Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Early Translational II
Grant Number: 
TR2-01785
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 614 441
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Injuries to the spinal cord commonly result from motor vehicle accidents, traumatic falls, diving, surfing, skiing, and snowboarding accidents, other forms of sports injuries, as well as from gunshot injuries in victims of violent crimes. Injuries to the anatomically lowest part of the spinal cord, the lumbosacral portion and its associated nerve roots commonly cause paralysis, loss of sensation, severe pain, as well as loss of bladder, bowel, and sexual function. Lumbosacral injuries represent approximately one-fifth of all traumatic lesions to the human spinal cord. As a result of the direct injury to the lumbosacral portion of the spinal cord, there is degeneration and death of spinal cord nerve cells, which control muscles in the legs as well as bladder, bowel, and sexual function. No treatments are presently available in clinical practice to reverse the effects of these devastating injuries. In order to reverse the loss of function after lumbosacral spinal cord injury, replacement of the lost nerve cells is required. Recent research studies have identified some properties that are shared by spinal cord neurons responsible for muscle and bladder control. Human embryonic stem cells can now be prepared in research laboratories to develop properties that are shared between nerve cells controlling muscle and bladder function. Such nerve cells are particularly at risk of degeneration and death as a result of injuries to the lumbosacral spinal cord. Human embryonic stem cells, which have undergone treatment to obtain properties of muscle and bladder controlling nerve cells, are now very attractive development candidates for new cell replacement therapies after lumbosacral spinal cord injuries. The proposed feasibility studies will study the properties of such cells in a clinically relevant rat model for lumbosacral spinal cord injuries. In Specific Aim 1, we will determine whether ACUTE transplantation of human embryonic stem cells, which have been treated to develop properties of specific lumbosacral spinal cord neurons, may replace lost nerve cells and result in a return of bladder function in a rat model of lumbosacral spinal cord injury and repair. In Specific Aim 2, we will determine whether DELAYED transplantation of human embryonic stem cells, which have been treated to develop properties of specific lumbosacral spinal cord neurons, may replace lost nerve cells and result in a return of bladder function in a rat model of lumbosacral spinal cord injury and repair. A variety of functional studies will determine the effect of the cell transplantation on bladder function, walking, and pain. We will also use detailed anatomical studies to determine in microscopes whether the transplanted cells have grown processes to connect with pelvic target tissues, including the lower urinary tract. If successful, the proposed experiments may lead to a new treatment strategy for patients with lumbosacral spinal cord injuries.
Statement of Benefit to California: 
There are presently about 250,000 patients living with neurological impairments from spinal cord injuries (SCIs) in the United States, and approximately 11,000 new cases present every year. SCIs typically result in paralysis, loss of sensation, pain as well as bladder, bowel, and sexual dysfunction. No successful treatments are available to reverse the neurological deficits that result from SCI. Common causes for SCIs include car and motorcycle accidents, skiing, diving, surfing, and snow boarding injuries, traumatic falls, sports injuries, and acts of violence. California medical centers encounter a large proportion of the overall cases in the U.S. because of our large population, extensive network of freeways, and an active life style with recreational activities taking place both along the Californian coastline and in the mountains. The proposed development candidate feasibility project will capitalize on recent progress in human stem cell science and surgical repair of conus medullaris/cauda equina (CM/CE) forms of SCI. Human embryonic stem cell-derived neurons and neuronal progenitors, which express the transcription factor Hb9, will be transplanted into the conus medullaris in attempts to replace lost motor and autonomic neurons after a lumbosacral ventral root avulsion injury in rats. Surgical replantation of avulsed lumbosacral ventral roots into the spinal cord will also be performed in this clinically relevant model for CM/CE injury and repair. If successful, our development candidate may reinnervate muscles and pelvic organs, including the lower urinary tract after CM/CE forms of SCI. Return of functional bladder control represents one of the absolute top priorities among the spinal cord injured population (Anderson, J Neurotrauma, 2004; 21, 1371-83). Successful recovery of bladder function after SCI is expected to have very significant impact on the quality of life of spinal cord injured subjects and markedly reduce health care costs. Recovery of bladder function in spinal cord injured subjects would markedly reduce or eliminate the need for intermittent bladder catheterizations and indwelling bladder catheters. The number of visits in physicians’ offices and already over-crowded California emergency rooms for bladder infections and other complications would be markedly reduced, thereby significantly reducing health care costs for both patients and our state. Improved neurological function among the SCI population is also expected to reduce care giver needs, thereby further reducing health care costs. The increased independence that will result from improved bladder control and concomitant possible recovery of other neurological functions, for instance in transfers and locomotion, will promote return to and participation in the work force for many individuals with SCI. These effects are also expected to bring a very positive effect to the California economy and increased quality of life for those living with an SCI.
Progress Report: 
  • Injuries to the lowest portion of the spine and the spinal cord commonly results in paralysis and impairment of bladder , bowel, and sexual functions. These injuries are usually referred to as conus medullaris and cauda equina forms of spinal cord injuries. Presently, no treatments are available to reverse the neurological deficits that result from these injuries.
  • In this project, we aim to reverse neurological deficits, including bladder function, in a rat model of spinal cord injury, which affects the lowermost portion of the spinal cord. This part of the spinal cord and the associated nerve roots are called the conus medullaris and cauda equina. In our experimental model, nerve roots carrying fibers that control muscle function and pelvic organs, such as the bladder and bowel, are injured at the surface of the spinal cord. This injury mimics many of the neurological deficits encountered in human cases.
  • For treatment purposes, we transplant human derived embryonic stem cells, which have been prepared to acquire properties of motor neurons, into the lowermost portion of the rat spinal cord after injury and surgical repair of nerve roots carrying motor fibers. The studies will evaluate both acute and delayed transplantation of human embryonoic
  • During the first year of the studies, we have developed improved protocols to increase our ability to produce large number of motor neurons from human embryonic stem cells. We have also developed improved methods to detect motor neurons during the neuron production process by using fluorescent reporters inside of the cells. The latter development is of great help when sorting and preparing cells with desired properties for transplantation studies. In addition, we have refined our surgical methods to make it less invasive, using a one-sided injury model instead of lesions on both sides of the spinal cord in rats. Specifically, bladder dysfunction can be assess after a one sided injury of nerve roots and be evaluated using a combination of bladder pressure recorings and electrical recordings referred to as electromyography (EMG) from muscles along the urethra. The revised procedure is well tolerated by the rats and is a suitable approach for studies of chronic injury and cell-based long-term treatments. A research manuscript describing this improved experimental method and refinement has been submitted to a scientific journal and reviewed, and the manuscript is currently undergoing our revisions before being considered for publication. The experimental refinement will greatly assist with our long-term studies on the effects of transplanted motor neurons derived from human embryonic stem cells. We have also begun experiments using our refined model and cells, which now can be produced in high numbers and be identifiable during both the cell culture steps and during the animal studies. Initial tissues have been harvested and are being processed for morphological analyses.
