Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders

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.

Developmental Regulation of Human Embryonic Stem Cells by microRNAs

Funding Type: 
SEED Grant
Grant Number: 
RS1-00409
ICOC Funds Committed: 
$0
Disease Focus: 
Multiple Sclerosis
Neurological Disorders
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Stem cells are remarkable cells which have two unique properties: self-renewal (self replication) and pluripotency (the ability to regenate a wide range of tissue-specific cells). These properties and the associated potential to use these cells to cure a wide range of degenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease, and heart disease, have made stem cells a subject of intense scientific and medical interest. The recent discovery of cancer stem cells implies that stem cell research will also have important implications for cancer therapy, since cancer stem cells must be targeted by cancer drugs to prevent relapse of tumors. The mechanisms by which stem cells self-renew and differentiate are poorly understood. Several genes have been isolated and are thought to be essential to mammalian stem cell renewal and differentiation. Recent evidence has identified a new and potentially important molecular mechanism for regulating these genes in ESCs. These molecules are small bits of RNA, of approximately 22 nucleotides (nt) in length, and often termed microRNAs (miRNAs). miRNAs appear to play an important part in regulating gene activity. These small RNAs “turn off” genes by directly binding to them and preventing the production of proteins based on the gene’s genetic code. Several recent studies indicate that the types of miRNAs present in stem cells (miRNA “expression profiles”) are different from other cells and tissues. We propose to identify candidate miRNAs that are playing key roles in hESCs and to characterize the effects of their actions on self-renewal and differentiation. Our preliminary data also indicates that miRNA “expression profiles” differ depending on whether a stem cell is differentiating, self-renewing, or quiescent. This project will employ a wide range of techniques in molecular biology, including microarrays, bioinformatics, and bioluminescent reporter gene assays, to determine which specific miRNAs show differential expression between stem cell states and we will identify the genes they target. Having identified candidate miRNAs that play an important role in determining stem cell fate, we will manipulate their expression levels using biotechnology techniques known as RNA oligos and plasmid or viral expression vectors. We will then determine if these manipulations change the cell’s decision-making process in regard to differentiation or self-renewal. This will be done using molecular biology and biochemical assays on undifferentiated and differentiated cells. Ultimately we will investigate the underlying regulatory mechanisms in hESCs that control miRNA expression. We anticipate that our results will contribute to understanding the transitions between stem cells and differentiated cells, as well as normal and cancer cells. This type of information can be invaluable in designing new therapeutic approaches for stem cell replacement or cancer treatment.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) hold the potential to revolutionize human medicine by making cell replacement therapies, drug delivery, or in vivo modifications of cell populations a reality. In degenerative conditions we may be able to replace dead cells with functional new ones derived from hESCs. This approach could be used to treat Parkinson’s Disease, Alzheimer’s Disease, heart disease, diabetes, or paralytic spinal cord injuries. There is also potential for new cancer treatments, as cancer stem cells share many properties of hESCs. This type of medical technology would benefit the citizens of California in several general ways. It could offer hope to Californians suffering from these diseases. It could help relieve the pain and suffering for affected individuals and their families and loved ones. Finally, the economic impact of hESC-based therapies is likely to be significant. Chronic diseases will no longer incapacitate patients and they can return to productive work lives. State government and private expenditures on health care will actually decrease. Companies will organize to commercialize these hESC-based therapies and this will stimulate the California economy by providing new jobs and tax revenue. Most medical economists believe that significant revenues from patents, royalties, and licenses will flow from scientific discoveries pioneered in California based stem cell research centers. This project will also enhance California’s competitive position in biomedical research and push the State to the forefront of research not only in America, but also the world. It will be a reflection of the enormous ongoing investment in science on the part of the state and private institutions, fueled in recent decades by the high-tech and biotechnology industries. All of the research will be done in California. The project focuses on the role of microRNA (miRNA) molecules in controlling the fate of hESCs. miRNAs are recently discovered molecules that play an important part in gene regulation. The expression profiles of miRNAs in stem cells are different from other tissues, and miRNAs may play an essential role in stem cell self-renewal and differentiation. Many potential target genes for miRNAs are essential players in stem cell renewal and differentiation. Some of the most exciting and innovative work on miRNAs has taken place in California and this project will help confirm the State’s leading role in miRNA research and California’s role as a place where miRNA researchers’ specific application to stem cell biology is being studied.
Progress Report: 
  • Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that results in demyelination and axonal loss, culminating in extensive disability through defects in neurologic function. The demyelination that defines MS pathology is progressive over time; however, studies indicate that myelin repair can occur during the course of disease in patients with MS and in animal models designed to mimic the immunopathogenesis of MS. While it is generally thought that endogenous oligodendrocyte precursor cells (OPCs) are largely responsible for spontaneous remyelination, it is unclear why these cells are only able to transiently induce myelin repair in the presence of ongoing disease. Along these lines, two therapies for demyelinating diseases look promising; implanting OPCs into sites of neuroinflammation that are directly capable of inducing remyelination of the damaged axons and/or modifying the local environment to stimulate and support remyelination by endogenous OPCs. Indeed, we have shown that human embryonic stem cell (hESC)-derived oligodendrocytes surgically implanted into the spinal cords of mice with virally induced demyelination promoted focal remyelination and axonal sparing. We are currently investigating how the implanted OPCs positionally migrate to areas of on-going demyelination and the role these cells play in repairing the damaged CNS. The purpose of this research is to identify the underlying mechanism(s) responsible for hESC-induced remyelination.
  • Oligodendrocyte progenitor cells (OPCs) are important in mediating remyelination in response to demyelinating lesions. As such, OPCs represent an attractive cell population for use in cell replacement therapies to promote remyelination for treatment of human demyelinating diseases. High-purity OPCs have been generated from hESC and have been shown to initiate remyelination associated with improved motor skills in animal models of demyelination. We have previously determined that engraftment of hESC-derived OPCs into mice with established demyelination does not significantly improve clinical recovery nor reduce the severity of demyelination. Importantly, remyelination is limited following OPC transplantation. These findings highlight that the microenvironment is critical with regards to the remyelination potential of engrafted cells. In addition, we have determined that human OPCs are capable of migrating in response to proinflammatory molecules often associated with human neuroinflammatory diseases such as multiple sclerosis. This is an important observation in that it will likely be necessary for engrafted OPCs to be able to positionally navigate within tissue in order to move from the site of surgical transplantation to areas of damage to initiate repair and tissue remodeling. Finally, we have also made a novel discovery of a unique signaling pathway that protects OPCs from damage/death in response to treatment with proinflammatory cytokines. We believe this is an important and translationally relevant observation as OPCs are critical in contributing to remyelination and remyelination failure is an important clinical feature for many human demyelinating diseases inclusing spinal cord injury and MS. We have identified a putative protective ligand/receptor interaction affords protection from cytokine-induced apoptosis. These findings may reveal novel avenues for therapeutic intervention to prevent damage/death of OPCs and enhance remyelination.

