Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

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.

Role of Notch signaling in human embryonic stem cell differentiation to neuronal cell fates

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: 
Human embryonic stem cells (HESCs) are capable of giving rise to a variety of differentiated human cell types that in principle could be used therapeutically to treat tissue damage that arises in human disease. The promise of HESCs is still quite limited because of technical limitations in our ability to propagate these cells in culture, while retaining their potency to become many different types of cells, and to guide them to become the right type of cell needed for clinical use. The proposed work will develop the tools to address these issues, by focusing on the Notch signaling pathway. Studies of the Notch pathway in model organisms like mice has shown that it plays a pivotal role in regulating the development of embryonic cells, by activating critical target genes that maintain cells in a proliferative, undifferentiated state. The proposed experiments will examine the activity of the Notch pathway in HESCs, as they are experimentally induced to form the precursors to nerve cells. The long-term goal of this work is to develop the information and tools needed to manipulate HESCs in culture via the Notch pathway, allowing one to better control their proliferation and differentiation into defined cell types.
Statement of Benefit to California: 
The goal of the proposed research is to develop tools that can be used to manipulate human embryonic stem cells, thus allowing them to be more effectively used as therapeutic agents. The process we are studying will help define optimal procedures to encourage human embryonic stem cells to produce homogeneous populations of specific neural cell types that are needed to replace damaged neural tissues for patients with Parkinson’s and other neural diseases.
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.

Brain Aging and hESC-derived Neural Stem Cell Transplantation

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: 
Aging is an important risk factor for human diseases. In addition to cognitive decline in the elderly, brain aging also increases the risk for neurological diseases like Alzheimer’s disease (AD), Parkinson's disease (PD), and stroke, which are major causes of disability and mortality in the elderly. Current therapeutic approaches typically fail to treat the underlying cause of these disorders. The potential capacity of human neural stem cells to replace cells and tissues damaged due to aging and age-realted diseases is, therefore, of great importance. Activation or transplantation of these cells has already shown promise in animal models of these disorders. However, little is known about how aging affects host receptivity to hESC-derived cell transplants, or about the proliferation, survival, differentiation, migration and functionality of the donor cells. This is partly because in vivo studies of aged-related neurological diseases have relied almost universally on experimental models using young adult animals.We hypothesize that the ability of hESC-derived transplants to proliferate, survive, differentiate, migrate and function may be compromised in the aged brain, requiring that new strategies be devised to overcome this deficiency. The experiments we propose are designed to increase our understanding of how the age of the recipient alters the ability of transplanted neural stem cells to differentiate into mature, functional neurons and integrate into local neuronal circuits. Our first Specific Aim will examine these issues using immunocytochemistry and electrophysiological methods, after transplanting neural stem cells into young adult, middle aged and aged rats. In addition, because brain aging produces progressive changes in learning and memory, a critical area to examine is whether neural stem cell therapy can produce functional improvement in learning and memory deficits in aged rats. Our second Specific Aim will address this question by using a battery of behavioral tests to assess the ability of transplanted neural stem cells to reverse age-related losses of cognitive function. The use of neural stem cells to repair age-related neurological diseases will require an increased understanding of stem cell biology, the environment of the aged tissue, and the interaction between the two, which this proposed work will focus on. If the aims of the application are achieved, significant advances will be made in establishing a new paradigm of brain aging and the biological behaviors of transplanted neural stem cells, and substantial progress will be made in developing basic science knowledge that can be translated fairly rapidly into clinical treatment of aged-related neurological diseases.
Statement of Benefit to California: 
Aging is destined to become a serious public health problem for California over the next several decades. California’s elderly population is expected to grow more than twice as fast as the total population from 1990 to 2020, and will reach 12.5 million by 2040. One in five Californians will be 60 years of age or older beginning in 2010. Consequently, age-related diseases, including neurological diseases, will dramatically increased in parallel, presenting enormous social and economic challenges. Determining whether transplanted human neural stem cells can differentiate into functional neurons in the aging brain, and thereby slow or prevent cognitive decline in the aged, could have great social and economic impact in our state.
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.

Developing chicken embryos as an experimental microenvironment for human embryonic stem cells