  • Injuries to the lowest portion of the spine and the spinal cord commonly results in paralysis and impairment of bladder , bowel, and sexual functions. These injuries are usually referred to as conus medullaris and cauda equina forms of spinal cord injuries. Presently, no treatments are available to reverse the neurological deficits that result from these injuries.
  • In this project, we aim to reverse neurological deficits, including bladder function, in a rat model of spinal cord injury, which affects the lowermost portion of the spinal cord. This part of the spinal cord and the associated nerve roots are called the conus medullaris and cauda equina. In our experimental model, nerve roots carrying fibers that control muscle function and pelvic organs, such as the bladder and bowel, are injured at the surface of the spinal cord. This injury mimics many of the neurological deficits encountered in human cases.
  • For treatment purposes, we transplant human derived embryonic stem cells, which have been prepared to acquire properties of motor neurons, into the lowermost portion of the rat spinal cord after injury and surgical repair of nerve roots carrying motor fibers. The studies will evaluate both acute and delayed transplantation of human embryonic stem cells, which have acquired properties of motor neurons.
  • During the second year of the studies, we have developed improved protocols to increase our ability to produce large number of motor neurons from human embryonic stem cells. We have also developed improved methods to detect motor neurons during the neuron production process by using fluorescent reporters inside of the cells. The latter development is of great help when sorting and preparing cells with desired properties for transplantation studies. In addition, we have refined our surgical methods to make it less invasive, using a one-sided injury model instead of lesions on both sides of the spinal cord in rats. Specifically, bladder dysfunction can be assessed after a one sided injury of nerve roots and be evaluated using a combination of bladder pressure recordings and electrical recordings referred to as electromyography (EMG) from muscles along the urethra. The revised procedure is well tolerated by the rats and is a suitable approach for studies of chronic injury and cell-based long-term treatments. A research manuscript describing this improved experimental method and refinement has been published. The experimental refinement will greatly assist with our long-term studies on the effects of transplanted motor neurons derived from human embryonic stem cells. We have also performed transplantations of embryonic human stem cell derived motor neurons into the rat spinal cord and demonstrated surgical feasibility as well as survival of large numbers of neurons in the rat spinal cord. Some of the transplanted cells also demonstrate anatomical markers for motor neurons after transplantation.
  • During the reporting period, we have contined to demonstrate that human embryonic stem cell derived motor neurons and motor neuron progenitors can be produced in vitro. These motor neurons and motor neuron progenitors are transplanted into the rat spinal cord after a lumbosacral ventral root avulsion injury and repair of injured roots in the form of surgical re-attachment of the roots to the spinal cord surface. The lumbosacral ventral root avulsion injury mimics cauda equina and conus medullaris forms of spinal cord injury, an underserved patient population with paralysis of the legs and loss of bladder and bowel funcion. In this clinically relevant injury and repair model in rats, we have during the past several months demonstrated that transplanted human embryonic stem cell-derived motor neurons and motor neuron progenitors are able to survive in the spinal cord of rats over extended periods of time with large numbers of neurons being detectable in the spinal cord grey matter at 1, 2, and 10 weeks after the injury, surgical root repair, and transplantation of the cells. The long term viability of translanted cells suggests integration of the transplanted cells in the host tissues. Some of the cells show expression of motor neuron markers, such as the transcription factor Hb9, as demonstrated by immunohistochemistry and light microscopy.
  • Additional studies have been performed during this reporting period to address whether the transplanted cells may extend axons into the replanted lumbosacral ventral roots. Interestingly, many human axons were detected in the replanted ventral roots using immunohistochemitry and light microscopy for the detection of human processes. Additional immunohistochemistry demonstrated that these processes contained neurofilaments, which are characteristic for axons. In control experiments, we showed that avulsed roots, which had not been replanted into the spinal cord, did not exhibit any human axons. As expected, surgical reconnection of lesioned ventral roots to the spinal cord is needed in order for the axons of the transplanted human embryonic stem cell derived motor neurons and motor neuron progenitors to be extended into avulsed ventral roots. Furthermore, in a series of sham operated animals without ventral root lesions, human motor neurons and motor neuron progenitors were also transplanted into the rat spinal cord. Interestingly, the transplanted human motor neurons and motor neuron progenitors were here also able to extend axons into ventral roots, even though the ventral roots had never been lesions. We conclude that transplanted human embryonic stem cell derived motor neurons are capable of extending axons into both intact ventrl roots and into ventral roots, which had been avulsed and surgically reattached to the spinal cord using a replantation procedure.
  • In functional studies, we have performed urodynamic studies and voiding behavioral studies in rats after the transplantation of human embryonic stem cell derived motor neurons and motor neuron progenitors. These studies are still ongoing with additional experiments being performed. However, preliminary studies suggest that the combination of acute repair of avulsed ventral roots and cell transplantation results in a gradual improvement of voiding reflexes. Ongoing studies are addressing the relative contribution that may be provided by the replantation of avulsed ventral roots and by the transplantation of human motor neurons and motor neuron progenitors into the rat spinal cord.
Funding Type: 
Strategic Partnership III Track A
Grant Number: 
SP3A-07552
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$14 323 318
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The proposed project is designed to assess the safety and preliminary activity of escalating doses of human embryonic stem cell derived oligodendrocyte progenitor cells (OPCs) for the treatment of spinal cord injury. OPCs have two important functions: they produce factors which stimulate the survival and growth of nerve cells after injury, and they mature in the spinal cord to produce myelin, the insulation which enables electrical signals to be conducted within the spinal cord. Clinical testing of this product initiated in 2010 after extensive safety and efficacy testing in more than 20 nonclinical studies. Initial clinical safety testing was conducted in five subjects with neurologically complete thoracic injuries. No safety concerns have been observed after following these five subjects for more than two years. The current project proposes to extend testing to subjects with neurologically complete cervical injuries, the intended population for further clinical development, and the population considered most likely to benefit from the therapy. Initial safety testing will be performed in three subjects at a low dose level, with subsequent groups of five subjects at higher doses bracketing the range believed most likely to result in functional improvements. Subjects will be monitored both for evidence of safety issues and for signs of neurological improvement using a variety of neurological, imaging and laboratory assessments. By completion of the project, we expect to have accumulated sufficient safety and dosing data to support initiation of an expanded efficacy study of a single selected dose in the intended clinical target population.