The role of HER2 signaling in differentiation and maintenance of human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00409
ICOC Funds Committed: 
$0
Disease Focus: 
Multiple Sclerosis
Neurological Disorders
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The ability to propagate high quality human embryonic stem cells (hESCs) that are capable of giving rise to multiple cell types is essential in using hESCs to treat human diseases. Understanding the signaling pathways that control the maintenance of pluripotency and differentiation of hESCs is necessary for endowing such ability. The goal of this project is to determine whether HER2, a receptor tyrosine kinase, is essential in maintaining hESCs. HER2 is a multiple functional protein. De-regulation of HER2 expression or signaling is implicated in several human diseases. For example, over-expression of HER2 is found in 20-30% of breast cancer patients. Herceptin, a humanlized blocking monoclonal antibody, has been approved by the Food and Drug Agency to treat breast cancer patients. HER2 is required as an essential partner for the response of cells to multiple growth factors, including neuregulin (NRG). Mutation of the NRG1 gene is associated with Schizophrenia in several populations. Studies in mice suggest that HER2 may play a role in the etiology of Hirschsprung disease and in preventing dilated cardiomyopathy and muscular dystrophy. Interestingly, some breast cancer patients treated with Herceptin develop cardiac dysfunction, revealing multiple functions of HER2 and the complication of designing an ideal therapy. NRG1 has been shown to play a role in the formation of the conduction system (pacemaker) of the heart in mice. Improper processing of NRG1 may play a role in Alzheimer's Disease and neuropathy. We found that NRG1 plays a role in the recovery rate of rat neural stem cells. HER2 is highly expressed in undifferentiated hESCs. These data demonstrate that HER2 has pleitropic effects on multiple cell types and organs and suggest that HER2 is capable of integrating diverse signaling pathways that are essential in the control of maintenance and/or differentiation of hESCs where HER2 is highly expressed. Understanding whether and how HER2 regulates the maintenance and/or differentiation of hESCs may provide insight into harnessing the strategy to grow and use hESCs to treat human disease.
Statement of Benefit to California: 
The ability to propagate high quality human embryonic stem cells (hESCs) that are capable of giving rise to multiple cell types is essential in using hESCs to treat human diseases. Understanding the signaling pathways that control the maintenance of pluripotency and differentiation of hESCs is necessary for endowing such ability. HER2 is a receptor tyrosine kinase and has been shown to play essential roles or is implicated in breast cancer, dilated cardiomyopathy, muscular dystrophy, heart pace maker, peripheral neuropathy, Schizophrenia and Alzheimer's Disease. For example, over-expression of HER2 is found in 20-30% of breast cancer patients. Herceptin, a humanlized blocking monoclonal antibody, has been approved by the Food and Drug Agency to treat breast cancer patients. Several lines of evidence suggest that HER2 is a multiple functional protein and may play essential roles in the maintenance and/or differentiation of hESCs. Understanding whether and how HER2 regulates the maintenance and differentiation of hESCs may provide insight into harnessing the strategy to grow and use high quality hESCs to treat human disease.
Progress Report: 
  • Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that results in demyelination and axonal loss, culminating in extensive disability through defects in neurologic function. The demyelination that defines MS pathology is progressive over time; however, studies indicate that myelin repair can occur during the course of disease in patients with MS and in animal models designed to mimic the immunopathogenesis of MS. While it is generally thought that endogenous oligodendrocyte precursor cells (OPCs) are largely responsible for spontaneous remyelination, it is unclear why these cells are only able to transiently induce myelin repair in the presence of ongoing disease. Along these lines, two therapies for demyelinating diseases look promising; implanting OPCs into sites of neuroinflammation that are directly capable of inducing remyelination of the damaged axons and/or modifying the local environment to stimulate and support remyelination by endogenous OPCs. Indeed, we have shown that human embryonic stem cell (hESC)-derived oligodendrocytes surgically implanted into the spinal cords of mice with virally induced demyelination promoted focal remyelination and axonal sparing. We are currently investigating how the implanted OPCs positionally migrate to areas of on-going demyelination and the role these cells play in repairing the damaged CNS. The purpose of this research is to identify the underlying mechanism(s) responsible for hESC-induced remyelination.
  • Oligodendrocyte progenitor cells (OPCs) are important in mediating remyelination in response to demyelinating lesions. As such, OPCs represent an attractive cell population for use in cell replacement therapies to promote remyelination for treatment of human demyelinating diseases. High-purity OPCs have been generated from hESC and have been shown to initiate remyelination associated with improved motor skills in animal models of demyelination. We have previously determined that engraftment of hESC-derived OPCs into mice with established demyelination does not significantly improve clinical recovery nor reduce the severity of demyelination. Importantly, remyelination is limited following OPC transplantation. These findings highlight that the microenvironment is critical with regards to the remyelination potential of engrafted cells. In addition, we have determined that human OPCs are capable of migrating in response to proinflammatory molecules often associated with human neuroinflammatory diseases such as multiple sclerosis. This is an important observation in that it will likely be necessary for engrafted OPCs to be able to positionally navigate within tissue in order to move from the site of surgical transplantation to areas of damage to initiate repair and tissue remodeling. Finally, we have also made a novel discovery of a unique signaling pathway that protects OPCs from damage/death in response to treatment with proinflammatory cytokines. We believe this is an important and translationally relevant observation as OPCs are critical in contributing to remyelination and remyelination failure is an important clinical feature for many human demyelinating diseases inclusing spinal cord injury and MS. We have identified a putative protective ligand/receptor interaction affords protection from cytokine-induced apoptosis. These findings may reveal novel avenues for therapeutic intervention to prevent damage/death of OPCs and enhance remyelination.

