Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

Differentiation of Human Embryonic Stem Cells to Heart Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00428
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Public Abstract California researchers will investigate how human embryonic stem cells can be transformed into working heart cells. This will, advance us toward a cure for chronic heart disease – for which there is now no cure other than heart transplantation. In so doing, they will broaden our understanding of how human embryonic stem cells, in general, can be transformed into functional cells that repair and regenerate parts of the body that are damaged by disease or injury. Each year there are 1.2 million heart attacks in the US. Although most do not result in immediate death, the cumulative damage leads to 500,000 deaths per year. When the oxygen supply to parts of the heart is shut off, heart muscle cells die leaving only scar tissue. Repeated small heart attacks build up this damage until the heart can no longer pump in response to exertion or excitement. At that stage, patients can die of congestive heart failure. It was previously thought that the scarring damage that leads to heart attacks was permanent. In recent years, however, researchers have shown how the scar tissue may be repaired with stem cells from bone marrow. However it is not known if the bone marrow stem cells become new heart cells or stimulate existing cells in the heart to perform better. This project’s researchers have one of the few laboratories world wide that are able to grow cardiac cells in large numbers and test them. This research project will build from the investigators’ ongoing stem cell research experience and study human embryonic stem cells because of the remarkable ability of such stem cells to turn into almost any type of adult cell. To convert the human embryonic stem cells into cardiac specific cells before they are delivered into the heart it is essential that we understand the molecular and biochemical process for differentiating human embryonic stem cells Researchers plan to study human embryonic stem cells with a chemical inducers to drive the transformation from stem cell to heart cells. The investigators will use a new technique which they have developed to label heart specified cells. They will study the molecular changes that underly the changes as the stem cell transforms. To understand this process fully, they will intensely analyze with computers, changes in newly discovered molecules that control the DNA code of life The resulting heart cells will be administer into the hearts to test if they are, in fact, turning into effective new cells that replace scar tissue in the heart ventricle as theorized. The fate of the transformed stem cells will be followed with a new magnetic imaging method to measure simultaneously the change in scar size, heart performance and the longevity of the stem cells.
Statement of Benefit to California: 
Benefits to Californians The citizens of California have a right to expect many benefits from their forward looking approval of stem cell research. The initiative to fund human embryonic stem cell research will bring breakthroughs in treatments for previously untreatable diseases and create unprecedented new business opportunities. This proposal is directed to saving the thousands of Californians who die or suffer from heart failure each year. The only known cure available now is a heart transplant, but because the demand for hearts is greater than the number of donors, an alternative source of heart cells is needed. We are engaged in using the potentiality of human embryonic stem cells to turn them into heart cells that could be injected into damaged hearts and make them heal. To do this we have to build our understanding of the fundamental process of making a stem cell into a heart cell and then testing how effectively such cells can repair injured hearts. Knowing the underlying scientific principles at work in such a process will create the potential to benefit not only heart disease patients, benefits to Californians The citizens of California have a right to expect many benefits from their forward looking approval of stem cell research. The initiative to fund human embryonic stem cell research will bring breakthroughs in treatments for previously untreatable diseases and create unprecedented new business opportunities. This proposal is directed to saving the thousands of Californians who die or suffer from heart failure each year. The only known cure available now is a heart transplant, but because the demand for hearts is greater than the number of donors, an alternative source of heart cells is needed. We are engaged in using the potentiality of human embryonic stem cells to turn them into heart cells that could be injected into damaged hearts and make them heal. To do this we have to build our understanding of the fundamental process of making a stem cell into a heart cell and then testing how effectively such cells can repair injured hearts. Knowing the underlying scientific principles at work in such a process will create the potential to benefit not only heart disease patients, but also others suffering from diseases for which treatment is inadequate or not available. We anticipate that the entrepreneurial business spirit for which California is famous, will generate new jobs and businesses that will build upon the discoveries made in this type of stem cell research. This will leverage the economy because to have an inexhaustible supply of heart cells (or any other type of organ cell) will require significant commercial manufacturing processes. Biologicals for growing cells will be an industry. Newly discovered drugs can be tested on such cells by pharmaceutical companies. California will become the world’s provider of human cells, for transplantation treatments and cures, and it will reap the rewards of its investment.