Statement of Benefit to California: 
The proposed project has the potential to benefit the state of California by improving medical outcomes for California residents with spinal cord injuries (SCIs), building on California’s leadership position in the field of stem cell research, and creating high quality biotechnology jobs for Californians. Over 12,000 Americans suffer an SCI each year, and approximately 1.3 million people in the United States are estimated to be living with a spinal cord injury. Although specific estimates for the state of California are not available, the majority of SCI result from motor vehicle accidents, falls, acts of violence, and recreational sporting activities, all of which are common in California. Thus, the annual incidence of SCI in California is likely equal to or higher than the 1,400 cases predicted by a purely population-based distribution of the nationwide incidence. The medical, societal and economic burden of SCI is extraordinarily high. Traumatic SCI most commonly impacts individuals in their 20s and 30s, resulting in a high-level of permanent disability in young and previously healthy individuals. At one year post injury, only 11.8% of SCI patients are employed, and fewer than 35% are employed even at more than twenty years post-injury (NSCISC Spinal Cord Injury Facts and Figures 2013). Life expectancies of SCI patients are significantly below those of similar aged patients with no SCI. Additionally, many patients require help with activities of daily living such as feeding and bathing. As a result, the lifetime cost of care for SCI patients are enormous; a recent paper (Cao et al 2009) estimated lifetime costs of care for a patient obtaining a cervical SCI (the population to be enrolled in this study) at age 25 at $4.2 million. Even partial correction of any of the debilitating consequences of SCI could enhance activities of daily living, increase employment, and decrease reliance on attendant and medical care, resulting in substantial improvements in both quality of life and cost of care for SCI patients. California has a history of leadership both in biotechnology and in stem cell research. The product described in this application was invented in California, and has already undergone safety testing in five patients in a clinical study initiated by a California corporation. The applicant, who has licensed this product from its original developer and recruited many of the employees responsible for its previous development, currently employs 17 full-time employees at its California headquarters, with plans to significantly increase in size over the coming years. The successful performance of the proposed project would enable significant additional jobs creation in preparation for pivotal trials and product registration.
Funding Type: 
Early Translational III
Grant Number: 
TR3-05669
Investigator: 
ICOC Funds Committed: 
$1 673 757
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Over 6 million people in the US suffer from AD. There are no drugs that prevent the death of nerve cells in AD, nor has any drug been identified that can stimulate their replacement. Even if nerve cells could be replaced, the toxic environment of the brain will kill them unless they are protected by a drug. Therefore, drugs that stimulate the generation of new neurons (neurogenesis) alone will not be effective; a drug with both neurogenic and neuroprotective properties is required. With the ability to use cells derived from human embryonic stem cells (hESCs) as a screen for neurogenic compounds, it should now be possible to identify and tailor drugs for therapeutic use in AD. Our laboratory has developed a drug discovery scheme based upon using hESCs to screen drug candidates. We have recently identified a very potent drug that is exceptionally effective in rodent models of AD. However, this molecule needs to be optimized for human use. In this proposal, we will harness the power of hESCs to develop derivatives of J147 specifically tailored to stimulate neurogenesis and be neuroprotective in human cells. This work will optimize the chances for its true therapeutic potential in AD, and presents a unique opportunity to expand the use of hESCs for the development of a therapeutic for a disease for which there is no cure. This work could lead to a paradigm shift in the treatment of neurodegenerative disease.
Statement of Benefit to California: 
Over 6 million people in the US suffer from Alzheimer’s disease (AD). Unless a viable therapeutic is identified it is estimated that this number will increase to 16 million by 2050, with a cost of well over $1 trillion per year, overwhelming California and national health care systems. Among the top 10 causes of death, AD (6th) is the only one with no treatment available to prevent, cure or slow down the condition. An enormous additional burden to families is the emotional and physical stress of having to deal with a family member with a disease which is going to become much more frequent with our aging population. In this application we use new human stem cell technologies to develop an AD drug candidate based upon a strong lead compound that we have already made that stimulates the multiplication of nerve precursor cells derived from human embryonic stem cells. This approach presents a unique opportunity to expand the use of human embryonic stem cells for the development of a therapeutic for a disease for which there is no cure, and could lead to a paradigm shift in the treatment of neurodegenerative disease. Since our AD drug discovery approach is fundamentally different from the unsuccessful approaches used by the pharmaceutical industry, it could also stimulate new biotech. The work in this proposal addresses one of the most important medical problems of California as well as the rest of the world, and if successful would benefit all.
Progress Report: 
  • Introduction: Over 6 million people in the US suffer from AD. There are no drugs that prevent the death of nerve cells in AD, nor has any drug been identified that can stimulate their replacement. Even if nerve cells could be replaced, the toxic environment of the brain will kill them unless they are protected by a drug. Therefore, drugs that stimulate the generation of new neurons (neurogenesis) alone will not be effective; a drug with both neurogenic and neuroprotective properties is required. With the ability to use cells derived from human embryonic stem cells (hESCs) as a screen for neurogenic compounds, it should now be possible to identify and tailor drugs for therapeutic use in AD. This is the overall goal of this application.
  • Year One Progress: Using a novel drug discovery paradigm, we have made a very potent drug called J147 that is exceptionally effective in rodent models of AD and also stimulates neurogenesis in both young and very old mice. Very few, if any, drugs or drug candidates are both neuroprotective and neurogenic, particularly in old animals. In the first year of this application we harnessed the power of hESCs and medicinal chemistry to develop derivatives of J147 specifically tailored to stimulate neurogenesis and be neuroprotective in human cells. Using iterative chemistry, we synthesized over 200 new compounds, tested them for neurogenic properties in ES-derived neural precursor cells, assayed their ability to protect from the amyloid toxicity associated with AD, and determined their metabolic stability. All of the year one milestones we met and we now have the required minimum of six compounds to move into year two studies. In addition, we have made a good start on the work for year two in that some pharmacokinetics and safety studies has been completed.
  • This work will optimize the chances for its true therapeutic potential in AD, and presents a unique opportunity to expand the use of hESCs for the development of a therapeutic for a disease for which there is no cure. This work could lead to a paradigm shift in the treatment of neurodegenerative disease.
  • Introduction: Over 6 million people in the US suffer from Alzheimer’s disease (AD). There are no drugs that prevent the death of nerve cells in AD, nor has any drug been identified that can stimulate their replacement. Even if nerve cells could be replaced, the toxic environment of the brain will kill them unless they are protected by a drug. Therefore, drugs that stimulate the generation of new neurons (neurogenesis) alone will not be effective; a drug with both neurogenic and neuroprotective properties is required. With the ability to use cells derived from human embryonic stem cells (hESCs) as a screen to identify neurogenic compounds, we have shown that it is now be possible to identify and tailor drugs for therapeutic use in AD. This was the overall goal of this application, and to date we have made outstanding progress, making a drug that is both neurogenic for human cells and has therapeutic efficacy in a rigorous mouse model of AD.
  • Year 2 Progress: Using a novel drug discovery paradigm based upon human stem cell derived nerve precursor cells, we have made a very potent drug called CAD-31. CAD-31 potently stimulates neurogenesis in human cells in culture and in mice, and prevents nerve cell death in cell culture models of toxicities associated with old age and AD. Very few, if any, drugs or drug candidates are both neuroprotective and neurogenic, particularly in animals. In the first year of this project, we harnessed the power of hESCs and medicinal chemistry to develop CAD-31. All of the Year 1 milestones were met. In Year 2 we completed all of the required pharmacokinetics and safety studies on the six best compounds synthesized in Year 1. Of those six, one compound, CAD-31, was the best in terms of medicinal chemical, pharmacokinetic, neuroprotective and neurogenic properties. This compound underwent extensive testing for safety and passed with flying colors. It was then put into an AD mouse model where it stimulated neurogenesis, prevented behavioral deficits and some of the disease pathology. All Year 2 milestones were completed. In Year 3 of the project we will determine if CAD-31 is able to reverse AD symptoms in old AD mice that already have the disease. This is the most clinically relevant model of AD since therapies can only be initiated once the disease is identified.