Ovol genes and hES differentiation into hair-producing cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00377
ICOC Funds Committed: 
$0
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Public Abstract: 
Each year in America, there are 700,000 emergency visits and 45,000 hospitalizations for burn treatments, and 4,500 deaths due to fire and burns (http://www.ameriburn.org/resources_factsheet.php). Since twenty years ago, the use of artificial skin generated from human skin cells has tremendously improved the survival rate of severely burned patients. However, this technology suffers a major drawback: the artificial skin lacks the various structures that are part of the normal skin, such as hair follicles, sweat glands, and cells that make pigments or fight pathogens. Embryonic stem cells are cells that are isolated from early embryos and have the potential to make all kinds of cells in the human body. Conceivably, they hold great promises to help generate a new kind of artificial skin containing all residential structures and cell types that would function just like real skin. Hair follicles play many important roles in skin – they provide a passage for perspiration, are the source of cells that are used for natural wound repair, and the house of other important cell types. We therefore will focus our effort to first use hES cells to make hair follicle-containing skin on Petri dishes. Theoretically, a hES cell has to make three important, sequential fate choices during its path to become a hair follicle cell. First, it has to choose a fate that has the potential to be part of the nervous system or part of skin. After this, it has to then choose a skin fate over a neural fate. Last, it has to choose to become a hair follicle cell instead of just the skin between follicles. We will experimentally manipulate these fate choices by adding or removing key regulatory proteins (such as growth factors) that are known to be involved in these decisions based on studies in model systems such as mice. Clearly, this is a long-term process: we not only have to find conditions that would allow the generation of some hair-producing cells, but also have to optimize the conditions so that we can generate many of such cells and have them distribute in a pattern that resembles the distribution of hair follicles in normal skin. Our goal in the current grant application is to be able to generate some hair-producing cells from hES cells. This, if successful, will lay the ground for future work from our as well as other laboratories. Ultimately, we may be able to generate hair follicle-containing skin to be used for transplantation not only onto burn patients, but also onto healthy people with baldness.
Statement of Benefit to California: 
Wildfire has been an unfortunately frequent presence in the State of California. As the state continues to grow, more and more people live in forest areas, facing a high risk of physical (and property) damage from wildfire. For example, a total of 3500 houses were burned in the Oakland/Berkeley fire in 1988 and Painted Cave fire in 1990. This, together with other causes of fire and burn such as use of defective product and accidents, leaves Californian citizens suffering from a high incidence of burns and injuries. The studies we propose in this application may yield findings that will directly benefit the health and welfare of burn patients in California. Furthermore, these findings may also be used to help establish industrial entities in the State of California that perform research, development, and marketing of new therapeutic products. This will create new job opportunities for California citizens and increase revenues for the State of California.
Progress Report: 
  • We have shown that fetal human central nervous system derived stem cells (HuCNS-SC) transplanted into a mouse model of spinal cord injury (SCI) improve behavioral recovery. Transplanted human cells differentiated into myelinating oligodendrocytes and synapse forming neurons. These data suggest that efficacy is dependent upon successful cell engraftment and appropriate cell fate. The strain of mice (NOD-scid mice) are immunodeficient, which allows transplanted human cell populations to engraft and promote behavioral recovery in the absence of confounds due to a rejection response and allows us to avoid using immunosuppressant drugs. Clinically, however, it is clear that transplantation of therapeutic human cell populations will require administration of immunosuppressants (IS) such as CsA, FK506, or Rapamycin. These immunosuppressants work by altering signaling pathways which are also present within stem cells. Hence, in addition to promoting engraftment, IS have the potential to affect stem cell proliferation and/or differentiation. In Aim 1A, we tested this hypothesis in a cell culture model and found that HuCNS-SC fate and proliferation were altered by exposure to different IS. CsA and FK506 decreased the number of astrocytes in culture compared to control conditions, while Rapamyin increased the number of astrocytes. All three IS increased the number of ß-tubulin III positive neuron-like cells.
  • In Aim 1B, we tested whether cells of the inflammatory system (neutrophils and macrophages) could also directly influence stem cell proliferation and fate. To test this possibility, we exposed either fetal or embryonic neural stem cells to cell culture media from co-cultures of neutrophils or macrophages. We found that neutrophil-mediated release of inflammatory proteins promotes astrocyte differentiation of fetal derived neural stem cells but not embryonic derived neural stem cells. One way inflammatory cells might be working is via oxidative stress (e.g. hydrogen peroxide). Interestingly, excess hydrogen peroxide promoted more extensive cell death of embryonic derived versus fetal fetal derived neural stem cells, suggesting an intrinsic difference in the vulnerably of these two cell populations to oxidative stress. Conditioned media from neutrophils was found to reduce proliferation in fetal neural stem cells but not embryonic derived neural stem cells. In addition, we found neutrophil conditioned media promotes human fetal NSC astrocytic fate and migration towards sites of injury epicenter in an animal model of spinal cord injury; followup cell culture experiments enabled us to determine that neutrophil synthesized complement proteins were having a direct effect on stem cell fate and migration, resulting in a patent filing. These data demonstrate that fetal NSCs and ES-NSCs are very different by nature and nurture.
  • In Aim 2, we evaluated the hypothesis that IS could alter stem cell proliferation and/or fate in vivo, independent of rejection from the recipient’s immune system. HuCNS-SC were transplanted into NOD-scid mice, which have no immune system and hence cannot mount an immune response to the foreign cells. These animals received different immunosuppressants (CsA, FK506, Rapamycin, or vehicle) daily after transplantation until sacrifice 13 weeks later to determine if the total number of surviving human cells, or the end cell fate of the transplanted cells would be altered due to exposure to IS drugs compared to the vehicle control group. Behavioral recovery was assessed via open-field walking assessment, horizontal ladder beam testing, and video based “CatWalk” gait analysis. IS administration did not affect behavioral recovery by any of these measures compared to HuCNS-SC transplanted animals that received vehicle as an IS. Spinal cords were dissected, sectioned, and immunostained using human-specific markers in conjunction with cell lineage/fate and proliferation markers. Cell engraftment, proliferation, and fate were quantified using unbiased methods. The average number of engrafted human cells in uninjured animals was 319,700 vs 214,900 in vehicle treated injured controls. Human cell engraftment in any IS group was not significantly different than vehicle injured controls. Interestingly, 67% of human cells differentiated into Olig2+ oligodendrocyte-like cells in the uninjured controls, while 45% were Olig2 positive in vehicle treated injured controls. IS treatment did not alter Olig2 cell numbers in injured animals. 9% of human cells differentiated into GFAP positive astrocyte-like cells in the uninjured controls, compared with 9% in vehicle treated injured controls. IS treatment did not alter GFAP cell numbers in injured animals. Quantification of proliferation and other lineage markers is ongoing. The important finding thus far is that when administered to whole animals with a human stem cell transplant, a range of immunosuppressant drugs does not appear to significantly alter stem cell fate.

