Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

Engineering Bioactive Hydrogels for Neuronal Differentiation of hESCs

Funding Type: 
SEED Grant
Grant Number: 
RS1-00298
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Human embryonic stem cells (hESCs) have strong potential as sources of cells for the treatment for disease and injury (e.g. Parkinson’s Disease, amyotrophic lateral sclerosis, spinal cord injury, diabetes, congestive heart failure, etc.). The successful integration of hESC into such therapies will hinge upon three critical steps: their expansion without differentiation, their differentiation into a specific cell type or collection of cell types, and the promotion of their survival and functional integration at the site of disease or injury. Precisely controlling each of these steps will be essential to maximize hESC’s therapeutic efficacy, as well as to minimize potential side effects that can occur when the cells numbers and types are not properly controlled. However, hESCs are typically grown on murine or human feeder cells, in conditioned media derived from these cells, and/or within complex mixtures of animal or human proteins. Such growth conditions present major problems: there is a possibility of pathogen transmission from feeder cells or proteins, hESCs can acquire non-human antigens that will lead to immune rejection following implantation into a patient, and these growth conditions are difficult to precisely control and reproducibly scale up to a clinical process for the treatment of large patient populations. To achieve the intended goals of regenerative medicine, methods for the precise control of the proliferation, differentiation, and survival of stem cell populations in cell culture and in the body after cell implantation are necessary. We have made significant progress in developing a novel technology platform consisting of completely synthetic polymer-based synthetic matrices to support hESC proliferation and self-renewal. We now propose to create synthetic microenvironments to support hESC differentiation into two important neuronal lineages: dopaminergic neurons with potential for Parkinson’s Disease therapy and motor neurons with potential for Lou Gehrig’s Disease. Previous protocols have been developed for controlled differentiation into these lineages; however, they have typically involved culture conditions with animal and human proteins and ECM. Furthermore, after implantation into the site of injury or disease, the majority of neurons typically die. We hypothesize that implanting neurons differentiated from hESCs along with a supporting, bioactive matrix will enhance cell survival and therefore future efforts to utilize grafts for tissue engineering and repair. The result will be a technology platform that can be generally applied to numerous stem cell populations and used to investigate the basic biological/developmental mechanisms underlying cell differentiation. Therefore, this novel integration of stem cell biology, neurobiology, bioengineering, and materials science has the potential to overcome a major challenge in regenerative medicine.
Statement of Benefit to California: 
Stem cell research in general, and this proposed research in particular, have great potential for enhancing the scientific and economic development of the state of California. First, this project is highly integrative in that it melds expertise and investigators from a number of scientific fields including tissue engineering, materials science, chemical engineering, stem cell biology, neurobiology, electrophysiology, and genomics. It therefore represents a model project for the development of interdisciplinary research teams, since success in research increasingly relies upon taking the initiative to draw from numerous fields of science and engineering. Furthermore, this project will represent a highly valuable and unique interdisciplinary training environment. Two trainees will be able to draw from leading scientific expertise in five research groups in five departments and two institutes to make progress in this high impact work. Finally, the collaborative expertise that this group develops as a result of this funding will be in place to continue this and other research areas, with the aid of numerous additional trainees, in the future. In addition, the field of stem cells represents a unique economic opportunity for the state of California. Both Northern and Southern California are dominant areas for biotechnology research and companies. We anticipate that the products of this research will be of interest to numerous sectors of biotech, not only for its potential in neuronal differentiation but in its generality for both embryonic and adult stem cell culture and differentiation into numerous lineages. First, the use of stem cells for in vitro pharmacology and toxicology screening will rely upon the development of scaleable and reproducible systems for stem cell expansion and differentiation, which this work can provide. Second, research product companies may be interested in providing reproducible, synthetic culture systems for stem cell experimentalists. Finally, this work potentially has its largest promise in the development of scaleable systems to support stem cell differentiation in vitro and cell transplantation in vivo for therapeutic application in tissue engineering and repair.
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.