  • This work has produced a novel AD drug candidate that is developed based upon a set of assays never before used by pharmaceutical companies. It presents a unique opportunity to expand the use of hESCs for the development of a therapeutic for a disease for which there is no cure. This work could lead to a paradigm shift in drug discovery for the treatment of neurodegenerative disease.
Funding Type: 
Early Translational III
Grant Number: 
TR3-05606
Investigator: 
ICOC Funds Committed: 
$1 623 251
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Transplantation of neuronal precursors into the central nervous system offers great promise for the treatment of neurological disorders including spinal cord injury (SCI). Among the most significant consequences of SCI are bladder spasticity and neuropathic pain, both of which likely result from a reduction in those spinal inhibitory mechanisms that are essential for normal bladder and sensory functions. Our preliminary data show that embryonic inhibitory neuron precursor cells integrate in the adult nervous system and increase inhibitory network activity. Therefore inhibitory nerve cell transplants could be a powerful way to establish new inhibitory circuits in the injured spinal cord that will reduce bladder spasticity and attenuate central neuropathic pain. We already have proof-of-principle data that murine inhibitory nerve cells integrate in the adult spinal cord and improve symptoms in an animal model of chronic spinal cord injury. We have also recently developed methods to create human inhibitory interneurons from embryonic stem cells. This proposal will capitalize on these recent developments and determine whether our human embryonic stem cell-derived inhibitory cells can be successfully transplanted into the grey matter of the injured spinal cord and reduce neurogenic bladder dysfunction and neuropathic pain, two major causes of suffering in chronic SCI patients. If successful, our studies will lay the groundwork for a potential novel therapy for chronic SCI.
Statement of Benefit to California: 
There are an estimated 260,000 individuals in the United States who currently live with disability associated with chronic spinal cord injury (SCI). Symptoms of chronic SCI include bladder dyssynergia reflected by incontinence coincident with asynchronous contraction of internal and external sphincters, and central neuropathic pain, both of which severely impede activities of daily living, reduce quality of life, and contribute to the very high medical costs of caring for the Californians who suffer from chronic spinal cord injury. The Geron trial for SCI, as well as other cell-based approaches, aim to treat acute SCI. This proposal considers a different potentially complementary cell-transplantation strategy that is directed to more chronic SCI with the goal of improving bladder function and reducing pain. We propose to use cell grafts of inhibitory interneurons that we have derived from human stem cells in order to provide a novel treatment. If successful, we will have defined a therapeutic option that targets the most prevalent population of spinal cord injured patients. As the country's most populous state, California has the largest number of patients with chronic SCI, approximately 12,000. The estimated economic cost to California in lost productivity and medical expenses amounts to $400,000,000 annually. The potential savings in medical care costs, and improvement in quality of life will therfore have a disproportional benefit to the state of California.
Progress Report: 
  • From the past six months of work, we report considerable progress toward our aims of investigating the safety and efficacy of human inhibitory nerve precursor (MGE) cell transplantation for the treatment of spinal cord injury-induced bladder spasticity and neuropathic pain. Our first aim details the injection of human MGE cells into the uninjured rodent spinal cord and investigation of cell fate and potential adverse side effects from their transplantation. During the reporting period, we completed histological analyses for the two-month time point post-injection, and we found that the human MGE cells, derived from human embryonic stem cells (hESCs), appropriately matured into forebrain-type inhibitory interneurons in the rodent spinal cord. Also, we initiated histological examination of animals six months post-injection and detected robust human cell survival, dispersal into the spinal cord grey matter, and neuronal maturation, but no evidence of tumor formation. In addition, we completed behavioral analyses of animals injected with hESC-derived MGE cells at two and six months post-injection. Thus far, we have not observed any adverse side effects when human MGE cells are transplanted into the uninjured animal as determined by measures of body weight, locomotion, bladder function, and pain sensitivity.
  • Since the beginning of this project, we report considerable progress toward our aims of investigating the safety and efficacy of human inhibitory nerve precursor (MGE) cell transplantation for the treatment of spinal cord injury-induced bladder spasticity and neuropathic pain. In year one of this award we completed the major objectives of Aim1, namely to explore the survival, integration, and cell fate of stem cell-derived MGE cell transplants in the uninjured rodent spinal cord. We have now obtained preliminary efficacy results from Aim 2, namely the effects of hESC-MGE cells injected in spinal cord injured animals. Behavioral testing has been obtained to assess pain thresholds for all injected animals up to the six month endpoint, and measures of bladder spasticity have been obtained at six months post cell injection. We are evaluating whether the unblinded data demonstrates amelioration of neuropathic pain and bladder spasticity. Our preliminary histological analysis shows robust human cell survival, distribution, and neuronal differentiation, and we have electrophysiological data indicating functional integration of the transplanted cells. We are on track to complete all aims by the end of the award period.
Funding Type: 
Alpha Stem Cell Clinics
Grant Number: 
AC1-07764
Investigator: 
ICOC Funds Committed: 
$8 000 000
Disease Focus: 
Diabetes
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Adult Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Adult Stem Cell
Public Abstract: 
The proposed alpha clinic will bring together an outstanding team of physician-scientists with substantial clinical trials experience including stem cell and other cellular treatments of blood diseases and others. This team will also draw on our unique regional competitive advantages derived from our history of extensive collaboration with investigators at many nearby first-class research institutions and biotech companies. We propose to include these regional assets in our plans to translate our successful research on basic properties of stem cells to stem cell clinical trials and ultimately to delivery of effective and novel therapies. We propose to build an alpha clinic that serves the stem cell clinical trial needs of our large region where we are the only major academic health center with the needed expertise to establish a high impact alpha clinic. Our infrastructure will initially be developed and then used to support two major high-impact stem cell clinical trials: one in type I diabetes and one in spinal cord injury. Both are collaborations with established and well known companies. The type I diabetes trial will test embryonic stem cell derived cells that differentiate to become the missing beta cells of the pancreas. The cells are contained in a semipermeable bag that has inherent safety because of restriction of cell migration while allowing proper control of insulin levels in response to blood sugar. These hybrid devices are implanted just beneath the skin in patients in these trials. In a second trial of stem cell therapy for spinal cord injury, neuronal stem cells that have been shown to have substantial safety and efficacy in animal models of spinal cord injury and other types of spinal cord trauma or disease will be tested in human patients with chronic spinal cord injury. Both of these trials have the potential to have very substantial and important impact on patients with these diseases and the families and society that supports them. Following on these two trials, we are planning stem cell clinical trials for heart failure, cancer, ALS, and other terrible deadly disorders. Our proposed alpha clinic also benefits from very substantial leveraged institutional commitments, which will allow for an alpha clinic that is sustainable well beyond the five-year grant, which is essential to continue to manage the patients who have participated in the first trials being planned since multi-year followup and tracking is essential scientifically and ethically. We have a plan for our proposed alpha clinic to be sustainable to 10 years and beyond to the point at which these therapies if successful will be delivered to patients in our healthcare system.