Cell-Cell Interactions Promote Differentiation of Human Embryonic Stem Cells to Insulin-Secreting Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00377
ICOC Funds Committed: 
$0
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Public Abstract: 
One million people in the United States have insulin dependent diabetes - a disease that elevates blood glucose and may result in kidney failure, blindness and amputation. Transplantation of insulin-producing beta cells can establish normal blood sugar levels without the need for insulin injections but multiple doses of cells are required and diabetes returns within 2-3 years in most islet transplant patients. This failure with time is thought to be primarily from “exhaustion” and other insults to an inadequate number of engrafted beta cells. If an abundant source of beta cells was available, long-term success would likely improve. Human embryonic stem cells are a promising source of beta cells for transplantation. A specific cell line of “pluripotential” stem cells differentiates into forerunners of beta cells. The forerunner cells do not make insulin but they can be identified and, when transplanted together with embryonic pancreas or blood vessel cells, will transform into insulin-producing cells. Prior studies have accomplished this transformation only after transplantation in rodents - in vivo - where the insulin-producing cells are not accessible for easy study or harvest for transplantation. We plan to isolate and grow human forerunner cells in the laboratory then mix them in a Petri dish with appropriate cells to coax them into becoming insulin-secreting cells. Once we have an ongoing colony of transformed insulin-producing cells, we will transplant them into diabetic mice (a strain that does not reject human tissue) to assess their ability to reverse insulin-dependent diabetes. These human embryonic stem cell investigations will deepen our understanding of stem cell biology and, potentially, lead to successful long-term treatment of insulin dependent diabetes.
Statement of Benefit to California: 
One million people in the United States have insulin dependent diabetes - a disease that elevates blood glucose and may result in kidney failure, blindness and amputation. Transplantation of insulin-producing beta cells can establish normal blood sugar levels without the need for insulin injections but multiple doses of cells are required and diabetes returns within 2-3 years in most islet transplant patients. This failure with time is thought to be primarily from “exhaustion” and other insults to an inadequate number of engrafted beta cells. If an abundant source of beta cells was available, long-term success would likely improve. Human embryonic stem cells are a promising source of beta cells for transplantation. A specific cell line of “pluripotential” stem cells differentiates into forerunners of beta cells. The forerunner cells do not make insulin but they can be identified and, when transplanted together with embryonic pancreas or blood vessel cells, will transform into insulin-producing cells. Prior studies have accomplished this transformation only after transplantation in rodents - in vivo - where the insulin-producing cells are not accessible for easy study or harvest for transplantation. We plan to isolate and grow human forerunner cells in the laboratory then mix them in a Petri dish with appropriate cells to coax them into becoming insulin-secreting cells. Once we have an ongoing colony of transformed insulin-producing cells, we will transplant them into diabetic mice (a strain that does not reject human tissue) to assess their ability to reverse insulin-dependent diabetes. These human embryonic stem cell investigations will deepen our understanding of stem cell biology and, potentially, lead to successful long-term treatment of insulin dependent diabetes. The proposed research will improve our understanding of stem biology; specifically, how stem cells become insulin-secreting cells. This work will enhance California's standing as a leader in cutting-edge stem cell research, a position that will translate into economic gains through the stimulation of biotechnology investment and scientific endeavor. For citizens of California with insulin dependent diabetes this work could ultimately led to an effective treatment through the transplantation of insulin-secreting cells or, perhaps, the regeneration of patients' own damaged beta cells.
Progress Report: 
  • We have shown that fetal human central nervous system derived stem cells (HuCNS-SC) transplanted into a mouse model of spinal cord injury (SCI) improve behavioral recovery. Transplanted human cells differentiated into myelinating oligodendrocytes and synapse forming neurons. These data suggest that efficacy is dependent upon successful cell engraftment and appropriate cell fate. The strain of mice (NOD-scid mice) are immunodeficient, which allows transplanted human cell populations to engraft and promote behavioral recovery in the absence of confounds due to a rejection response and allows us to avoid using immunosuppressant drugs. Clinically, however, it is clear that transplantation of therapeutic human cell populations will require administration of immunosuppressants (IS) such as CsA, FK506, or Rapamycin. These immunosuppressants work by altering signaling pathways which are also present within stem cells. Hence, in addition to promoting engraftment, IS have the potential to affect stem cell proliferation and/or differentiation. In Aim 1A, we tested this hypothesis in a cell culture model and found that HuCNS-SC fate and proliferation were altered by exposure to different IS. CsA and FK506 decreased the number of astrocytes in culture compared to control conditions, while Rapamyin increased the number of astrocytes. All three IS increased the number of ß-tubulin III positive neuron-like cells.
  • In Aim 1B, we tested whether cells of the inflammatory system (neutrophils and macrophages) could also directly influence stem cell proliferation and fate. To test this possibility, we exposed either fetal or embryonic neural stem cells to cell culture media from co-cultures of neutrophils or macrophages. We found that neutrophil-mediated release of inflammatory proteins promotes astrocyte differentiation of fetal derived neural stem cells but not embryonic derived neural stem cells. One way inflammatory cells might be working is via oxidative stress (e.g. hydrogen peroxide). Interestingly, excess hydrogen peroxide promoted more extensive cell death of embryonic derived versus fetal fetal derived neural stem cells, suggesting an intrinsic difference in the vulnerably of these two cell populations to oxidative stress. Conditioned media from neutrophils was found to reduce proliferation in fetal neural stem cells but not embryonic derived neural stem cells. In addition, we found neutrophil conditioned media promotes human fetal NSC astrocytic fate and migration towards sites of injury epicenter in an animal model of spinal cord injury; followup cell culture experiments enabled us to determine that neutrophil synthesized complement proteins were having a direct effect on stem cell fate and migration, resulting in a patent filing. These data demonstrate that fetal NSCs and ES-NSCs are very different by nature and nurture.
  • In Aim 2, we evaluated the hypothesis that IS could alter stem cell proliferation and/or fate in vivo, independent of rejection from the recipient’s immune system. HuCNS-SC were transplanted into NOD-scid mice, which have no immune system and hence cannot mount an immune response to the foreign cells. These animals received different immunosuppressants (CsA, FK506, Rapamycin, or vehicle) daily after transplantation until sacrifice 13 weeks later to determine if the total number of surviving human cells, or the end cell fate of the transplanted cells would be altered due to exposure to IS drugs compared to the vehicle control group. Behavioral recovery was assessed via open-field walking assessment, horizontal ladder beam testing, and video based “CatWalk” gait analysis. IS administration did not affect behavioral recovery by any of these measures compared to HuCNS-SC transplanted animals that received vehicle as an IS. Spinal cords were dissected, sectioned, and immunostained using human-specific markers in conjunction with cell lineage/fate and proliferation markers. Cell engraftment, proliferation, and fate were quantified using unbiased methods. The average number of engrafted human cells in uninjured animals was 319,700 vs 214,900 in vehicle treated injured controls. Human cell engraftment in any IS group was not significantly different than vehicle injured controls. Interestingly, 67% of human cells differentiated into Olig2+ oligodendrocyte-like cells in the uninjured controls, while 45% were Olig2 positive in vehicle treated injured controls. IS treatment did not alter Olig2 cell numbers in injured animals. 9% of human cells differentiated into GFAP positive astrocyte-like cells in the uninjured controls, compared with 9% in vehicle treated injured controls. IS treatment did not alter GFAP cell numbers in injured animals. Quantification of proliferation and other lineage markers is ongoing. The important finding thus far is that when administered to whole animals with a human stem cell transplant, a range of immunosuppressant drugs does not appear to significantly alter stem cell fate.