Progress Report: 
  • Human embryonic stem cells (hESCs) originate directly from human embryos, whereas induced pluripotent stem cells (iPSCs) originate from body (somatic) cells that are re-programmed by producing or introducing proteins that control the process making specific RNAs. Together, both these pluripotent cell types are referred to as human pluripotent stem cells (hPSCs). Several reports have observed that in hESCs grown for long times, their genetic material, DNA, is unstable. The stable maintenance of DNA is performed by groups of proteins functioning in different systems globally known as DNA repair pathways. Since the development of aneuploidy is closely linked to cancer and to deficiencies in DNA repair, we have studied the propensity of hPSCs to repair their DNA efficiently by 4 major known DNA repair pathways. In addition, we are also investigating if specific damage to DNA in either hPSCs or somatic cells is processed differently and could lead to deleterious mutations.
  • One major goal of the CIRM SEED grant mission is to bring new researchers into the hPSC field. The results we obtained during the funding period indicate that we have succeeded in that objective, since initially our laboratory had little experience with hESC culture. However, through courses and establishing critical collaborations with other hESC laboratories, we developed expertise in hPSC culture techniques. Most conditions for hPSCs growth require cells (feeder cells) that serve as a matrix and provide some factors needed for the pluripotent cells to divide. In accomplishing this aim, we perfected a method to generate reproducible feeder cells that significantly reduces the time and cost of feeder cell maintenance, and also developed a non-enzymatic and non-mechanical way to expand hPSCs. We now have experience with at least 5 hPSC lines and have methods to introduce foreign DNAs into hESCs and iPSCs to monitor DNA repair in hPSCs.
  • In Aim II of our grant, we used our accumulated knowledge of hPSCs and DNA repair to investigate 4 DNA repair mechanisms in hPSCs and in somatic cells. Depending on the DNA damage, there is often a preferred DNA repair pathway that cells use to alleviate potential harm. We initiated our investigation by treating hPSCs using different DNA damaging agents, including ultraviolet light and gamma radiation. However, we found that hPSCs exposed to these agents rapidly died compared to treatments that allowed somatic cells to continue growing. Therefore, we developed methods to study DNA repair in hPSCs without directly treating the cells with external agents. We treated closed, circular DNA (plasmids) with damaging agents separately, outside the hPSCs and then introduced them into the hPSCs. The plasmid DNA has a sequence that codes for a protein that is produced only when the damage is repaired. The length of time for repair both in hPSCs and in somatic cells was followed by determining the protein production. We have shown superior DNA repair ability and elevated protection against DNA damage in hPSCs compared to somatic cells for ultraviolet light and oxidative damage, two common sources of damage in cells. A major pathway for joining double-strand DNA breaks in mammalian cells, non-homologous end-joining (NHEJ) repair (error prone), is greater in H9 cells than in iPSCs. Another way to repair double-strand DNA breaks that uses similar (i.e., homologous) sequences is lower in iPSCs compared to hESCs and somatic cells. Further study of these repair pathways is warranted, since several methods can be used to form iPSCs. Therefore, the genomic stability for iPSCs could depend on the method used for their generation.
  • DNA repair analysis is critical to understanding how hPSCs protect against damage, but if left unrepaired, cells can turn damage into mutations when the damage is copied by enzymes (DNA polymerases) before repair occurs. Therefore, to monitor the mutations that ultimately lead to cancer or alter hPSC biology, we are using a plasmid that is damaged outside the cells and will make copies in hPSCs and somatic cells. That plasmid is introduced into cells and then the copies are recovered. The number of mutations found in the plasmid DNA indicates the likelihood of observing mutations in hPSCs compared to mutations in somatic cells. Together, these results will yield data on the stability of hPSCs and also a basis to monitor cells for stability which could serve as an indicator of safety for clinical use.

