Walter C. Low - US grants

The loss of cholinergic neurons in the septal nucleus, diagonal band, and nucleus basalis has been implicated in the neuropathology of senile dimentia of the Alzheimer's type. Patients with this disorder exhibit profound memory and learning impairment and progressive intellectual deterioration. Experimental animals with lesions of the cholinergic septo-hippocampal pathway have also been shown to exhibit memory disorders. This behavioral deficit can be rectified with transplants containing cholinergic neurons from embryonic septal nucleus-diagonal band tissue. The recovery of behavioral function is associated with a transplant-derived reinnervation of the host brain along with a recovery of choline acetyltransferase activity, the enzyme involved in the synthesis of acetylcholine. The success of neural transplantation to the central nervous system has been confined primarily to the use of embryonic material as a source of donor tissue. Recently, new neuronal survival factors have been identified, and calcium free media have been developed that aid in the regeneration of axotomized neurons. We propose to investigate the use of these survival factors and media in conjunction with recently developed methods of dissociated cell suspensions to transplant adult neurons. Nerve cells in the septal nucleus-diagonal band region from adult rats will be dissociated in a calcium free regeneration-promoting media. The cell suspension will then be injected into the hippocampal formation of adult rats that have previously received lesions to denervate the intrinsic cholinergic fiber projections. After transplantation, parameters of fiber ingrowth as determined by acetylcholinesterase histochemistry and choline acetyltransferase activity will be assessed. These studies will provide information needed to assess the use of central cholinergic neurons from adult donors for transplantation. If such neurons are capable of innervating the hippocampus of the host brain, then they may eventually provide an invaluable source of tissue for transplantation to aid in the restoration of neural function lost through stroke, trauma, or progressive degenerative disorders.

The transplantation of human fetal tissue has been proposed for the treatment of a variety of devastating diseases and disorders. The potential clinical applications of cell transplantation therapies, however, have been significantly hampered by the limited availability of such tissue for research. We proposed to assess the quality and quantity of human fetal tissues obtained solely from spontaneous abortions and ectopic pregnancies to serve as sources for transplantation therapy. We further propose to develop and test methods that will be essential for the establishment of effective human fetal tissue banks. These proposals will be addressed by the following projects and specific aims. Project 1 will develop and evaluate methods for procuring, processing, and distributing human fetal tissue which will ensure tissue of the highest quality for cellular transplantation. Project 2 will assess the availability of human fetal tissue for tissue banking in a prospective study involving local hospitals in the Minneapolis/St. Paul metropolitan area, and in retrospective studies involving local hospitals and health maintenance organizations (HMOs). Project 3 will evaluate new ways of storing and preserving human fetal tissue for tissue banking. Project 4 will characterize human hematopoietic progenitors cells for use in transplantation. Project 5 will evaluate the development of fetal cells of the humoral immune system. Project 6 will determine markers for fetal pancreatic islet progenitor cells, and determine the growth potential of these cells under the influence of growth factors. Finally, Project 7 will examine the effects of epidermal growth factor on the proliferation of fetal dopamine nerve cells, and assess the ability of transplanted mesencephalic neurons to release dopamine, innervate the host brain, and restore locomotor function. Together these projects will assess the viability of a variety of cells obtained from spontaneous abortions or ectopic pregnancies, and determine the feasibility of such tissue to serve as sources for transplantation therapies.

Studies of bone marrow cells have shown that they can restore neurologic function when transplanted into animals with ischemic brain injury. Recently, our laboratories have isolated a specific multi-potent stem cell population from human bone marrow that is capable of differentiating into neurons, astrocytes, and oligodendroglia in vitro under appropriate culture conditions. We postulate that these bone marrow-derived stem cells can provide an autologous source of donor cells for transplantation and repair in conditions of ischemic brain injury and stroke. In Specific Aim 1 we will determine the neural phenotypes generated from bone marrow-derived human stem cells when transplanted into the brain of spontaneously hypertensive rats. Human stem cells will be transduced to express eGFP to label the donor cells. Bone marrow-derived stem cells will then be transplanted into the striatum, hippocampal formation, or sensorimotor cortex. At various time periods after transplantation, brain tissue from host rats will be studied to identify presence of eGFP labeled cells. eGFP labeled cells will also be examined to determine whether they develop into neurons, glial cells, and/or oligodendroglia. Site-specific differentiation will also be evaluated to determine whether grafted neurons also co-express neurotransmitter markers that are characteristic of neurons that found in the striatum, hippocampus, and cortex. In Specific Aim 2 we will determine the efficacy of transplanted bone-marrow stem cells in ameliorating neurologic deficits associated with ischemic brain injury. The middle cerebral artery will be occluded unilaterally by ligation distal to the branching striatal vessels. This occlusion produces a discrete ischemic lesion of the cortex and results in permanent sensory and motor deficits in the forelimb contralateral to the side of injury. One week after the ischemic event animals will receive transplants of bone marrow-derived stem cells into regions of the neocortex surrounding the area of injury. In preliminary studies we have found that these transplants are capable of ameliorating sensorimotor deficits. The extent of the functional recovery will be assessed over time using a battery of behavioral tests. Also as part of Specific Aim 2 we will determine the time window after ischemic brain injury for stem cell transplantation that will result in an amelioration of neurologic deficits. In these studies ischemic rats will receive transplants at various time periods after ischemic injury and assessed functionally over time using a defined set of sensory and motor tests. In Specific Aim 3 we will investigate possible mechanisms underlying bone marrow-graft induced recovery of neurologic function. Possible mechanisms include trophic effects on neural stem cells of the host brain, the induction of growth factor release by host brain cells, and the reorganization of host fiber connections. The results of these studies will provide information regarding the efficacy of using specific bone marrow-derived stem cells as an autologous source of cells for transplantation in ischemic brain injury, and information regarding possible mechanisms of graft-induced neural plasticity.