Exploring the Therapeutic Potential of Human Embryonic Stem Cells in Pediatric Neurotrauma

Funding Type: 
SEED Grant
Grant Number: 
RS1-00377
ICOC Funds Committed: 
$0
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Public Abstract: 
Traumatic brain injury (TBI) is a major cause of death (50,000/yr) and disability. The Centers for Disease Control and Prevention (CDC) has estimated that approximately 1.4-1.5 million Americans survive TBI annually. The economic burden of TBI in the United States has been estimated at approximately $56.3 billion in 1995. In children TBI has lifelong cognitive, physical, psychosocial/ behavioral/ emotional impairments since it interferes with the developing brain. In fact, in children deficits may not be fully revealed until later in life. TBI is among the most frequent pediatric neurological conditions (400,000 emergency department visits/yr; Langlois, 2001), occurring more frequently than cerebral palsy (10,000/yr), global developmental delay (80,000/yr) and epilepsy (30,000/yr). TBI continues to pose a serious health concern and adequate treatment for these injuries is seriously lacking. For example, the majority of TBI patients are discharged without subsequent health-care assistance. Among children aged 0-4 years 91% are unattended clinically post discharge (compare to 66% for all injuries). This is particularly discordant with data showing that at least 15% of TBI patients continue to experience negative consequences 1yr after injury (Guerrero et al, 2000). Optimistically, the neonatal brain may be more amenable than the adult brain to the therapeutic potential of stem cells. Transplantation studies using human embryonic stem cells and other stem cells have demonstrated their ability to facilitate functional recovery after brain and spinal cord injury. The proposed studies will determine the therapeutic capacity of neuron-enriched human embryonic stem cells (hESCs) to attenuate histological damage, normalize brain metabolite profiles, generate functional neural circuits and recover sensorimotor and memory functions following transplantation into postnatal rats subjected to lateral fluid percussion injury (FPI). The lateral FPI model most closely mimics postlesional events associated with TBI in humans (Dietrich et al, 1996). In Aim 1 we will evaluate whether hESCs (previously differentiated into neurons and labeled ferromagnetically) transplanted into the traumatically injured pediatric brain migrate to the site of injury and compensate for lesion area and brain metabolite changes using brain imaging methods. In Aim 2 accelerating rotarod and Morris water maze tests will be used to investigate whether grafted hESCs can ameliorate sensorimotor and cognitive deficits. In Aim 3 we will use immunohistochemical methods to label neural and glial phenotypes and synaptic contacts to determine if grafted hESCs form functional neuronal networks in injured brain. Our findings should eventually help promote the development of novel strategies to alleviate the effects of TBI. The proof of concept obtained in these studies will provide the necessary preliminary data and expertise necessary to be competitive for NIH funding.
Statement of Benefit to California: 
Traumatic brain injury (TBI) is a major cause of death (50,000/yr) and disability. The Centers for Disease Control and Prevention (CDC) has estimated that approximately 1.4-1.5 million Americans survive TBI annually. In contrast, the incidence of breast cancer and HIV/AIDS is 8 and 34 times lower (National Center for Injury Prevention and Control, CDC, http://www.cdc.gov/ncipc/factsheets/tbi.htm). The economic burden of TBI in the United States has been estimated at approximately $56.3 billion in 1995. In addition, TBI imposes enormous losses to individuals, their families, and society that cannot be completely enumerated. TBI is among the most frequent pediatric neurological conditions (400,000 emergency department visits; Langlois, 2001), occurring more frequently than cerebral palsy (10,000/year), global developmental delay (80,000/yr) and epilepsy (30,000/yr). TBI also accounts for significant neurological and neuropsychological morbidity in children. Data from 12 states, including California, indicate that the age-adjusted rate for TBI-related hospitalizations in 2002 was 79.0 per 100,000 population. While this estimate is ~20% lower than annual estimates for 1994-1995 from the National Hospital Discharge Survey (NHDS), TBI continues to leave an estimated 80,000-90,000 persons with long-term disability (Langlois et al, 2004). This apparent decline should be interpreted cautiously as estimates exclude non-residents and brain injuries not treated at hospitals. Most TBI cases are mild and deficits are subtle. In children deficits may not be fully revealed until later in life. TBI, which produces cognitive, physical and psychosocial/ behavioral/ emotional impairments, that often continue to develop for up to months and years after impact, continues to pose a serious health concern and adequate treatment for these injuries is seriously lacking. For example, the majority of TBI patients are discharged without subsequent health-care assistance. Among children aged 0-4 years 91% are unattended clinically post discharge (compare to 66% for all injuries). This is particularly discordant with data showing that at least 15% of TBI patients continue to experience negative consequences 1yr after injury (Guerrero et al, 2000). Optimistically, neonatal brain injury may be more amenable than the adult brain to the therapeutic potential of stem cells (Santner-Nanan et al, 2005). Transplantation studies using non-embryonic stem cells from human fetus, mouse embryonic stem cells have demonstrated their ability to facilitate functional recovery after brain and spinal cord injury. hESCs offer greater ability of pluropotency and can prevent any cross-species consequences. To date a very limited number of studies have explored the ability of hESCs to reverse or protect against detrimental and permanent effects of TBI, especially as measured noninvasively using brain imaging.
Progress Report: 
  • We have shown that fetal human central nervous system derived stem cells (HuCNS-SC) transplanted into a mouse model of spinal cord injury (SCI) improve behavioral recovery. Transplanted human cells differentiated into myelinating oligodendrocytes and synapse forming neurons. These data suggest that efficacy is dependent upon successful cell engraftment and appropriate cell fate. The strain of mice (NOD-scid mice) are immunodeficient, which allows transplanted human cell populations to engraft and promote behavioral recovery in the absence of confounds due to a rejection response and allows us to avoid using immunosuppressant drugs. Clinically, however, it is clear that transplantation of therapeutic human cell populations will require administration of immunosuppressants (IS) such as CsA, FK506, or Rapamycin. These immunosuppressants work by altering signaling pathways which are also present within stem cells. Hence, in addition to promoting engraftment, IS have the potential to affect stem cell proliferation and/or differentiation. In Aim 1A, we tested this hypothesis in a cell culture model and found that HuCNS-SC fate and proliferation were altered by exposure to different IS. CsA and FK506 decreased the number of astrocytes in culture compared to control conditions, while Rapamyin increased the number of astrocytes. All three IS increased the number of ß-tubulin III positive neuron-like cells.