Treatment of Lung Disease with Inhaled Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00428
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Incurable lung diseases pose a major challenge to medical science. Cystic fibrosis, asthma, pulmonary fibrosis, cancer, hyaline membrane disease and emphysema are examples of diseases that may be eventually conquered by stem cell therapies. However, due to its geometrical complexity it is difficult to deliver therapeutic agents to the diseased regions of the lung where they may effect cures. Fortunately, the use of other inhaled medicines has progressed to the point where stem cell delivery can now be considered. Aerosol treatments are typically painless and very effective. The challenges to therapy with inhaled stem cells are many, including; cell survival during the process of aerosolization, delivery of cells to the proper disease- specific region(s) of the lungs, and providing support for the stem cells, post deposition, that will allow them to survive and become properly established. Our project systematically attacks these challenges by establishing a team with competence in aerosol science, inhaled aerosol deposition, and clinical pulmonary medicine. Completion of this pilot project will lay the foundation for developing specific new therapies for lung diseases.
Statement of Benefit to California: 
Many Californians suffer from incurable and even untreatable lung diseases. This project has the goal of developing aerosol delivery systems for human embryonic stem cells so that the advances in stem cell biology can be used to treat lung disease. California could become a world leader in treating patients with lung diseases such as cystic fibrosis, asthma, pulmonary fibrosis, cancer, hayline membrane disease, and emphysema. Another benefit is that aerosol treatments for lung diseases are typically, painless, do not require anesthesia, and tend to have fewer side effects than do alternative forms of administration. Projecting into the future, microgravity medicine will permit the administering of stem cells deeper into the lung because the deposition in bronchial airways by the sedimentation mechanism will be absent. California's significant role in space may thus be enhanced.
Progress Report: 
  • Human embryonic stem cells (hESCs) originate directly from human embryos, whereas induced pluripotent stem cells (iPSCs) originate from body (somatic) cells that are re-programmed by producing or introducing proteins that control the process making specific RNAs. Together, both these pluripotent cell types are referred to as human pluripotent stem cells (hPSCs). Several reports have observed that in hESCs grown for long times, their genetic material, DNA, is unstable. The stable maintenance of DNA is performed by groups of proteins functioning in different systems globally known as DNA repair pathways. Since the development of aneuploidy is closely linked to cancer and to deficiencies in DNA repair, we have studied the propensity of hPSCs to repair their DNA efficiently by 4 major known DNA repair pathways. In addition, we are also investigating if specific damage to DNA in either hPSCs or somatic cells is processed differently and could lead to deleterious mutations.
  • One major goal of the CIRM SEED grant mission is to bring new researchers into the hPSC field. The results we obtained during the funding period indicate that we have succeeded in that objective, since initially our laboratory had little experience with hESC culture. However, through courses and establishing critical collaborations with other hESC laboratories, we developed expertise in hPSC culture techniques. Most conditions for hPSCs growth require cells (feeder cells) that serve as a matrix and provide some factors needed for the pluripotent cells to divide. In accomplishing this aim, we perfected a method to generate reproducible feeder cells that significantly reduces the time and cost of feeder cell maintenance, and also developed a non-enzymatic and non-mechanical way to expand hPSCs. We now have experience with at least 5 hPSC lines and have methods to introduce foreign DNAs into hESCs and iPSCs to monitor DNA repair in hPSCs.
  • In Aim II of our grant, we used our accumulated knowledge of hPSCs and DNA repair to investigate 4 DNA repair mechanisms in hPSCs and in somatic cells. Depending on the DNA damage, there is often a preferred DNA repair pathway that cells use to alleviate potential harm. We initiated our investigation by treating hPSCs using different DNA damaging agents, including ultraviolet light and gamma radiation. However, we found that hPSCs exposed to these agents rapidly died compared to treatments that allowed somatic cells to continue growing. Therefore, we developed methods to study DNA repair in hPSCs without directly treating the cells with external agents. We treated closed, circular DNA (plasmids) with damaging agents separately, outside the hPSCs and then introduced them into the hPSCs. The plasmid DNA has a sequence that codes for a protein that is produced only when the damage is repaired. The length of time for repair both in hPSCs and in somatic cells was followed by determining the protein production. We have shown superior DNA repair ability and elevated protection against DNA damage in hPSCs compared to somatic cells for ultraviolet light and oxidative damage, two common sources of damage in cells. A major pathway for joining double-strand DNA breaks in mammalian cells, non-homologous end-joining (NHEJ) repair (error prone), is greater in H9 cells than in iPSCs. Another way to repair double-strand DNA breaks that uses similar (i.e., homologous) sequences is lower in iPSCs compared to hESCs and somatic cells. Further study of these repair pathways is warranted, since several methods can be used to form iPSCs. Therefore, the genomic stability for iPSCs could depend on the method used for their generation.