Statement of Benefit to California: 
Many terrible diseases that afflict the citizens of California and cause substantial economic and emotional disruption to California families can potentially be treated with novel stem cell therapies. These therapies need to be tested in a rigorous and unbiased fashion in clinical trials, which is the focus of our proposed alpha clinic. Our clinic proposes to begin with clinical trials in two major diseases in need of improved treatment: type I diabetes and spinal cord injuries. The type I diabetes clinical trial will test a novel hybrid embryonic stem cell-derived pancreatic cell/encapsulation technology that is implanted just beneath the skin in an out-patient procedure, and is inherently safe because the cells are confined to a semi-permeable bag. The spinal cord injury trial will test the benefit of neural stem cells delivered to the site of injury. Both have substantial positive evidence in animal models and have the potential of leading to major breakthroughs. In addition to providing the infrastructure for these two trials, our proposed alpha clinic will also take advantage of very substantial regional expertise at our partner institutions to test stem cells in other diseases of importance in California including heart failure, ALS, cancer, and many others. Our proposed alpha clinic will also be a major economic as well as medical driver as it leverages substantial institutional and private sector commitment, and has the potential to deliver breakthrough therapies that will be marketed either in a health care system or by private sector companies.
Funding Type: 
Basic Biology IV
Grant Number: 
RB4-05855
Investigator: 
ICOC Funds Committed: 
$1 387 800
Disease Focus: 
Neuropathy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The use of stem cells or stem cell-derived cells to treat disease is one important goal of stem cell research. A second, important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. Existing means of generating genetically modified stem cells are not ideal, as they do not preserve the normal gene regulation, are inefficient, and do not permit removal of foreign genes. We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. We are adapting this approach for use with human embryonic stem cells, so that these cells can be better understood and harnessed for modeling, or even treating, human diseases. We will use this approach to create a human stem cell model of Charcot-Marie-Tooth (CMT) disease, an inherited neuropathy. How gene dysfunction leads to nerve defects in CMT is not clear, and there is no cure or specific therapy for this neurological disease. Thus, we will use our genetic tools to investigate how gene function is disrupted to cause CMT. By developing these tools and using them to gain understanding of CMT, we will illustrate how this system can be used to gain insight into other important diseases.
Statement of Benefit to California: 
Although human stem cells hold the potential to generate new understanding about human biology and new approaches to important diseases, the inability to efficiently and specifically modify stem cells currently limits the pace of research. Also, there is presently no safe means of changing genes compatible with the use of the stem cells in therapies. We are developing new genetic tools to allow for the tractable manipulation of human stem cells. By accelerating diverse other stem cell research projects, these tools will enhance the scientific and economic development of California. We will use these tools to create cellular models of Charcot-Marie-Tooth (CMT), a neurological disease with no cure that affects about 15,000 Californians. This model will facilitate understanding of the etiology of CMT, and may lead to insights that can be used to develop specific therapies. Beyond gaining insight into CMT, the ability to engineer specific genetic changes in human stem cells will be useful for many applications, including the creation of replacement cells for personalized therapies, reporter lines for stem cell-based drug screens, and models of other diseases. Thus, our research will assist the endeavors of the stem cell community in both the public and private arenas, contributing to economic growth and new product development. This project will also train students and postdoctoral scholars in human stem cell biology, who will contribute to the economic capacity of California.
Progress Report: 
  • An important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. During the first year of this project, we adapted this approach for use with human embryonic stem cells. We have also created gene trap mutations in a diversity of human embryonic stem cell genes that can be used to better harness human embryonic stem cells for modeling, or even treating, human diseases.
  • An important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. During the second year of this project, we took advantage of new methods using the CRISPR/Cas9 system to develop novel approaches to modifying human embryonic stem cells. We have also created reversible gene trap mutations in a diversity of human embryonic stem cell genes that can be used to better harness human embryonic stem cells for modeling, or even treating, human diseases.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01880
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 619 627
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The objective of this study is to develop a new, optimized technology to obtain a homogenous population of midbrain dopaminergic (mDA) neurons in a culture dish through neuronal differentiation. Dopaminergic neurons of the midbrain are the main source of dopamine in the mammalian central nervous system. Their loss is associated with one of the most prominent human neurological disorders, Parkinson's disease (PD). There is no cure for PD, or good long-term therapeutics without deleterious side effects. Therefore, there is a great need for novel drugs and therapies to halt or reverse the disease. Recent groundbreaking discoveries allow us to use adult human skin cells, transduce them with specific genes, and generate cells that exhibit virtually all characteristics of embryonic stem cells, termed induced pluripotent stem cells (iPSCs). These cell lines, when derived from PD patient skin cells, can be used as an experimental pre-clinical model to study disease mechanisms unique to PD. These cells will not only serve as an ‘authentic’ model for PD when further differentiated into the specific dopaminergic neurons, but that these cells are actually pathologically affected with PD. All of the current protocols for directed neuronal differentiation from iPSCs are lengthy and suboptimal in terms of efficiency and reproducibility of defined cell populations. This hinders the ability to establish a robust model in-a-dish for the disease of interest, in our case PD-related neurodegeneration. We will use a new, efficient gene integration technology to induce expression of midbrain specific transcription factors in iPSC lines derived from a patient with PD and a sibling control. Forced expression of these midbrain transcription factors will direct iPSCs to differentiate into DA neurons in cell culture. We aim at achieving higher efficiency and reproducibility in generating a homogenous population of midbrain DA neurons, which will lay the foundation for successfully modeling PD and improving hit rates of future drug screening approaches. Our study could also set a milestone towards the establishment of efficient, stable, and reproducible neuronal differentiation using a technology that has proven to be safe and is therefore suitable for cell replacement therapies in human. The absence of cellular models of Parkinson’s disease represents a major bottleneck in the scientific field of Parkinson’s disease, which, if solved, would be instantly translated into a wide range of clinical applications, including drug discovery. This is an essential avenue if we want to offer our patients a new therapeutic approach that can give them a near normal life after being diagnosed with this progressively disabling disease.