The Neurological Services Core for this program project grant will provide support for the surgical delivery of therapeutic gene constructs into the brain of MPS I, MPS VII, and SCA1 mice. In addition, this core will be involved in the neurologic assessment of cognitive and motor functions in these animals, and neurohistological assessments after the targeted surgical delivery of the therapeutic gene constructs.

[unreadable] DESCRIPTION (provided by applicant): Human umbilical cord blood (hUCB) is a rich source of stem cells that exhibit multipotent properties. Previous studies have shown that hUCB cells can differentiate into neural cells under the appropriate environment conditions. SANERON CCEL, Inc. is focusing on the use of hUCB as a therapeutic approach for treating a variety of neurological disorders. In this Phase I STTR study we will evaluate the efficacy of using hUCB in treating experimental hemorrhagic brain injury. [unreadable] Intracerebral hemorrhage (ICH) is a major cause of morbidity and mortality. Patients who survive are often left severely disabled, and the costs for caring for these individuals are substantial. [unreadable] Improvements in function for patients with ICH would add significantly to their quality of life and reduce the cost for care. We will evaluate the effects of administering hUCB intravenously into rats with collagenase-induced ICH of the striatum. hUCB will be given at various time periods after the induction of the hemorrhage. Animals will then be evaluated periodically to assess their neurological status over time. Comparisons will be made between hUCB treated animals and those receiving other types of sham therapy. After the neurological assessments, brain tissue from each animal will be studied histologically to determine whether hUCB cells differentiated into neural cells within the environment of the hemorrhagic brain. The results from this study will provide information on whether hUCB can be used as a therapy for treating hemorrhagic brain injury. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal is for a training grant, "Translational Research in Neurobiology of Disease", at the University of Minnesota. Trainees of this program are 1) graduate students who are pursuing a Ph.D. degree through the Graduate Program in Neuroscience, 2) postdoctoral fellows, and 3) clinical fellows. The challenge is to train basic neuroscientists who will have an appreciation for clinically relevant problems, and to train clinicians who have an appreciation of the fundamental principals of tools of basic science so that they can work together and accelerate the pace of translational neuroscience. The proposed training program funds predoctoral students during their second year in the Graduate Program in Neuroscience after they have committed to the translational neuroscience track. Postdoctoral fellows and clinical fellows can be funded at any point in their training, but must be committed to training in translational neuroscience. The training program is built around a core of didactic course work that includes a course in Neurobiology of Disease, and a Neuroscience Laboratory summer course for all trainees. These courses are designed to produce a dialog and interactions among basic and clinical neuroscientists to facilitate translational research. A group of over 37 trainers are proposed that reflects the diversity of research techniques in basic and clinical neuroscience, but with a thematic focus on neural degeneration and repair. Research interests range from molecular mechanisms underlying neurodegenerative disorders to therapies involving stem cells and recombinant DNA. Each trainer directs a productive research program and has demonstrated commitment to teaching and training. Representing 11 departments throughout the university, the trainers are united by their participation in the Graduate Program in Neuroscience and translational neuroscience research. The trainees will be provided with a strong, broad foundation in basic and clinical neurosciences upon which to build their translational research careers.