Anesthetic Effects on Neural Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00298
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The long-term objectives of this research are to identify safe and effective anesthetics to be used for human stem cell transplantation and to define the effect anesthetics have on stem cells in vivo. To achieve this goal we will identify the effect of several common anesthetic drugs on stem cells in culture and in animals. Specifically, we will determine whether anesthetics change the rate of growth of stem cells or limit the type of cell they may eventually become.
Statement of Benefit to California: 
Human embryonic stem cell based therapies will likely be attempted for multiple diseases in many different organ systems in the next few decades. Stem cell transplant in humans and experimentation in animal models will require sedation or complete general anesthesia for many therapies. Very little research has been done on the role that common anesthetics may play in the biology of human stem cells, and how such anesthetics may affect the function or differentiation of these cells once transplanted. Choosing the correct anesthetic may impact the success or failure of early animal and human clinical trials. This proposal focuses specifically on neural stem cells which have been proposed as a potential treatment for many different pathologic states including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, stroke, spinal cord injury and traumatic brain injury. Some stem cell transplants will be performed under general anesthesia and some will be performed in individuals likely to undergo multiple or long duration anesthetics around the time of their injury and potential transplant (i.e. traumatic brain injury, spinal cord injury, and neonatal stroke) leading to more anesthetic exposure after the transplant. Understanding the role of anesthetics in stem cell biology is imperative and will provide the basis for developing appropriate anesthetic techniques for stem cell based clinical applications.
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.

Derivation of Customized Stem Cells for Regenerative Medical Therapy

Funding Type: 
SEED Grant
Grant Number: 
RS1-00249
ICOC Funds Committed: 
$0
Disease Focus: 
Solid Tumor
Cancer
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Embryonic stem cells hold great promise in regenerative medicine for the treatment of numerous diseases, injuries, and disabilities. Despite recent clinical successes, there remain significant hurdles to establishing ethically sound, scientifically feasible, and practically realistic human stem cells that engender broad public support and exhibit convincing therapeutic effectiveness. Among these hurdles are the source (embryo vs adult) of stem cells and immune rejection following transplantation. To overcome these two hurdles, our long-term goal is to develop and apply efficient technologies for deriving pluripotent, embryonic-like stem cells from a patient's own tissues for the purpose of providing "customized", patient-specific regenerative therapy. The rationale behind our long-term goal is that the destruction of human embryos to derive new embryonic stem cell lines, and the clinical complications associated with rejection of transplanted stem cells that are not recognized as "self", prevent full realization of the enormous potential of regenerative therapies using stem cells. Therefore, the overall objective of this SEED grant, which is the first step in achieving our long-term goal, is to extend and enhance existing technology for efficiently and reliably using adult human somatic cell nuclei to derive pure, pluripotent human embryonic-like stem cells. Our central hypothesis is that customized and therapeutically-useful embryonic-like stem cells can be derived from adult, human fibroblast nuclei reprogrammed in mouse oocytes. The justification for this project is that this methodology would eliminate the creation and/or destruction of human oocytes and embryos to derive patient-specific embryonic-like stem cells, preclude the need for immunosuppresvie therapy in patients receiving stem cells, and allow for the possibility of correcting inherited genetic mutations.
Statement of Benefit to California: 
The research proposed here will significantly advance the field of stem cell biology, thereby promoting translational research applications that drive achievements of basic research to the patient's bedside faster and more effectively than before. By doing so, and by improving the health of the citizenry in need of regenerative medicine using stem cells, then this project will be of benefit to the State of California. In addition, this area of research will attract a broader and more diverse array of scientific experts in the field of stem cell biology to California, thereby also contributing to the advancement and development of the State's biomedical research enterprise.
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.

Therapeutic Potential of Human Embryonic Stem Cells: Cardiovascular Tissue Engineering