  • In Aim 1B, we tested whether cells of the inflammatory system (neutrophils and macrophages) could also directly influence stem cell proliferation and fate. To test this possibility, we exposed either fetal or embryonic neural stem cells to cell culture media from co-cultures of neutrophils or macrophages. We found that neutrophil-mediated release of inflammatory proteins promotes astrocyte differentiation of fetal derived neural stem cells but not embryonic derived neural stem cells. One way inflammatory cells might be working is via oxidative stress (e.g. hydrogen peroxide). Interestingly, excess hydrogen peroxide promoted more extensive cell death of embryonic derived versus fetal fetal derived neural stem cells, suggesting an intrinsic difference in the vulnerably of these two cell populations to oxidative stress. Conditioned media from neutrophils was found to reduce proliferation in fetal neural stem cells but not embryonic derived neural stem cells. In addition, we found neutrophil conditioned media promotes human fetal NSC astrocytic fate and migration towards sites of injury epicenter in an animal model of spinal cord injury; followup cell culture experiments enabled us to determine that neutrophil synthesized complement proteins were having a direct effect on stem cell fate and migration, resulting in a patent filing. These data demonstrate that fetal NSCs and ES-NSCs are very different by nature and nurture.
  • In Aim 2, we evaluated the hypothesis that IS could alter stem cell proliferation and/or fate in vivo, independent of rejection from the recipient’s immune system. HuCNS-SC were transplanted into NOD-scid mice, which have no immune system and hence cannot mount an immune response to the foreign cells. These animals received different immunosuppressants (CsA, FK506, Rapamycin, or vehicle) daily after transplantation until sacrifice 13 weeks later to determine if the total number of surviving human cells, or the end cell fate of the transplanted cells would be altered due to exposure to IS drugs compared to the vehicle control group. Behavioral recovery was assessed via open-field walking assessment, horizontal ladder beam testing, and video based “CatWalk” gait analysis. IS administration did not affect behavioral recovery by any of these measures compared to HuCNS-SC transplanted animals that received vehicle as an IS. Spinal cords were dissected, sectioned, and immunostained using human-specific markers in conjunction with cell lineage/fate and proliferation markers. Cell engraftment, proliferation, and fate were quantified using unbiased methods. The average number of engrafted human cells in uninjured animals was 319,700 vs 214,900 in vehicle treated injured controls. Human cell engraftment in any IS group was not significantly different than vehicle injured controls. Interestingly, 67% of human cells differentiated into Olig2+ oligodendrocyte-like cells in the uninjured controls, while 45% were Olig2 positive in vehicle treated injured controls. IS treatment did not alter Olig2 cell numbers in injured animals. 9% of human cells differentiated into GFAP positive astrocyte-like cells in the uninjured controls, compared with 9% in vehicle treated injured controls. IS treatment did not alter GFAP cell numbers in injured animals. Quantification of proliferation and other lineage markers is ongoing. The important finding thus far is that when administered to whole animals with a human stem cell transplant, a range of immunosuppressant drugs does not appear to significantly alter stem cell fate.

Telomerase and self-renewal in human embyronic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00333
ICOC Funds Committed: 
$0
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Embryonic stem cells are unique in their ability to give rise to all mammalian tissues. Their potential application to human disease is enormous because they could be employed to repair or replaced damaged tissue. Although tremendous strides have been made in recent years in treating human disease, replacing damaged tissue remains almost completely beyond our grasp. Harnessing human embryonic stem cells for this purpose will open completely new areas of regenerative medicine. One shared characteristic of embryonic stem cells and adult stem cells that reside in many of our tissues is the ability to self-renew. Self-renewal is the ability of a stem cell to divide and give rise to a daughter cell that is undifferentiated and capable of giving rise to all the same lineages as the parent stem cell. Understanding how embryonic stem cells self-renew is critical for determining how to maintain these cells, how to differentiate them toward specific tissue lineages and how to expand more committed stem cells or progenitor cells in cell culture. A small number of genes that control embryonic stem cell self-renewal have been discovered, but our understanding of this process remains in its infancy. In this proposal, we investigate the molecular mechanism by which telomerase contributes to embryonic stem cell self-renewal. Telomerase is an enzyme complex expressed in embryonic stem cells, some tissue stem cells and in almost all human cancers. Most differentiated cells lack telomerase expression. Telomerase adds DNA repeats to structures at the ends of our chromosomes, termed telomeres. Telomeres are very important in protecting chromosome ends and in preventing chromosome ends from breaking down or sticking to other ends inappropriately. By maintaining telomeres, telomerase supports the ability of stem cells to divide a large number of times. In addition to its function in telomere synthesis, we recently discovered a second role for telomerase. We expressed the TERT protein component of telomerase in mouse skin and unexpectedly found that TERT activated resting tissue stem cells. Activation of skin stem cell by TERT triggered a regenerative program in skin leading to robust hair growth. We used a rigorous technique to show that this new activity does not involve TERT’s other role in telomere lengthening. It is absolutely essential to understand how telomerase carries out its functions in stem cells. Therefore, we have isolated proteins that strongly interact with TERT. In this proposal, we investigate the function of TERT in embryonic stem cells and determine the role of several TERT associated proteins in embryonic stem cell self-renewal.
Statement of Benefit to California: 
This proposal will benefit California and its citizen in two general ways. First, I have recruited two new scientists to California from Texas to work on this proposal. These are new taxpayers and consumers, which will benefit local businesses. They would have been less likely to come to California in the absence of the CIRM grant program. Second, this novel grant will generate new intellectual property in the form of patents. These patents may in fact be licensed to California companies or be used to support the formation of new start-up companies. The growth of such companies has historically fueled much of the profound growth in California. The future of California is linked to new technologies in the stem cell, biotechnology and other technology sectors.