Statement of Benefit to California: 
The proposed research could lead to a robust model in-a-dish for Parkinson’s disease (PD)-related neurodegeneration. This outcome would deliver a variety of benefits to the state of California. First, there would be a profound personal impact on patients and their families if the current inevitable decline of PD patients could be halted or reversed. This would bring great happiness and satisfaction to the tens of thousands of Californians affected directly or indirectly by PD. Progress toward a cure for PD is also likely to accelerate the development of treatments for other degenerative disorders. The technology for PD modeling in-a-dish could be applied to other cell types such as cardiomyocytes (for heart diseases) and beta-cells (for diabetes). The impact would likely stimulate medical progress on a variety of conditions in which stem cell based drug screening and therapy could be beneficial. An effective drug and therapy for PD would also bring economic benefits to the state. Currently, there is a huge burden of costs associated with the care of patients with long-term degenerative disorders like PD, which afflict tens of thousands of patients statewide. If the clinical condition of these patients could be improved, the cost of maintenance would be reduced, saving billions in medical costs. Many of these patients would be more able to contribute to the workforce and pay taxes. Another benefit is the effect of novel, cutting-edge technologies developed in California on the business economy of the state. Such technologies can have a profound effect on the competitiveness of California through the formation of new manufacturing and health care delivery facilities that would employ California citizens and bring new sources of revenue to the state. Therefore, this project has the potential to bring health and economic benefits to California that is highly desirable for the state.
Progress Report: 
  • Dopaminergic (DA) neurons of the midbrain are the main source of dopamine in the mammalian central nervous system. Their loss is associated with a prominent human neurological disorder, Parkinson's disease (PD). There is no cure for PD, nor are there any good long-term therapeutics without deleterious side effects. Therefore, there is a great need for novel therapies to halt or reverse the disease. The objective of this study is to develop a new technology to obtain a purer, more abundant population of midbrain DA neurons in a culture dish. Such cells would be useful for disease modeling, drug screening, and development of cell therapies.
  • Recent discoveries allow us to use adult human skin cells, introduce specific genes into them, and generate cells, termed induced pluripotent stem cells (iPSC), that exhibit the characteristics of embryonic stem cells. These iPSC, when derived from PD patient skin cells, can be used as an experimental model to study disease mechanisms that are unique to PD. When differentiated into DA neurons, and these cells are actually pathologically affected with PD.
  • The current methods for directed DA neuronal differentiation from iPSC are inadequate in terms of efficiency and reproducibility. This situation hinders the ability to establish a robust model for PD-related neurodegeneration. In this study, we use a new, efficient gene integration technology to induce expression of midbrain-specific genes in iPSC lines derived from a patient with PD and a normal sibling. Forced expression of these midbrain transcription factor genes directs iPSC to differentiate into DA neurons in cell culture. A purer population of midbrain DA neurons may lay the foundation for successfully modeling PD and improving hit rates in drug screening approaches.
  • The milestones for the first year of the project were to establish PD-specific iPSC lines that contain genomic “docking” sites, termed “attP” sites. In year 2, these iPSC/attP cell lines will be used to insert midbrain-specific transcription factors with high efficiency, mediated by enzymes called integrases. We previously established an improved, high-efficiency, site-specific DNA integration technology in mice. This technology combines the integrase system with newly identified, actively expressed locations in the genome and ensures efficient, uniform gene expression.
  • The PD patient-specific iPSC lines we used were PI-1754, which contains a severe mutation in the SNCA (synuclein alpha) gene, and an unaffected sibling line, PI-1761. The SNCA mutation causes dramatic clinical symptoms of PD, with early-onset progressive disease. We use a homologous recombination-based procedure to place the “docking” site, attP, at well-expressed locations in the SNCA and control iPSC lines (Aim 1.1). We also included a human embryonic stem cell line, H9, to monitor our experimental procedures. The genomic locations we chose for placement of the attP sites included a site on chromosome 22 (Chr22) and a second, backup site on chromosome 19 (Chr19). These two sites were chosen based on mouse studies, in which mouse equivalents of both locations conferred strong gene expression. In order to perform recombination, we constructed targeting vectors, each containing an attP cassette flanked by 5’ and 3’ homologous fragments corresponding to the human genomic location we want to target. For the Chr22 locus, we were able to obtain all 3 targeting constructs for the PI-1754, PI-1761 and H9 cell lines. For technical reasons, we were not able to obtain constructs for the Chr19 location Thus, we decided to focus on the Chr22 locus and move to the next step.
  • We introduced the targeting vectors into the cells and selected for positive clones by both drug selection and green fluorescent protein expression. For the H9 cells, we obtained 110 double positive clones and analyzed 98 of them. We found 8 clones that had targeted the attP site precisely to the Chr22 locus. For the PI-1761 sibling control line, we obtained 44 clones, and 1 of them had the attP site inserted at the Chr22 locus. The PI-1754 SNCA mutant line, on the other hand, grows slowly in cell culture. We are in the process of obtaining enough cells to perform the recombination experiment in that cell line.
  • In summary, we demonstrated that the experimental strategy proposed in the grant indeed worked. We were successful in obtaining iPSC lines with a “docking” site placed in a pre-selected human genomic location. These cell lines are the necessary materials that set the stage for us to fulfill the milestones of year 2.
  • Parkinson's disease (PD) is caused by the loss of dopaminergic (DA) neurons in the midbrain. These DA neurons are the main source of dopamine, an important chemical in the central nervous system. PD is a common neurological disorder, affecting 1% of those at 60 years old and 4% of those over 80. Unfortunately, there is no cure for PD, nor are there any long-term therapeutics without harmful side effects. Therefore, there is a need for new therapies to halt or reverse the disease. The goal of this study is to develop a new technology that helps us obtain a purer, more abundant population of DA neurons in a culture dish and to characterize the resulting cells. These cells will be useful for studying the disease, screening potential drugs, and developing cell therapies.
  • Due to recent discoveries, we can introduce specific genes into adult human skin cells and generate cells similar to embryonic stem cells, termed induced pluripotent stem cells (iPSC). These iPSC, when derived from PD patients, can be used as an experimental model to study disease mechanisms that are unique to PD, because when differentiated into DA neurons, these cells are actually pathologically affected with PD. We are using a PD iPSC line called PI-1754 derived from a patient with a severe mutation in the SNCA gene, which encodes alpha-synuclein. The SNCA mutation causes dramatic clinical symptoms of PD, with early-onset progressive disease. For comparison we are using a normal, unaffected sibling iPSC line PI-1761. We are also using a normal human embryonic stem cell (ESC) line H9 as the gold standard for differentiation.
  • The current methods for differentiating iPSC into DA neurons are not adequate in terms of efficiency and reliability. Our hypothesis is that forced expression of certain midbrain-specific genes called transcription factors will direct iPSC to differentiate more effectively into DA neurons in cell culture. We use transcription factors called Lmx1a, Otx2, and FoxA2, abbreviated L, O, and F. In this project, we have developed a new, efficient gene integration technology that allows us rapidly to introduce and express these transcription factor genes in various combinations, in order to test whether they stimulate the differentiation of iPSC into DA neurons.
  • In the first year of the project, we began establishing iPSC and ESC lines that contained a genomic “landing pad” site for insertion of the transcription factor genes. We carefully chose a location for placement of the genes based on previous work in mouse that suggested that a site on human chromosome 22 would provide strong and constant gene expression. We initially used ordinary homologous recombination to place the landing pad into this site. By the end of year 1 of the project, this method was successful in the normal iPSC and in the ESC, but not in the more difficult-to-grow PD iPSC. To solve this problem, in year 2 we introduced a new and more powerful recombination technology, called TALENs, and were successful in placing the landing pad in the correct position in all three of the lines, including the PD iPSC.