Funding Type: 
SEED Grant
Grant Number: 
RS1-00249
ICOC Funds Committed: 
$0
Disease Focus: 
Solid Tumor
Cancer
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Cardiovascular diseases are the leading cause of death in the United States. Blood vessel replacement is a common treatment for vascular diseases such as atherosclerosis, restenosis and aneurysm, with over 300,000 artery bypass procedures performed each year. However, vein grafts are limited to their availability and the additional cost and surgeries, and small-diameter synthetic vascular grafts have frequent clogging due to thrombogenesis. Tissue engineering is a promising approach to the fabrication of non-thrombogenic vascular grafts, but the reliable and expandable cell sources for tissue-engineered vascular graft (TEVG) have not been established. Our long-term objectives are to engineer stem cells and nanostructured biomaterials for the repair and regeneration of cardiovascular tissues. In this project, we will investigate the differentiation of human embryonic stem cells (hESCs) into vascular cells, and use hESC-derived cells and nanostructured scaffolds to construct TEVGs that are non-thrombogenic, are capable of self-remodeling, and have long-term patency. This study will generate insights into the differentiation and regeneration potential of hESCs and their derived cells in vascular microenvironment, and help to establish a stable cell source for cardiovascular repair and therapies, which will benefit our health care in the near future.
Statement of Benefit to California: 
Cardiovascular diseases are the leading cause of death in the United States. Our long-term objectives are to engineer stem cells and nanostructured biomaterials for the repair and regeneration of cardiovascular tissues. In this project, we will investigate the differentiation of human embryonic stem cells (hESCs) into vascular cells, and use hESC-derived cells and nanostructured scaffolds to construct tissue-engineered vascular grafts (TEVGs) that are non-thrombogenic, are capable of self-remodeling, and have long-term patency. This study will generate insights into the differentiation and regeneration potential of hESCs and their derived cells in vascular microenvironment, and help to establish a stable cell source for cardiovascular repair and therapies. TEVGs will benefit patients and reduce our cost for health care. For example, the additional surgeries, cost and morbidity for harvesting autologous blood vessels can be avoided, and the clogging of synthetic vascular grafts can be minimized. Furthermore, hESC-derived vascular progenitors could be used to fabricate TEVGs that are available off-the-shelf.
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.

Therapeutic Potential of Transplanted human Embryonic Stem Cells Overexpressing Soluble APP in Treating Alzheimer's Disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00249
ICOC Funds Committed: 
$0
Disease Focus: 
Solid Tumor
Cancer
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Alzheimer disease (AD) afflicts over 5 million elderly Americans and is characterized by deposits of insoluble protein aggregates (amyloid plaques) and neurofibillary tangles) as well as massive neuronal loss in selected regions affecting learning and memory. Stem cell therapy represents a promising strategy for treating the chronic central nervous system (CNS) diseases such as AD by replacing damaged and lost neurons and thus restoring defective cognitive behaviors. Human embryonic neuronal stem cells (hES) transplanted into aged rodent brains are found to differentiate into neuronal cells and significantly improve the cognitive functions of the animals. However, ethical and practical issues remain which compel us to seek alternative strategies. Using a well-characterized human ES line in transplantation is an option which can be greatly enhanced by some potent neurotrophic factors to nourish neurons. In this application, we propose to combine hES with a natural soluble factor, the N-terminal portion of the amyloid precursor protein (sAPP) to create a superior stem cell agent for treating AD. sAPP is present normally in the cerebral spinal fluid (CSF) and its level is found to dramatically decline in AD patients, suggesting that this protein plays a critical role in preventing AD. Indeed, this is the best-characterized natural molecule that displays potent neuroprotective and neurotrophic actions on cultured neurons as well as in CNS cells upon infusion. We thus propose to engineer two human ES lines to secrete sAPP via lentivirus infection and to characterize these established lines for the effects of sAPP on differentiation and migration features of the transduced hES. Subsequently, we will transplant these cells into mouse brains at various ages to optimize a transplantation procedure. Finally, the efficacy of the transplanted hES secreting sAPP will be tested in reducing AD pathology in a selected mouse model that displays massive neuronal death/loss and impaired synaptic function. We hope this study will provide proof-of-concept for an established human ES line with a superior ability to differentiate and to stimulate neighboring neurons to proliferate into new neurons which can be further validated and used in future therapeutics.
Statement of Benefit to California: 
California's population is aging, and as people live longer the incidence of diseases caused by aging increases. This has an enormous economic impact on California, since the caregivers for the elderly are usually their children, who are in the peak of their productive years. Alzheimer's disease (AD), the number one dementia among the elderly, is especially devastating because the disease develops and worsens over a long period of time with no available cure . Stem cell replacement by transplantation represents a promising therapeutic option for treating AD. However, both the ethical and practical issues compel neuroscientists to seek alternative approaches (e.g., using human embryonic stem cell lines, hES lines herein) in addition to using primary human stem cells. The proposed studies to fully characterize and establish well-behaved hES lines with superior ability to replace damaged/lost neurons in AD brains upon transplantation will provide proof-of-concept for future transplantation feasibility in patients. Nationwide, an estimated 5 million Americans have AD. The number of Americans with AD has more than doubled since 1980 and continues to grow at an accelerated rate. California, as a paradise to retirees, accommodates the largest aging population and is estimated to have nearly 1 million people with AD. Additionally California farmers use approximately 250 million pounds of pesticides which is about a quarter of all pesticides used in the entire country. Pesticides have been proven to be neural toxic and linked to higher incidences of Parkinson’s disease and AD. Not to mention curing the disease, finding a treatment that could delay the onset of AD by five years alone could reduce the number of individuals with AD by nearly 50% after 50 years and thus greatly reduce the government’s medicare costs (which are expected to increase 75% from $11 billion in 2005 to $19 billion in 2010 in California).
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.