  • DNA repair analysis is critical to understanding how hPSCs protect against damage, but if left unrepaired, cells can turn damage into mutations when the damage is copied by enzymes (DNA polymerases) before repair occurs. Therefore, to monitor the mutations that ultimately lead to cancer or alter hPSC biology, we are using a plasmid that is damaged outside the cells and will make copies in hPSCs and somatic cells. That plasmid is introduced into cells and then the copies are recovered. The number of mutations found in the plasmid DNA indicates the likelihood of observing mutations in hPSCs compared to mutations in somatic cells. Together, these results will yield data on the stability of hPSCs and also a basis to monitor cells for stability which could serve as an indicator of safety for clinical use.

Treating Stress Urinary Incontinence with Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00413
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Neurological Disorders
Skeletal Muscle
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Urinary incontinence (UI) is a major health issue that affects more than 200 million people worldwide. Stress urinary incontinence (SUI), which accounts for half of all UI cases, is the involuntary loss of urine in the absence of a detrusor contraction. SUI occurs as a result of weakened muscles of the pelvic floor and urethra, producing urine loss whenever there is an increase of intra-abdominal pressure, such as coughing, sneezing, and laughing. Currently there is no effective treatment for SUI. Because weakened muscles and nerves in the urethra are the underlying cause of SUI, this proposed study seeks to correct such deficiencies by replenishing the affected urethra with human embryonic stem cells (hESC). Human ESC are capable of differentiating into various cell types including smooth muscle, striated muscle, and nerves. These cell types are also found in the urethra and are affected during the disease progression of SUI. In Specific Aim 1 of this proposed project we will investigate whether hESC can be induced to turn into cell types found in the healthy urethra. In Specific Aim 2 we will test the therapeutic efficacy of hESC. Because it is unethical to conduct this research in patients, we will employ a rat SUI model that was developed in our laboratory 11 years ago. We have shown in several publications that this SUI model closely mimics human SUI in both the disease progression and pathology. We are therefore confident that this rat model will allow us to assess the therapeutic effectiveness of hESC. This assessment will then help us to decide whether hESC is suitable for human therapy.
Statement of Benefit to California: 
Urinary incontinence (UI) is a major health problem worldwide; therefore, this proposed study will not just benefit California but the whole world. If there is anything specifically Californian, that would be the research team and the use of human embryonic stem cells (hESC) that are federally restricted. In other words, the research has the potential to strengthen California's leadership in both the UI and hESC research fields. In the long term this enhanced leadership may translate into economic gains for California such as investment in the biotech industry and health care system. If permitted by regulatory agencies at the federal and state levels, clinical trials for this stem cell therapy could perhaps be initiated in California and therefore benefit Californians firsthand.
Progress Report: 
  • We have undertaken an extensive series of studies to delineate the radiation response of human embryonic stem cells (hESCs) and human neural stem cells (hNSCs) both in vitro and in vivo. These studies are important because radiotherapy is a frontline treatment for primary and secondary (metastatic) brain tumors. While radiotherapy is quite beneficial, it is limited by the tolerance of normal tissue to radiation injury. At clinically relevant exposures, patients often develop variable degrees of cognitive dysfunction that manifest as impaired learning and memory, and that have pronounced adverse effects on quality of life. Thus, our studies have been designed to address this serious complication of cranial irradiation.
  • We have now found that transplanted human embryonic stem cells (hESCs) can rescue radiation-induced cognitive impairment in athymic rats, providing the first evidence that such cells can ameliorate radiation-induced normal-tissue damage in the brain. Four months following head-only irradiation and hESC transplantation, the stem cells were found to have migrated toward specific regions of the brain known to support the development of new brain cells throughout life. Cells migrating toward these specialized neural regions were also found to develop into new brain cells. Cognitive analyses of these animals revealed that the rats who had received stem cells performed better in a standard test of brain function which measures the rats’ reactions to novelty. The data suggests that transplanted hESCs can rescue radiation-induced deficits in learning and memory. Additional work is underway to determine whether the rats’ improved cognitive function was due to the functional integration of transplanted stem cells or whether these cells supported and helped repair the rats’ existing brain cells.