  • We were now in a position to insert the midbrain-specific transcription factor genes with high efficiency. For this step, we developed a new genome engineering methodology called DICE, for dual integrase cassette exchange. In this technology, we use two site-specific integrase enzymes, called phiC31 and Bxb1, to catalyze precise placement of the transcription factor genes into the desired place in the genome.
  • We constructed gene cassettes carrying all pair-wise combinations of the L, O, and F transcription factors, LO, LF, and OF, and the triple combination, LOF. We successfully demonstrated the power of this technology by rapidly generating a large set of iPSC and ESC that contained all the above combinations of transcription factors, as well as lines that contained no transcription factors, as negative controls for comparison. Two examples of each type of line for the 1754 and 1761 iPSC and the H9 ESC were chosen for differentiation and functional characterization studies. Initial results from these studies have demonstrated correct differentiation of neural stem cells and expression of the introduced transcription factor genes.
  • In summary, we were successful in obtaining ESC and iPSC lines from normal and PD patient cells that carry a landing pad in a pre-selected genomic location chosen and validated for strong gene expression. These lines are valuable reagents. We then modified these lines to add DA-associated transcription factors in four combinations. All these lines are currently undergoing differentiation studies in accordance with the year two and three timelines. During year three of the project, the correlation between expression of various transcription factors and the level of DA differentiation will be established. Furthermore, functional studies with the PD versus normal lines will be carried out.
  • The objective of this project is to develop approaches and technologies that will improve neuronal differentiation of stem cells into midbrain dopaminergic (DA) neurons. DA neurons are of central importance in the project, because they are that cells that are impaired in patients with Parkinson’s disease (PD). Current differentiation methods typically produce low yields of DA neurons. The methods also give variable results, and cell populations contain many types of cells. These impediments have hampered the study of disease mechanisms for PD, as well as other uses for the cells, such as drug screening and cell replacement therapy. Our strategy is to develop a novel method to introduce genes into the genome at a specific place, so we can rapidly add genes that might help in the differentiation of DA neurons. The genes we would like to add are called transcription factors, which are proteins involved differentiation of stem cells into DA neurons. We have placed the genes for three transcription factors into a safe, active position on human chromosome 22 in the cell lines we are studying. These cells, called pluripotent stem cells, have the potential to differentiate into almost any type of cell. We are using embryonic stem cells in our study, as well as induced pluripotent stem cells (iPSC), which are similar, but are derived from adult cells, rather than an embryo. We are using iPSC derived from a PD patient, as well as iPSC from a normal person, for comparison. By forced expression of these neuronal transcription factors, we may achieve more efficient and reproducible generation of DA neurons. The effects of expressing different combinations of the three transcription factors called Lmx1a, FoxA2, and Otx2 on DA neuronal differentiation will be evaluated in the context of embryonic stem cells (ESC) as the gold standard, as well as in iPSC derived from a PD patient with a severe mutation in alpha-synuclein and iPSC derived from a normal control. Comparative functional assays of the resulting DA neurons will complete the analysis.
  • To date, this project has created a novel technology for modifying the genome. The strategy developed out of the one that we originally proposed, but contains several innovations that make it more powerful and useful. The new methodology, called DICE for Dual Integrase Cassette Exchange, allowed us to generate “master” or recipient cell lines for ESC, normal iPSC, and PD iPSC. These recipient cell lines contain a “landing pad” placed into a newly-identified actively-expressed location on human chromosome 22 called H11 that permits robust expression of genes placed into it. We then generated a series of cell lines by "cassette exchange" at the H11 locus. In cassette exchange, the new genes we want to add take the place of the landing pad we originally put into the cells. Cassette exchange is a good way to introduce various genes into the same place in the chromosomes. We created cell lines expressing three neuronal transcription factors suspected to be involved in DA neuronal differentiation, in all pair-wise combinations, including lines with expression of all three factors, and negative control lines with no transcription factors added. This collection of modified human pluripotent stem cell lines is now being used to study neural differentiation. The modified ESC have undergone differentiation into DA neurons and are being evaluated for the effects of the different transcription factor combinations on DA neuronal differentiation. During the final year of the project, this differentiation analysis will be completed, and we will also analyze functional properties of the differentiated DA neurons, with special emphasis on disease-related features of the cells derived from PD iPSC.
  • The objective of this project is to develop technologies and approaches that will improve differentiation of stem cells into midbrain dopaminergic (DA) neurons. DA neurons are of central importance in the project, because they are the cells that are impaired in patients with Parkinson’s disease (PD). It appears that midbrain dopaminergic neurons have an enormous energy requirement, which might help explain their vulnerability to degeneration in PD. Current differentiation methods typically produce low yields of DA neurons. The methods also give variable results, and cell populations contain many types of cells. These impediments have hampered the study of disease mechanisms for PD, as well as other uses for the cells, such as drug screening and cell replacement therapy. Our strategy is to develop a novel method to introduce genes into the genome at a specific place, so we can rapidly add genes that might help in the differentiation of DA neurons. The genes we would like to add are called transcription factors, which are proteins involved in differentiation of stem cells into DA neurons. We have placed the genes for three transcription factors into a safe, active position on human chromosome 22 in the cell lines we are studying. These cells, called pluripotent stem cells, have the ability to differentiate into almost any type of cell. We are using embryonic stem cells in our study, as well as induced pluripotent stem cells (iPSC), which are similar, but are derived from adult cells, rather than an embryo. We are using iPSC derived from a PD patient, as well as iPSC from a normal person, for comparison. By forced expression of neuronal transcription factors, we may achieve more efficient and reproducible generation of DA neurons. We want to evaluate the effects of expressing different combinations of three transcription factors called Lmx1a, FoxA2, and Otx2 on DA neuronal differentiation in the context of embryonic stem cells (ESC) as the gold standard, as well as in iPSC derived from a PD patient with a severe mutation in alpha-synuclein, and in iPSC derived from a normal person without PD. Comparative functional assays of the resulting DA neurons will complete the analysis.
  • To date, this project created a novel technology for modifying the genome. The strategy developed out of the one that we originally proposed, but contains several innovations that make it more powerful and useful. The new methodology, called DICE for Dual Integrase Cassette Exchange, allowed us to generate “master” or recipient cell lines for ESC, normal iPSC, and PD iPSC. These recipient cell lines contain a “landing pad” placed into a newly-identified actively-expressed location on human chromosome 22 called H11 that permits robust expression of genes placed into it. We then generated a series of cell lines by "cassette exchange" at the H11 locus. In cassette exchange, the new genes we want to add take the place of the landing pad we originally put into the cells. Cassette exchange is a good way to introduce various genes into the same place in the chromosomes.
