T cells. Importantly, stem cells can persist for the life of the individual, and thus have the potential to produce genetically modified T cells for many years. In addition, these new tumor specific cells should expand in the body in response to the presence of the tumor, thus a large supply of tumor-fighting cells should be available as long as needed. This project proposes to develop novel means to introduce the anti-cancer gene into human embryonic stem cells. These stem cells will then be differentiated to generate tumor specific T cells utilizing animal model systems. We will then use several laboratory and mouse models to determine if the T cells derived from these genetically modified stem cells have anti-tumor activity. If successful, we will have provided proof-of principle that long-lived stem cells have the potential be utilized as a means of producing anti-cancer T cells. In the long run, these results could provide important information for design of future clinical trials designed to produce life-long improved anti-cancer immune responses.
Statement of Benefit to California: 
We propose to use human embryonic stem cells to develop a novel, yet potentially very effective method to treat invasive melanoma. Melanoma is a serious type of skin cancer which, if not removed early, spreads internally and is usually fatal. Overall melanoma is the 6th most common cancer in males and 7th in females and the incidence of this form of cancer is currently increasing at an epidemic rate. Although melanomas may occur in areas of skin that are not normally exposed to sunlight, sun exposure is believed to be a factor in about 70% of new cases. California’s mild winters and high number of sunny days provide opportunities for a number of occupational and recreational outdoor activities, and people in California are exposed to more than average levels of solar radiation. Consequently, there is a higher risk of developing this disease. As a matter of fact, California is one of the five US states with the highest predicted incidence of new cases of melanoma. According to the California Cancer Registry, each year 4,700 new cases of invasive melanoma and over 800 deaths related to this disease are reported in California, with the incidence rate increasing by 15% over the last decade. While the white population is at the greatest risk of developing this disease, it was recently reported that the rates of invasive melanoma have risen substantially in Hispanic people living in California as well. If our proposal is successful, our work could pave the way to the development of a new and effective form of melanoma therapy, one which would clearly benefit all the people of California affected by this disease.
Progress Report: 
  • In this grant we proposed to genetically engineer human embryonic stem cells (hESC) and hematopoietic stem cells (HSC) and to use them to produce T cells with enhanced ability to kill melanoma cells. Our proposal consists of several steps. In the first year of the grant, we completed the first step and introduced the genes for a melanoma specific T cell receptor (TCR) into hESC and HSC. In this, second year of funding we were able to generate genetically modified T cells from hESC and HSC and to characterize the HSC-derived cells in more details. We found that HSC-derived T cells carrying the new TCR are indistinguishable from normal T cells, based on the cell surface expression of other T cell specific proteins. Also, we found that they can kill human melanoma cells in a Petri dish. We are currently evaluating their ability to destroy tumors in experimental animals transplanted with human melanoma cells. This is a more relevant approach as it mimics the potential treatment of melanoma patients. We are also trying to obtain larger numbers of the genetically modified hESC-derived T cells and analyze them in the same types of assays. Our data are encouraging and suggestive of possible clinical application of these cells in future.

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