Progress Report: 
  • A main goal of research in our laboratory is to identify strategies to promote neural repair in spinal cord injury and related neurological conditions. On the one hand, we have been using mouse models of spinal cord injury to study a long-standing puzzle in the field, namely, why axons, the fibers that connect nerve cells, do not regenerate after injury to the brain and the spinal cord. On the other hand, relevant to this CIRM SEED grant, we have started to explore the developmental and therapeutic potential of human embryonic stem cells (hESCs) for neural repair. We do this by first developing a method to genetically manipulate a HUES line of hESCs. The advent of hESCs has offered enormous potential for regenerative medicine and for basic understanding of human biology. To attain the full potential of hESCs as a tool both for therapeutic development and for basic research, we need to greatly enhance and expand our ability to genetically manipulate hESCs. A major goal for our SEED grant-sponsored research is to establish methods to genetically manipulate the HUES series of hESC lines, which are gaining wide utility in the research community due to the advantages on their growth characteristics over previously developed hESC lines. The first gene that we targeted in HUES cells, Fezf2, is critical for the development of the corticospinal tract, which plays important roles in fine motor control in humans and hence represents an important target for recovery and repair after spinal cord injury. By introducing a fluorescent reporter to the Fezf2 locus, we are now able to monitor the differentiation of hESCs into Fezf2-expressing neuronal lineages. This work has been published. A second goal is to start to explore the developmental and therapeutic potential of these cells and cells that derived from these cells in the brain and spinal cord. We are currently utilizing the cell line genetically engineered above to develop an efficient method to differentiate HUES cells into subcerebral neurons. Results so far have been encouraging. Efforts are also underway to overexpress Fezf2 as a complementary approach to drive the differentiation of HUES cells into specific neuronal types. Together, these studies will lay down the foundation for therapeutic development with HUES cells and their more differentiated derivatives for neurological disorders including spinal cord injury where neural regeneration can be beneficial. The CIRM SEED grant has allowed us to pursue a new, exciting path of research that we would have not pursued had we not been awarded the grant. Furthermore, the CIRM funded research has opened a new window of opportunity for us to explore genetic engineering of hESCs to model human neurological conditions in future.

Micropallet Arrays to Screen and Select Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00333
ICOC Funds Committed: 
$0
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The goal of this CIRM seed grant is to extend the potential of a new technology for stem cell studies by bringing to bear state-of-the-art microengineering techniques to the challenges of stem cell screening and selection. The completed system will provide a practical and flexible device with far-ranging applications. The capability to sort cells after experimental manipulation will fuel basic research by providing a means to establish new cell lines for stem cell study. The capabilities of the new instrumentation will enable greater precision and flexibility in the exposure of cells to growth factors to maintain cells in a sem-cell-like state of to differentiate cells into desired tissue types for regenerative medicine. Experiments are envisioned to more accurately recreate developmental events or the functions of stem cell in the living organism. Precise control of the cellular environment along with isolation of precursor cells for the treatment of a particular disorder would have a dramatic impact for medical applications.
Statement of Benefit to California: 
The research to be funded by this CIRM Seed Grant has to potential to directly benefit the citizens of California. The funds will be used to develop a new instrument that stem cell researchers will utilize to test and select unique stem cells for either further study or to grow and apply to medical therapies. The technology has practical and widely applicable uses, so that many researchers in the academic and industrial laboratories of the state will receive benefit through increased efficiency of their studies and the ability to perform new types of experiments to better understand the biology of stem cells and how to use them for regenerative medicine. The research will also stimulate economic development through creation of intellectual property which will be owned by citizens of the California through the state university where the research will take place. This research and that of other investigators funded by these grants will enhance the state’s competitiveness in biotechnology research and development, thus furthering California’s lead in this important area of economic growth.
Progress Report: 
  • A main goal of research in our laboratory is to identify strategies to promote neural repair in spinal cord injury and related neurological conditions. On the one hand, we have been using mouse models of spinal cord injury to study a long-standing puzzle in the field, namely, why axons, the fibers that connect nerve cells, do not regenerate after injury to the brain and the spinal cord. On the other hand, relevant to this CIRM SEED grant, we have started to explore the developmental and therapeutic potential of human embryonic stem cells (hESCs) for neural repair. We do this by first developing a method to genetically manipulate a HUES line of hESCs. The advent of hESCs has offered enormous potential for regenerative medicine and for basic understanding of human biology. To attain the full potential of hESCs as a tool both for therapeutic development and for basic research, we need to greatly enhance and expand our ability to genetically manipulate hESCs. A major goal for our SEED grant-sponsored research is to establish methods to genetically manipulate the HUES series of hESC lines, which are gaining wide utility in the research community due to the advantages on their growth characteristics over previously developed hESC lines. The first gene that we targeted in HUES cells, Fezf2, is critical for the development of the corticospinal tract, which plays important roles in fine motor control in humans and hence represents an important target for recovery and repair after spinal cord injury. By introducing a fluorescent reporter to the Fezf2 locus, we are now able to monitor the differentiation of hESCs into Fezf2-expressing neuronal lineages. This work has been published. A second goal is to start to explore the developmental and therapeutic potential of these cells and cells that derived from these cells in the brain and spinal cord. We are currently utilizing the cell line genetically engineered above to develop an efficient method to differentiate HUES cells into subcerebral neurons. Results so far have been encouraging. Efforts are also underway to overexpress Fezf2 as a complementary approach to drive the differentiation of HUES cells into specific neuronal types. Together, these studies will lay down the foundation for therapeutic development with HUES cells and their more differentiated derivatives for neurological disorders including spinal cord injury where neural regeneration can be beneficial. The CIRM SEED grant has allowed us to pursue a new, exciting path of research that we would have not pursued had we not been awarded the grant. Furthermore, the CIRM funded research has opened a new window of opportunity for us to explore genetic engineering of hESCs to model human neurological conditions in future.

Establishment of human embryonic stem cell lines using re-constructed human embryos derived from polyspermic eggs

Funding Type: 
SEED Grant
Grant Number: 
RS1-00333
ICOC Funds Committed: 
$0
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Public Abstract Using human embryos produced from in vitro fertilization laboratories is always a major ethical concern. There is a great resource neglected in this field, which is the eggs containing two sperms. This type of egg, called tripronuclear zygotes, will be routinely discarded in the in vitro fertilization laboratory. Approximately 7% of fertilized eggs contain more than one sperm, with tripronuclear zygotes as a most common phenomenon. There is some evidence that tripronuclear zygotes could develop into normal embryos. Recently, a normal, live human birth was achieved by removing the extra male pronucleus in the zygote. Previously, in pigs, the normal piglets were delivered by transferring the polyspermic eggs confirmed by microscope. The principal investigator for this proposal also reported in 2001 that polyspermy in pigs could be a physiological phenomenon if extra sperm did not affect the embryonic genome. Therefore, in this proposal, the extra male pronucleus from the tripronuclear zygotes will be removed by a microsurgical procedure and the resulting zygotes will be cultured to the blastocyst stage when the inner cell mass can be used for establishing human embryonic stem cell lines. Once the technique is established, the scientists will have a valuable resource in using the ESC lines for therapeutic applications and regenerative medicine. The most important aspect of this work is to further study the biology and differentiation of human embryonic stem cells. To date, none of the fertility clinics in the Northern California reserves or freezes these tripronuclear zygotes for research studies. Hence, there is an urgent need to bring attention to this unique resource in the field of human embryonic stem cells.