  • The application of stem cell therapies to reduce radiation-induced normal tissue damage is still in its infancy. Our finding that transplanted hESCs can rescue radiation-induced cognitive impairment is significant in this regard, and provides evidence that similar types of approaches hold promise for ameliorating normal-tissue damage throughout other target tissues after irradiation.
  • A comprehensive series of studies was undertaken to determine if/how stem cell transplantation could ameliorate the adverse effects of cranial irradiation, both at the cellular and cognitive levels. These studies are important since radiotherapy to the head remains the only tenable option for the control of primary and metastatic brain tumors. Unfortunately, a devastating side-effect of this treatment involves cognitive decline in ~50% of those patients surviving ≥ 18 months. Pediatric patients treated for brain tumors can lose up to 3 IQ points per year, making the use of irradiation particularly problematic for this patient class. Thus, the purpose of these studies was to determine whether cranial transplantation of stem cells could afford some relief from the cognitive declines typical in patients afflicted with brain tumors, and subjected to cranial radiotherapy. Human embryonic (hESCs) and neural (hNSCs) stem cells were implanted into the brain of rats following head only irradiation. At 1 and 4 months later, rats were tested for cognitive performance using a series of specialized tests designed to determine the extent of radiation injury and the extent that transplanted cells ameliorated any radiation-induced cognitive deficits. These cognitive tasks take advantage of the innate tendency of rats to explore novelty. Successful performance of this task has been shown to rely on intact spatial memory function, a brain function known to be adversely impacted by irradiation. Our data shows that irradiation elicits significant deficits in learning and spatial task recognition 1 and 4-months following irradiation. We have now demonstrated conclusively, and for the first time, that irradiated animals receiving targeted transplantation of hESCs or hNSCs 2-days after, show significant recovery of these radiation induced cognitive decrements. In sum, our data shows the capability of 2 stem cell types (hESC and hNSC) to improve radiation-induced cognitive dysfunction at 1 and 4 months post-grafting, and demonstrates that stem cell based therapies can be used to effectively to reduce a serious complication of cranial irradiation.

NOVEL REAGENTS TO CONTROL STEM CELL DIFFERENTIATION

Funding Type: 
SEED Grant
Grant Number: 
RS1-00298
ICOC Funds Committed: 
$0
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
In diseases of the nervous system such as Parkinson’s disease (PD) and Lou Gehrig’s disease very specific groups of nerve cells die. At least in the case of PD, the surgical methods exist for the implantation of new cells into the area of the brain where the nerve cells are dying. However, since fetal brain cells are almost impossible to obtain, a viable and untested potential source of brain neurons are human embryonic stem (ES) cells. In order to get these cells to function in the brain, it is mandatory that the ES cells be converted to nerve cells before they can be surgically implanted. In our past work with rat and mouse stem cells we have been able to identify and purify factors that are made by nerve precursor cells that cause stem cells to become neurons. In addition, we have a very large potential source of these types of factors that is unique to our laboratory. Finally, we have identified a new family of drugs that may help keep the ES cell-derived neurons alive when they are transplanted into the brain, a situation in which most of them normally die. Therefore, the goal of this proposal is to identify new factors that convert human ES cells to specific types of neurons and to test the new family of compounds to determine if they promote the survival of ES cell-derived neurons in the brain.
Statement of Benefit to California: 
Our work will benefit the State in a number of ways. 1) There could be a tremendous health benefit for individuals with diseases of the brain such as Parkinson’s and Lou Gehrig’s diseases as well as damage due to stroke or trauma. 2) Support for this work will provide current employment within the State and help educate scientists in the stem cell field. 3) The advancement of work on novel growth factors and drugs will require the collaboration with commercial (for profit) companies. Most of early stage preclinical development is done in small biotech companies, many of which are within the State. Therefore, there is an economic benefit to the State as well as a health benefit to the State and the world if some of the most debilitating human diseases could be cured.
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.

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.

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