  • We created cell lines expressing three neuronal transcription factors suspected to be involved in DA neuronal differentiation, in all pair-wise combinations, including lines with expression of all three factors, and negative control lines with no transcription factors added. This collection of modified human pluripotent stem cell lines is being used to study neural differentiation. The modified ESC were used to form embryoid bodies, which are spherical aggregations of stem cells similar to an embryo that are favorable for producing differentiated cells. We found that the embyroid bodies underwent a rapid process of spontaneous differentiation into DA neurons. The differentiation was stimulated in the cells that expressed inserted transcription factors, and some combinations of transcription factors were better than others in bringing about DA neuronal differentiation. We obtained the best differentiation in the lines that expressed the LMX1A and OTX2 transcription factors. In continuing studies, we will analyze functional properties of the differentiated DA neurons, with special emphasis on disease-related features of the cells derived from PD iPSC.
Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06530
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$3 031 737
Disease Focus: 
Neuropathy
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The applicant is an MD/PhD trained physician scientist, whose clinical expertise is neuromuscular disorders including peripheral nerve disease. The proposal is aimed at providing a research proposal and career development plan that will allow the applicant to develop an independent research program, which attempts to bring stem cell based therapies to patients with peripheral nerve diseases. The proposal will use “adult stem cells” derived from patients with an inherited nerve disease, correct the genetic abnormality in those cells, and determine the feasibility of transplanting the genetically engineered cells back into peripheral nerve to slow disease progression.
Statement of Benefit to California: 
The proposed research will benefit the State of California as it will support the career development of a uniquely trained physician scientist to establish an innovative translational stem cell research program aimed toward direct clinical application to patients. The cutting edge technologies proposed are directly in line with the fundamental purpose of the California Initiative for Regenerative Medicine. If successful, both scientific and patient advocate organizations would recognize that these advances came directly from the unique efforts of CIRM and the State of California to lead the world in stem cell research. Finally, as a result of funding of this award, further financial investments from private and public funding organizations would directly benefit the State in the years to come.
Progress Report: 
  • During this award period we have made significant progress. We have established induced pluripotent stem cell (iPSC) lines from four patients with Charcot-Marie-Tooth disease type 1A (CMT1A) due to the PMP22 duplication. We have validated our strategy to genetically engineer induced pluripotent stem cells from patients with inherited neuropathy, and have genetically engineered several patient lines. We further have begun to differentiate these iPSCs into Schwann cell precursors, to begin to investigate cell type specific defects that cause peripheral neurodegeneration in patients with CMT1A. Finally we have imported and characterized a transgenic rat model of CMT1A in order to begin to investigate the ability to inject iPSC derived Schwann cell precursors into rodent nerves as a possible neuroprotective strategy.
  • During this reporting period we developed genetically corrected induced pluripotent stem cell lines from patients with CMT1A. We improved and validated a novel method for differentiating Schwann cells from iPSCs, and used this to generate human Schwann cells from patients and controls. Finally we have initiated pilot studies injecting human iPSC derived Schwann cells into the peripheral nerves of rats with myelin diseases to determine whether cell replacement therapy is a viable strategy in these disorders.
Funding Type: 
Early Translational III
Grant Number: 
TR3-05577
Investigator: 
ICOC Funds Committed: 
$1 857 600
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
We propose to discover new drug candidates for Alzheimer’s Disease (AD), which is common, fatal, and for which no effective disease-modifying drugs are available. Because no effective AD treatment is available or imminent, we propose to discover novel candidates by screening purified human brain cells made from human reprogrammed stem cells (human IPS cells or hIPSC) from patients that have rare and aggressive hereditary forms of AD. We have already discovered that such human brain cells exhibit an unique biochemical behavior that indicates early development of AD in a dish. Thus, we hope to find new drugs by using the new tools of human stem cells that were previously unavailable. We think that human brain cells in a dish will succeed where animal models and other types of cells have thus far failed.
Statement of Benefit to California: 
Alzheimer’s Disease (AD) is a fatal neurodegenerative disease that afflicts millions of Californians. The emotional and financial impact on families and on the state healthcare budget is enormous. This project seeks to find new drugs to treat this terrible disease. If we are successful our work in the long-term may help diminish the social and familial cost of AD, and lead to establishment of new businesses in California using our approaches to drug discovery for AD.
Progress Report: 
  • We have made steady and significant progress in developing a way to use human reprogrammed stem cells to develop drugs for Alzheimer's disease. In the more recent project term we have further refined our key assay, and generated sufficient cells to enable screening of 50,000 different chemical candidates that might reveal potential drugs for this terrible disease. With a little bit of additional refinement, we will be able to begin our search in earnest in collaboration with the Sanford-Burnham Prebys Screening Center.
  • During the past year we completed screening of our Alzheimers “disease in a dish” cultured stem cell lines for response of a critical measure of Alzheimers disease in a dish to FDA approved drugs and other potentially promising drug like compounds. We found several reproducible and interesting categories of potential drugs some of which are already in common use in human patients and therefore might be readily available to the Alzheimer's disease population. We are conducting more careful analyses of these drugs for their mechanism and behavior in human neurons with different types of Alzheimer like behavior and we are beginning to test whether all human variants behave the same way as preparation for potential clinical trials. We are also initiating analysis of new chemical entities for possible modification to improve potency.
Funding Type: 
Research Leadership 14
Grant Number: 
LA1_C14-08015
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$6 368 285
Disease Focus: 
Heart Disease
Neurological Disorders
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Directly Reprogrammed Cell
Public Abstract: 
Tissues derived from stem cells can serve multiple purposes to enhance biomedical therapies. Human tissues engineered from stem cells hold tremendous potential to serve as better substrates for the discovery and development of new drugs, accurately model development or disease progression, and one day ultimately be used directly to repair, restore and replace traumatically injured and chronically degenerative organs. However, realizing the full potential of stem cells for regenerative medicine applications will require the ability to produce constructs that not only resemble the structure of real tissues, but also recapitulate appropriate physiological functions. In addition, engineered tissues should behave similarly regardless of the varying source of cells, thus requiring robust, reproducible and scalable methods of biofabrication that can be achieved using a holistic systems engineering approach. The primary objective of this research proposal is to create models of cardiac and neural human tissues from stem cells that can be used for various purposes to improve the quality of human health.
Statement of Benefit to California: 
California has become internationally renowned as home to the world's most cutting-edge stem cell biology and a global leader of clinical translation and commercialization activities for stem cell technologies and therapies. California has become the focus of worldwide attention due in large part to the significant investment made by the citizens of the state to prioritize innovative stem cell research as a critical step in advancing future biomedical therapies that can significantly improve the quality of life for countless numbers of people suffering from traumatic injuries, congenital disorders and chronic degenerative diseases. At this stage, additional investment in integration of novel tissue engineering principles with fundamental stem cell research will enable the development of novel human tissue constructs that can be used to further the translational use of stem cell-derived tissues for regenerative medicine applications. This proposal would enable the recruitment of a leading biomedical engineer with significant tissue engineering experience to collaborate with leading cardiovascular and neural investigators. The expected result will be development of new approaches to engineer transplantable tissues from pluripotent stem cell sources leading to new regenerative therapies as well as an enhanced understanding of mechanisms regulating human tissue development.

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