Human embryonic stem cell-derived neurons as a model to discover safer estrogens for hot flashes

Funding Type: 
SEED Grant
Grant Number: 
RS1-00249
ICOC Funds Committed: 
$0
Disease Focus: 
Solid Tumor
Cancer
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Menopause begins in women one year after the last menstrual period. The average age of menopause is 51 years. Because the average life-expectancy in the US is 80 years, most women will spend at least one-third of their life after menopause. Menopause is associated with a large drop in the levels of estrogens in the blood. The drop in estrogens during the menopausal transition leads to onset of hot flashes, night sweats, mood changes, and vagina dryness. Hot flashes are prevalent and extremely bothersome to many postmenopausal women. For over 50 years, women have been taking estrogens to prevent hot flashes, because their quality of life deteriorates due to lack of sleep, heat sensation and sweating. Recently many women have abandoned hormone therapy (HT) due to concerns about potential adverse effects, including breast cancer, strokes and blood clots. Currently, all the estrogenic drugs that are effective at treating menopausal symptoms are known to promote cancer. Because of the safety concerns, many clinicians prescribe non-estrogenic drugs, such as those you to treat depression and anxiety. These drugs are not as effective as estrogens for menopausal symptoms. They also produce adverse side-effects and do not have the beneficial effects of estrogens on preventing osteoporosis. Many postmenopausal women are anxiously waiting for new drugs that relieve menopausal symptoms, but do not promote cancer or other serious side-effects. A major problem that exists to discover safer drugs for menopausal symptoms is the lack of appropriate biological systems to screen estrogens for activity. For example, the cells used to test drugs are not involved in the generation of hot flashes and there is no good animal model to study hot flashes. The best system to study the effects of estrogens are neurons, which are involved in the generation of hot flashes. However, it has not been possible to obtain human neurons in sufficient amounts to test new drugs. The use of embryonic stem cells now makes it possible to generate enough human neurons to study. In this proposal, we will use human embryonic stem (hES) cells as a source for neurons that can be used as a model to identify estrogenic genes that could serve as markers to discover drugs for hot flash prevention.
Statement of Benefit to California: 
There are approximately 5 million postmenopausal women in California. Approximately, 80% of these women will experience hot flashes. During menopause the levels of the female hormone, estrogen, drop dramatically. This drop causes hot flashes to occur, which are most common during perimenopause, and usually last for one to five years after menopause. In some women hot flashes can extend extend through the 70s and beyond. A hot flash is a sudden feeling of warmth that is often associated with sweating, palpitations from an elevated heart rate, chills, and a sensation of anxiety. Although variable, hot flashes generally last for seconds or a few minutes and occur every 2-4 hours. Hot flashes are extremely debilitating to many women, because they often awaken many times during with night sweats. The daily and nightly hot flashes often cause women to be extremely tired and irritable and makes it more difficult to concentrate on daily tasks. In some cases job performance suffers. A desire to prevent hot flashes is the main reason women begin hormone therapy. Unfortunately, clinical trials have found that estrogens in hormone therapy can cause breast cancer, strokes and blood clots. The huge, potential beneficial impact of new drugs for treating hot flashes and menopausal symptoms is exemplified by the fact that hormone therapy was the most prescribed drug prior to recent clinical trials. The results of the clinical trials have created a huge need for millions of postmenopausal women in California who are anxiously waiting for safer estrogens for hot flashes and other menopausal symptoms. The goal of this proposal is use neurons that are derived from embryonic stems cells to discover safer estrogens to treat postmenopausal women who seek treatment for hot flashes.
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.