Statement of Benefit to California: 
Summary of benefit to California Obtaining human embryonic stem cell lines is the prerequisite for developing therapeutic approaches. The proposed research is to use the eggs with two sperm inside that are routinely discarded in the in vitro fertilization laboratory. There is evidence that these eggs could develop normally after removing the extra sperm. It is a valuable resource to generate human embryonic stem cell lines without ethical concern of using human embryos.
Progress Report: 
  • A main goal of research in our laboratory is to identify strategies to promote neural repair in spinal cord injury and related neurological conditions. On the one hand, we have been using mouse models of spinal cord injury to study a long-standing puzzle in the field, namely, why axons, the fibers that connect nerve cells, do not regenerate after injury to the brain and the spinal cord. On the other hand, relevant to this CIRM SEED grant, we have started to explore the developmental and therapeutic potential of human embryonic stem cells (hESCs) for neural repair. We do this by first developing a method to genetically manipulate a HUES line of hESCs. The advent of hESCs has offered enormous potential for regenerative medicine and for basic understanding of human biology. To attain the full potential of hESCs as a tool both for therapeutic development and for basic research, we need to greatly enhance and expand our ability to genetically manipulate hESCs. A major goal for our SEED grant-sponsored research is to establish methods to genetically manipulate the HUES series of hESC lines, which are gaining wide utility in the research community due to the advantages on their growth characteristics over previously developed hESC lines. The first gene that we targeted in HUES cells, Fezf2, is critical for the development of the corticospinal tract, which plays important roles in fine motor control in humans and hence represents an important target for recovery and repair after spinal cord injury. By introducing a fluorescent reporter to the Fezf2 locus, we are now able to monitor the differentiation of hESCs into Fezf2-expressing neuronal lineages. This work has been published. A second goal is to start to explore the developmental and therapeutic potential of these cells and cells that derived from these cells in the brain and spinal cord. We are currently utilizing the cell line genetically engineered above to develop an efficient method to differentiate HUES cells into subcerebral neurons. Results so far have been encouraging. Efforts are also underway to overexpress Fezf2 as a complementary approach to drive the differentiation of HUES cells into specific neuronal types. Together, these studies will lay down the foundation for therapeutic development with HUES cells and their more differentiated derivatives for neurological disorders including spinal cord injury where neural regeneration can be beneficial. The CIRM SEED grant has allowed us to pursue a new, exciting path of research that we would have not pursued had we not been awarded the grant. Furthermore, the CIRM funded research has opened a new window of opportunity for us to explore genetic engineering of hESCs to model human neurological conditions in future.

Defining Heterogeneity of Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00333
ICOC Funds Committed: 
$0
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Being pluripotent, human embryonic stem cells (hESCs) have the potential to act as a source of cells for regenerative therapies. But before these potential applications of hESCs can be effectively pursued, an understanding of the complex cellular relationships existing within hESC cultures and factors involved in the maintenance of hESC properties is required. hESC lines are known to be morphologically and phenotypically heterogeneous. We propose to select an antibody diversity library directly on live hESCs to identify a panel of monoclonal antibodies that recognize distinct hESC subpopulations, and to further characterize these subpopulations with regards to pluripotency and capacity for self-renewal. These antibodies will be of broad interest to laboratories that are either utilizing and optimizing the existing hESC lines or developing new hECS lines. These antibodies can be used in the future to ensure quality of hECS cultures, develop sublines that are more suitable for regenerative therapy, and identify corresponding antigens for functional studies of hESCs.
Statement of Benefit to California: 
This study aims to develop antibodies that recognize distinct subpopulations of human embryonic stem cells (hESCs), and to further characterize these subpopulations with regards to pluripotency and capacity for self-renewal. These antibodies will be of broad interest to laboratories that are utilizing or optimizing existing hESC lines, or developing new hESC lines. This study benefits California and its citizens because the knowledge and reagents generated can be used in the future to ensure quality of hESC cultures, and develop sublines that are more suitable for regenerative therapy.
Progress Report: 
  • A main goal of research in our laboratory is to identify strategies to promote neural repair in spinal cord injury and related neurological conditions. On the one hand, we have been using mouse models of spinal cord injury to study a long-standing puzzle in the field, namely, why axons, the fibers that connect nerve cells, do not regenerate after injury to the brain and the spinal cord. On the other hand, relevant to this CIRM SEED grant, we have started to explore the developmental and therapeutic potential of human embryonic stem cells (hESCs) for neural repair. We do this by first developing a method to genetically manipulate a HUES line of hESCs. The advent of hESCs has offered enormous potential for regenerative medicine and for basic understanding of human biology. To attain the full potential of hESCs as a tool both for therapeutic development and for basic research, we need to greatly enhance and expand our ability to genetically manipulate hESCs. A major goal for our SEED grant-sponsored research is to establish methods to genetically manipulate the HUES series of hESC lines, which are gaining wide utility in the research community due to the advantages on their growth characteristics over previously developed hESC lines. The first gene that we targeted in HUES cells, Fezf2, is critical for the development of the corticospinal tract, which plays important roles in fine motor control in humans and hence represents an important target for recovery and repair after spinal cord injury. By introducing a fluorescent reporter to the Fezf2 locus, we are now able to monitor the differentiation of hESCs into Fezf2-expressing neuronal lineages. This work has been published. A second goal is to start to explore the developmental and therapeutic potential of these cells and cells that derived from these cells in the brain and spinal cord. We are currently utilizing the cell line genetically engineered above to develop an efficient method to differentiate HUES cells into subcerebral neurons. Results so far have been encouraging. Efforts are also underway to overexpress Fezf2 as a complementary approach to drive the differentiation of HUES cells into specific neuronal types. Together, these studies will lay down the foundation for therapeutic development with HUES cells and their more differentiated derivatives for neurological disorders including spinal cord injury where neural regeneration can be beneficial. The CIRM SEED grant has allowed us to pursue a new, exciting path of research that we would have not pursued had we not been awarded the grant. Furthermore, the CIRM funded research has opened a new window of opportunity for us to explore genetic engineering of hESCs to model human neurological conditions in future.

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