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: 
It is expected that research funded by the California Institute of Regenerative Medicine will result in the development of many new human embryonic stem cells. The properties of these cell lines will have to be verified. One of the main attributes of embryonic stem cells is their capacity to differentiate toward an unlimited number of cell fates. This is what will make them a powerful tool in future regenerative medicine. Therefore, we need new methods capable of quickly evaluating the response of many cell lines to many different environments. In this proposal, we will develop and test a new test bed to evaluate the capabilities of potential human embryonic stem cells: the chicken embryo. Since chicken embryos develop outside the body in an egg, they are highly accessible to experimental manipulations. This enables us both to introduce reagents (ie., human embryonic stem cells) and to visualize the response of those reagents to their local environments as they happen. We propose to introduce human embryonic stem cells to six different organ systems during several stages of development. This will test the ability of these cells to respond to a large number of different environmental stimuli. Since different embryonic cell lines may have different capacities, we will test the abilities of seven different human embryonic stem cells. We will compare their response with that of partially differentiated cells that should have more limited differentiation capabilities. Transplanted cells will be fluorescently tagged so their migration can be traced by fluorescence microscopy. Antibodies and probes of molecular expression will be used to assess the response of these cell lines to different environments. Their origin (human or chicken) can also be confirmed with these methods using different antibodies and probes. This will help us to develop a set of formal criteria that to assess the response capability of hESC as they progressively become more differentiated. To further understand molecular aspects of the cellular response, we will begin to characterize changes in molecular expression that take place as cells progress toward specific cell fates. This profile will enable us to begin to understand molecular factors which regulate cellular differentiation, so they can be harnessed for effective future regenerative medical applications. This last goal will serve to show the power of this technology, but will have to await a later stage of funding to be completed.
Statement of Benefit to California: 
Human embryonic stem cells offer tremendous potential toward significant advances in the new age of regenerative medicine. These cells can be induced to differentiate along many different cell fates, providing the promise of tissue and/or organ replacement or supplementation. This approach offers great hope toward improving health care especially where tissues are damaged due to disease or injury. Ultimately, this approach could reduce health care costs and increase the well being of the general population. We expect that many new embryonic stem cell lines will be derived with support from the California Institute for Regenerative Medicine. We would not be surprised if the ability of these putative stem cells to differentiate toward specific cell fates differed from cell line to cell line. Additionally, some cell lines may lose their differentiative capacity as they are kept in in vitro culture conditions. Our research proposal aims to provide an easy and effective assay to test the pluripotentiality of these new putative human embryonic stem cell lines. In order for these cell lines to live up to their full potential and be useful in curing human diseases, we must understand their pluripotential properties. To date, assays of pluripotentiality have depended on 1) in vitro assays with limits in ascertaining the true developmental potential and 2) transplantation to mouse embryos which do not facilitate the high throughput analysis, essential to screen the myriad of generated cell lines. The latter assay also involves causing pain in a sentient being (the mother). Chicken embryos develop in an egg, outside of the body. Hence it is easily accessible to experimentation including the delivery and observation of putative embryonic stem cells. The chicken embryo is a classic model of development and has been very well documented through years of research. It offers a myriad of developmental microenvironments which can be utilized to test the responsiveness of these new cell lines. This research would contribute to the progress of stem cell research which ultimately could improve health care for everyone, worldwide. Since California is one of the first states to implement support for human embryonic stem cell research, our findings could also contribute to major economic advantages to the citizens of the state.
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.

EFFECTS OF SMALL MOLECULE LIBRARIES ON DIFFERENTIATION OF EMBRYONIC AND NERAL STEMS CELLS IN DOPAMINERGIC PHENOTYPE

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
PUBLIC ABSTRACT. Degenerative brain diseases account for a huge portion of the health burden for our society. Most are largely untreatable or, at best, inadequately treated. A number of devastating human neurological diseases are caused by abnormalities of the ways in which signals are transmitted from one brain cell to another and from one part of the brain to another. One of the most important signal systems in the brain and one that is most vulnerable to inherited or acquired damage is the system that is regulated by a special class of nerve cells (neurons) that use the molecule dopamine to transmit signals to other neurons and other cells in the brain. The most common of dopamine-deficiency diseases is the familiar Parkinson’s disease that afflicts millions of people throughout the US and hundred of thousands of Californians. Unfortunately, for many reasons, the causes of the dopamine degeneration in this disease are difficult to study, partly because most cases of this disorder seem to be caused by complex interactions among a number of genes or by mixtures of genetic and environmental factors. Only a few cases are caused by identified simple defects in the genes responsible for producing or maintaining the dopamine neurons. Fortunately for an understanding the genetics of the dopamine signaling system, another disorder of dopamine function, Lesch Nyhan Disease, is caused by defects in one single gene called HPRT. The disorder is associated with severe retardation, abnormal movements and a compulsive and untreatable self-mutilation behavior and is largely untreatable. Because the disease is a direct result of abnormalities in a single, well-understood gene, it is possible to study the ways in which genetic damage can cause defects in the dopamine systems, changes that are directly responsible for the severe neurological consequences. Because the damage to the dopamine pathways produces defects of the dopamine-dependent cells themselves, we propose to study the ways in which stem cells develop into mature normal dopamine nerve cells. There are a variety of cells that have developed along the pathway from stem cells to normal nerve cells but that have not reached the stage of functioning dopamine nerve cells. We wish to determine if we can force these cells as well as the stem cells themselves to become functional dopamine neurons by treating them with a collection of more approximately 50,000 chemicals. If these studies area successful, we may be able to understand the dopamine development process more thoroughly and possibly also to prepare large numbers of normal dopamine-producing cells for eventual transplantation approaches to these devastating diseases.
Statement of Benefit to California: 
BENEFIT TO CALIFORNIA Advances in modern molecular genetics are making possible new approaches to understanding the basis of normal and abnormal human biology and to improve treatment of human disease. Studies of human embryonic stem cells promise to become an important part of this new area of biomedicine, but many kinds of studies have been severely hampered in the U.S. by a restrictive national policy and inadequate funding mechanisms. Wisely, the people of California have taken steps to catalyze this field through the formation of the California Institute for regenerative Medicine (CIRM) and through the CIRM programs to support research in this area. Our studies, if successful, will contribute to an understanding of a basic process of brain function and will therefore strengthen the huge basic research effort in neurobiology at the California-based academic institutions. Furthermore, knowledge and techniques derived from this kind of study can be applied to the discovery and development of drugs that specifically affect neuron function and that potentially affect the movement, cognitive, mood, compulsive and aggressive behavioral aspects of brain disorders, all of which represent the central features of Lesch Nyhan Disease. Another potentially useful outcome with commercial implications could involve methods to produce large amounts of dopamine-producing cells for transplantation for treatment of some kinds of brain disorders and the development of similar approaches to other neurotransmitter CNS disorders. Such discoveries will constitute the basis for expanded and new pharmaceutical and biotechnology ventures in California, with the health and economic benefits that such progress carries with it.
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.