Titanium Oxide Nanotube Platforms for Bioartificial Livers and for Transplantation of Hepatocytes Derived from Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00249
ICOC Funds Committed: 
$0
Disease Focus: 
Solid Tumor
Cancer
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The liver is the largest organ in the human body. It is essential for life. The production of blood proteins involved in coagulation and the detoxification of poisons that enter the body are among the most important functions of the liver. Serious health consequences occur when the liver fails to perform these functions. For example, human beings born with defective coagulation proteins acquire Hemophilia (a genetic disease). Or humans with livers destroyed by incurable infections (hepatitis) or work-related chemicals often contract liver failure and fibrosis. When Hemophilia, liver failure and liver fibrosis are uncontrollable, these people often die of ‘end-stage liver disease’ (ESLD). ESLDs such as chronic hepatitis and liver cancer are rampant in California and clinical treatments are becoming increasingly strained. A recent study (2005) found that chronic liver disease ranked as one of the leading causes of death in California, resulting in 3,725 deaths in 2002; it was also found that minorities suffered disproportionately from ESLD. Liver organ transplantation is the current therapy for ESLD. However, it is very costly and complex, it depends upon the availablity of donor livers, and there are many associated problems. Donor livers are rare, transplant waiting lists are long, and transplant waiting times are long (so long that some patients die before a donor liver is available). The major problem -- unless the donor comes from an identical twin – is that patients reject the donor liver. To prevent rejection, patients are currently treated lifelong with drugs, but often these drugs fail or are themselves dangerously toxic and life-threatening. The research in this proposal will lay the groundwork for the development of a device that can replace a failing liver (without drugs), much as dialysis machines can save the lives of people with kidney failure. This device is called a BAL (bioartificial liver). A prototype BAL will be made by a team of biologists, physicians and bioengineers. Federally approved human embryonic stem cells (hESCs), which can be converted into liver cells, will be placed on ‘computer-like’ chips made from titanium (a metal harmless to the body) designed to simulate small livers. To see if hESC-derived liver cells-on-chips (LCOCs) maintain liver functions and survive transplantation, LCOCs will be put into special mice (which do not reject human cells) for up to a month. During this time, the LCOCs will be removed and tests for liver cell functions (e.g. production of blood proteins) will be made. If these experiments work, future research will be geared to (a) designing LCOCs that cure liver disease in animals, and (b) producing hESCs that resist rejection. If these problems are solved, studies will move into human trials. If human trials work, we hope to build universal, inexpensive, LCOCs to cure ESLD in California and worldwide, without resorting to liver transplantation and drugs.
Statement of Benefit to California: 
End-stage liver diseases (ESLDs) such as chronic hepatitis and liver cancer are rampant in California and therapeutic modalities are becoming increasingly strained. Many afflicted die of these conditions including those associated with alcoholic liver disease. According to the California DHS and Center for Health Statistics (Data Summary No. DS05-05000, May, 2005, pp. 1-11), in a study entitled “End Stage Liver Disease (ESLD): Morbidity, Mortality, and Transplantation California, 1999-2003”, chronic liver disease and cirrhosis ranked as one of the leading causes of death in California, resulting in 3,725 deaths statewide in 2002. Not surprisingly, minorities suffered disproportionately: American Indians, Alaska Natives, Hispanics and Latinos had significantly higher ESLD death and hospitalization rates, but lower liver transplant rates, despite many Adult Liver Transplant centers in the State (11 of 91 throughout the US, as determined 5/30/06 [https://www.cms.hhs.gov/ApprovedTransplantCenters/downloads/liver_list.pdf]). More surprisingly, the incidence of ESLD in California was higher than the incidence of newly diagnosed Parkinson’s disease cases as judged from a 1994–1995 study using information from Kaiser Permanente of Northern California (Van Den Eeden SK et al. Amer. J. Epidemiol. 2003;157:1015-1022). Current cures for ESLD depend mainly upon liver transplantation. However, liver donor organs are limited, matched organs rarely exist, and the medical costs for transplantation and post-operative care are prohibitive. Transplantation of suspensions of committed liver stem cells is one future option; but scientific controversy and technical issues plague isolation, culture, directed and stable differentiation of these cells, as well as the universal problems of (a) transplantation without rejection, and (b) provision of sufficient liver function to sustain normal life. State-of-the art materials science and nanotechnology, coupled with recent advances in the hepatocyte-directed differentiation of human embryonic stem cells (hESCs) in vitro, may provide tissue and biomedical engineering approaches that can lead to breakthroughs towards curing ESLDs without resorting to organ transplantation. These breakthroughs may well come from functional extracorporeal and transplantable hESC-based bioartificial livers (BALs), constructed from inexpensive TiO2 chips carrying liver acinar-like stacks of hESC-drived hepatocytes, to assist or cure human beings suffering from ESLDs of infectious (hepatitis), genetic (Hemophilia A and B) or chemical origin (alcohol abuse). Apart from therapeutic transplantation devices, significant benefits from these novel BALs would be quickly evident, as they would provide normal, homogeneous cell sources for robotic screening of potential specificities, metabolism, polymorphisms and toxicities of new or experimental drugs, chemicals, and therapeutics developed by pharmaceutical and chemical industries.
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.

The Mammalian Stress Response and Human Embryonic Stem Cell Survival

Funding Type: 
SEED Grant
Grant Number: 
RS1-00249
ICOC Funds Committed: 
$0
Disease Focus: 
Solid Tumor
Cancer
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Human embryonic stem cells (hESCs) are pluripotent stem cells that have the dual ability to self renew and to differentiate into multiple cell types in the body. Growth and expansion of pluripotent hESCs require a balance between survival, cell death, self-renewal and differentiation signals. Despite the identification of some of the growth factors believed to be involved in hESCs self-renewal and proliferation, hESCs are extremely difficult to propagate and their survival in continuous culture in vitro remains a major challenge. Without improvement in this critical research area, the growth and expansion of undifferentiated human stem cells, which have the highest potency in generating the differentiated cell types, will remain a major obstacle to attaining the goal of human stem cell transplantation. This is because for any human cell therapy to succeed, it is necessary to prepare sufficient amount of undifferentiated human cell stem cells to test out the experimental conditions as well as subsequent transplantation into patients. This critical step is currently the bottleneck for advancing stem cell biology and therapy. This proposal is aimed at discovering the cellular basis on why undifferentiated hESCs, in contrast to mouse embryonic stem cells and human embryonic carcinoma cells, grow so slowly and are so difficult to sustain. We will study and compare a major cellular defense system of human stem cells. Our study will utilize several undifferentiated hESCs, as well as hESCs that have been induced to differentiate into neural stem cells, the latter are critical for future transplantation studies for the cure of neurological disorders such as Alzheimer’s and Parkinson’s diseases. In this application, we also propose a simple procedure whereby upon modification of the growth medium the hESCs may be able to grow more rapidly with a higher survival rate. If successful, our discovery can be applied to the cure of many types of diseases amendable by stem cell therapy. The CIRM seed grant is most appropriate for this study as it is testing a novel concept, has direct translational potential into therapy, and will include human stem cell lines currently not approved by the NIH.
Statement of Benefit to California: 
This project has a broad benefit to Californians since we propose to evaluate basic biological functions of the human embryonic stem cells in culture. We hope to gain a better understanding of how the cells are responding to culture conditions and use this knowledge to improve culture conditions. Understanding the mechanisms governing human embryonic stem cell growth and stable propagation is vital to the success of this field. Our goal is to provide better and more reliable culture and scale-up preparations of human embryonic stem cells and their differentiated progeny. Unless we overcome the current limitations with current culture and scale-up protocols, the therapeutic potential of human embryonic stem cells will not be realized. This is an absolute prerequisite for achieving the goal of utilizing human stem cells for therapy of human diseases. Cost savings to California’s tax payers will be realized when human stem therapy can be achieved through stable growth and expansion of human embryonic stem cells.
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.