Generation of Inherited Disease Human Embryonic Stem Cell Lines

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The development of human embryonic stem (hES) cell lines that carry a disease causing mutation can provide insight into the mechanisms underlying disease progression as well as into the development of therapies that can ameliorate that pathology. The primary goal of this proposal will be the development of novel hES cell lines from embryos that will manifest a given genetic disease upon further development. This will be achieved following two distinct approaches. The first will be through the identification of embryos that are homozygous for a given mutation and the generation of novel cell lines from these embryos. These embryos will be identified by preimplantation genetic diagnosis (PDG). The afflicted embryos will then be grown according to established protocols known to generate human ES cell lines. The second approach will involve the generation of disease associated homozygote cell lines through a technique that will specifically modify a gene sequence and introduce a disease associated mutation. This technique, small fragment homologous replacement (SFHR), has been shown to be effective at modifying DNA sequences in human cells. SFHR-mediated changes are caused by small DNA fragments (SDFs) that are introduced into the cells. The SDFs are effectively the same as the gene target sequences except for the changes to be introduced. Initial studies will target genes on the X-chromosome of normal male hES cells that carry only one X-chromosome. The genetic diseases that are anticipated to be served by this proposal include, but are not limited to, cystic fibrosis, sickle cell disease, ?- and ?-thalassemia, Duchenne’s and Becker muscular dystrophy, X chromosome-linked severe combined immune deficiency (SCID-X1), spinal muscular atrophy (SMA), Guacher’s disease, Fanconi anemia, and Lesch-Nyhan syndrome.
Statement of Benefit to California: 
This proposal will provide benefit to the citizens of California by increase our knowledge of the basis of genetic diseases and by providing a means to develop new and more effective therapies for these diseases. This project is focused on improving the health and well being of the citizens of California and could have far reaching positive implications for health and economic factors influencing the quality of life for the citizens of this state.
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.

Targeting lentiviral vectors to modified hES derived dendritic cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
We hypothesize that human embryonic stem cells represent a potentially scalable source of human dendritic cells that could be used to treat a wide array of diseases including HIV/AIDS and cancer. In fact dendritic cell based therapies have shown some promise in early trials, but they are largely limited by the numbers of cells available for treatment protocols. We propose here to develop a potentially unlimited source of human dendritic cells from human embryonic stem cells. More importantly, we propose to modify these cells to express a novel cell surface molecule that can be used for targeted delivery of specific proteins. Ultimately we envision using these cells to direct primary immune responses. The studies described in this SEED proposal are directed towards HIV specific immune responses, but would be widely applicable to other pathogen-based diseases, as well to tumor associated antigens.
Statement of Benefit to California: 
Dendritic cell immunization, dendritic cell-based gene therapies and immunotherapies are being explored as treatments for a number of diverse human diseases, particularly in settings that are refractory to conventional therapies. Unfortunately, these protocols are directly limited by the ability to generate sufficient quantities of dendritic cells ex vivo. Human embryonic stem cells represent a potentially scalable source of dendritic cells that could be used in these settings. For instance, they could be used for the prevention and treatment of pathogen based diseases such as HIV/AIDS, Hepatitis C and Influenza. In addition, development of a source of self renewing dendritic cells could dramatically advance patient specific protocols for the treatment of cancer. Taken together these diseases and their treatments impact Californians personally and economically. The development of improved treatment and prevention modalities by harnessing the potential of human embryonic stem cells would represent a major benefit to the lives of all Californians.
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.

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