Therapeutic potential of genetically modified human ES cells in an Alzheimer's disease model: Contribution of IGF-1

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Alzheimer’s disease (AD) is a progressive and irreversible disease of the brain leading to deterioration of mental function and eventual morbidity and death. The major defining characteristic of AD brains is the excessive accumulation of amyloid plaques (composed of clumps of Abeta) outside of nerve cells and tangles (composed of clumps of tau) inside nerve cells. These lesions are toxic to nerve cells and likely explain the progressive degeneration seen in AD brains. Currently available treatments for AD provide only limited symptomatic relief and are unable to prevent, stop, or cure the disease. Even if next generation drugs prove to be more effective, they are unlikely to reverse the disease progression. Thus, it may be necessary to replace dead or dying nerve cells in order to reverse the course of the disease in many AD patients. The long-term objective of this proposal is to use genetically modified human embryonic stem cells (ESCs) as an inexhaustible source for replacing lost or damaged nerve cells, supplying the host brain with protection from further damage, and working against the underlying factors that promote amyloid and tangle lesions. Such objective ultimately may lead to a strategy for therapeutic intervention in AD patients who do not respond to available pharmacological treatments. It is well known that mouse embryonic stem cells exhibit the remarkable ability to respond to damaged nerve cells and home in on these degenerative environments in brain. At present, the capacity of human embryonic stem cells (ESCs) to integrate into the diseased brain such as those with amyloid and tangle lesions is unknown. In this proposal, we will use a mouse model of AD that develops both amyloid plaques and tangles to test the idea that transplantation of ESCs might be beneficial in treating AD. Our hypothesis is that human ESCs possess the inherent capacity to home in and integrate into sites surrounding plaques and tangles, where nerve cell damage is occurring. In addition, we hypothesize that human ESCs genetically modified to produce a protective factor called IGF-1 will further enhance this capability, help host nerve cells from further damage, and block the accumulation of plaques and tangles. It is known that IGF-1 promotes ESCs to become nerve cells, protects nerve cells from damage by Abeta, and decreases the levels of Abeta in brain. Furthermore, IGF-1 levels are reduced in AD, and loss of IGF-1 promotes tangle-like lesions in mice. If the above hypotheses can be even partially demonstrated, the current proposal is expected significantly advance our long-term objective of applying genetically modified human ESCs as a therapeutic technology for AD patients who are refractory to available pharmacological treatments.
Statement of Benefit to California: 
Alzheimer’s disease (AD) is an age-related debilitating disease of the brain characterized by progressive deterioration of mental function and accounts for more than 70% of all dementias of the brain. AD inflicts more than 465,000 residents in California alone and places substantial medical, social, psychological, and financial burden on the patients, their families, and social/medical institutions. The per capita cost of caring for an AD patient in California was estimated to be more than $65,000 per year in 1998. It was also projected at the time that the cost of caring for AD patients in California (in 1998 dollars) will be ~$25.9 billion in 2000, ~$47.5 billion in 2020, and ~$75.4 billion in 2040. During the same time period, the number of AD patients in California is projected to rise from ~395,000 in 2000 to ~1.2 million in 2040. At present, no effective treatment is available for AD. First generation drugs can temporarily mask symptoms of the disease but rapidly lose effectiveness during the progression of AD. Even if next generation drugs prove to be more effective, they will only help to slow down the progression of AD but not reverse it. As such, it may be necessary use an alternate therapeutic strategy to replace dead or dying nerve cells, especially in patients that do not respond to available drugs. Human embryonic stem cells have emerged in recent years to hold enormous potential for cell replacement therapy for wide variety of neurological disorders, including AD. As California continues to be at the forefront of new and innovative technologies, the passage of Proposition 71 to fund stem cell research further extends this spirit of innovation. The research proposed in this application attempts to generate genetically modified human embryonic stem cells capable of not only replacing lost nerve cells but also delivering protective factors that prevent further degeneration of existing nerve cells in an animal model of AD. Such kind of technological coupling between stem cell therapy and gene therapy poses therapeutic potential for application in AD where irreversible nerve cell damage cannot be treated with even the best of next generation drugs. If successful, this will also help to offset the enormous social and financial burden of caring for AD patients in California. Technologies and therapeutics derived from stem cell research funded by the California Institute for Regenerative Medicine (CIRM) are in part the contractual property of the state of California, and hence its residents. In the event that such intellectual property leads to commercialization or licensing down the line, a portion of the proceeds are contracted to enter the California state general fund, ensuring that all California residents benefit from potential successes of this research.
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.

Epigenetic regulation of AAVS1

Funding Type: 
SEED Grant
Grant Number: 
RS1-00228
ICOC Funds Committed: 
$0
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Development and differentiation is regulated by spatial and temporal regulation of genes. Genes in the nucleus are found associated with proteins and this is called chromatin, which regulates genes. Genes in stem cells are also regulated by chromatin and the structure of chromatin undergoes changes during differentiation. Understanding the sequence of events that occur in specific chromatin domains during stem cell self-renewal and differentiation becomes vital before we can begin to use these in regenerative medicine. Genetically modifying stem cells may be necessary prior to their use in therapy. The non-pathogenic virus AAV is employed as a vector in numerous gene therapy trials and holds promise for use in modifying stem cells. This virus establishes a latent infection by integrating into a specific region of the human genome called AAVS1. This is in contrast to other viruses used in gene therapy that randomly insert into the genome and thus can be mutagenic. We propose to investigate the chromatin structure at AAVS1 so that AAV based vectors can be used optimally in regenerative medicine. This proposal will improve our toolkit for modifying stem cells using gene therapy. One way to reverse the effects of dysfunctional genes is to deliver a corrected copy to the affected individual. By virtue of their ability to propagate indefinitely, stem cells offer an unlimited supply of healthy genes but undifferentiated stem cells transplanted into patients give rise to problems. These problems can potentially be circumvented by genetically manipulating stem cells in vitro to direct their differentiation into the lineage of choice prior to transplantation but will necessitate integrating transgenes into these cells. The proposed experiments will allow us to better genetically modify stem cells. The experiments outlined in this proposal will characterize the chromatin domains around the AAVS1 region in depth. We will determine how the AAVS1 genomic locus changes with respect to its chromatin structure as stem cells undergo differentiation into specific lineages. Furthermore, we will establish the chromatin determinants that (i) promote the stable integration of AAV into a specific region of the genome and (ii) allow stable expression of transgenes in stem cells. As our long-term goal we will study the changes that occur in the chromatin structure of the AAVS1 region in stem cells expressing an AAV-mediated transgene that induces these cells to differentiate along a specific lineage. These studies will enable the development of vectors for the expression of specific transgenes in stem cells that will direct their differentiation into specific cell types. Such a system could then be exploited to generate large cell banks with diverse histocompatibilities for use in patients with hereditary disorders.
Statement of Benefit to California: 
This proposal seeks to combine the potential of two of the most promising approaches in modern medicine: stem cell and gene therapy. Over 1800 genes have been determined to cause hereditary disorders and the most obvious way to reverse the effects of such dysfunctional genes is to deliver a corrected copy to the affected individual. By virtue of their ability to propagate indefinitely, stem cells offer an unlimited supply of healthy genes. However, when undifferentiated embryonic stem cells are transplanted into the patient they have the potential to form teratomas while adult stem cells can potentially give rise to tissues that are not desirable at the site of transplantation. These problems can potentially be circumvented by genetically manipulating stem cells in vitro to direct and control their differentiation into the lineage of choice prior to transplantation. In the future one can envision CA-based large therapeutic cell bank repositories of different lineages and immune characteristics that would enable physicians to find immunologically compatible cells for corrective cell therapy. Results from experiments in this proposal will allow the stable expression of proteins and growth factors that can direct stem cell differentiation without being subjected to position effects resulting from random integrations and can therefore be utilized for generating cell banks. A second application for the proposed research is in gene transfer therapy where stem cells derived from the patient are corrected for the defective gene, expanded, characterized and allowed to differentiate prior to re-transplantation into that patient thus avoiding immune rejection. Although this approach requires heavy logistics and might be limited to small numbers of patients, therapies such as these could be developed from the proposed research and will have the advantage that the integrated genes will not be subject to variations in expression by gene silencing and additionally will avoid the problems of histocompatibility mismatches and immune rejection. Knowledge from this research will also spur growth in new biotechnology firms to develop gene delivery vectors in stem cells thus offering a direct advantage to the state in terms of revenue and employment opportunities. This research will also put the state of California at the forefront of stem cell technology along with other nations.
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.

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