[unreadable] DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a neurological disorder caused by the progressive degeneration of dopamine neurons within the substantia nigra region of the brain. The causes of this degenerative process have yet to be clearly determined, however, many types of compounds such as growth factors, and anti-apoptotic agents have been proposed to protect against the loss of dopamine neurons. A major impediment to the assessment of these compounds is the lack of an in vivo model assay system for high through-put evaluation. Currently, the testing of these compounds requires that the brains of each animal be processed and analyzed over several weeks before the effects of these compounds can be determined by the quantification of dopamine neurons within the substantia nigra. We propose a new approach where the dopamine neurons within the substantia nigra can be visualized and quantified in individual animals be creating transgenic mice that express the firefly enzyme luciferase. The expression of this enzyme will be restricted to cells within the body that produce the enzyme tyrosine hydroxylase, the rate limiting enzyme in the synthesis of dopamine. This restricted distribution will be accomplished by using the tyrosine hydroxylase promoter to drive the expression of the luciferase gene. Cell specific expression of luciferase will enable us to visualize the quantify dopamine neurons in the substantia nigra of living mice, and provide a high through-put assay for screening compounds that affect dopaminergic neuron degeneration and survival. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a neurological disorder characterized by tremor, rigidity, and bradykinesia. This is a progressively deteriorating condition that currently affects about 1.5 million people in the United States. At the present time, there is no cure for this disease. In our proposal, we intend to develop a line of transgenic mice that will enable us to visualize dopamine neurons in the living animal. Using this animal, we will be able to evaluate compounds that protect against dopamine neuron loss in a high throughput manner that will allow us to quickly screen many possible compounds. In addition, the development of this transgenic animal will allow us to study inductive cues that might be used to produce dopamine neurons that can be transplanted into patients with Parkinson's disease. As a consequence, this transgenic mouse could play an important role in developing new therapies for treating Parkinson's disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Transcranial focused ultrasound (FUS) has been investigated for a range of applications in small and large animal models as well as humans. There is increased interest in using pulsed FUS (pFUS) in neurological applications (e.g. neurostimulation) due the two major advantages of non-invasiveness and high degree of localization. An exciting parallel development in neurology was triggered by the positive findings regarding the ability of human umbilical cord blood stem cells (UCBSCs) to restore limb functions even when transplanted days after the onset of ischemic stroke (in a small-animal stroke model). However, the therapeutic efficacy of the systemic administration of UCBSC is often limited by the inefficient homing and trafficking to the target tissue volumes. This limitatin may be addressed by the use of pFUS in conjunction with systemic administration of UCBSCs (specifically so-called non-hematopoietic umbilical cord blood stem cells (nh-UCBSCs)). This is motivated by recent results demonstrating that pFUS can improve cell-based therapies as noninvasive modality for homing and trafficking of therapeutic agents. We propose to design a dual-mode ultrasound array (DMUA) system capable of generating a range of localized sub-therapeutic pFUS exposures in the sensorimotor cortex of an in vivo rat stroke model. In addition, the system will be capable of generating imaging feedback with high specificity to the interaction between the pFUS exposure and the target tissue, e.g. thermal changes, acoustic radiation force (ARF) displacements and strains, cavitation. The successful implementation of the image-guided DMUA system will enhance the precision of homing and trafficking of the nh-UCBSCs to the target volume within and surrounding the ischemic tissue. Equally significantly, it will provide high specificity feedback on the nature of pFUS-tissue interactions thus allowing for better understanding of the key mechanisms leading to enhanced homing, with or without the use of microbubble ultrasound contrast agents. This will open the door for more efficient pre-clinical and clinical research on pFUS-enhanced homing and trafficking of UBCSCs in large-animal models of stroke and, subsequently, in human subjects.

ABSTRACT/SUMMARY In previous studies we identified novel non-hematopoietic umbilical cord blood stem cells (nh- UCBSCs) that could be expanded to high passages and exhibit restorative properties following ischemic brain injury repair. We found that the high passaged cells were equivalent, if not even better, than the low passaged nh-UCBSCs. This finding suggests that these cells can serve as a source of highly expandable cells that can be manufactured under controlled conditions for potential therapeutic applications. In this Phase I/II proposal we intend to scale-up production of nh-UCBSCs under good manufacturing practice (GMP) conditions at the GMP facility located on the campus of the University of Minnesota. These GMP manufactured cells will then be tested for efficacy using small animal (laboratory rat) and large animal (non-human primate) models of ischemic brain injury. In consultation with the FDA we are proposing IND-enabling studies to further qualifying these cells for use in a future clinical study. We will evaluate the safety and potential tumorigenicity of these cells, evaluate mechanisms of action and effects of cryopreservation. The results from this study will provide key data required by the Food and Drug Administration for an IND to conduct a clinical trial on the use of these cells in the treatment of ischemic brain injury.

SUMMARY/ABSTRACT Alzheimer?s disease (AD) is the leading cause of neurodegenerative disorders with over 5 million cases in the U.S. This represents one of the most compelling health-related burdens facing our society in the near future. Current approved therapies for AD include cholinesterase inhibitors to prevent the degradation of the neurotransmitter acetylcholine, although they have modest benefit and are effective only during the early stages of the disease process. The progressive dysfunction of the limbic system and loss of neurons in these regions of the brain are responsible for the dramatic cognitive decline seen in this disease. Loss of cholinergic neurons in the medial septal nucleus (MSN) and the nucleus basalis are observed in AD, in addition to the loss of glutamatergic pyramidal neurons in the hippocampal formation. Transplantation of cholinergic neurons and glutamatergic pyramidal neurons in rodents results in restoration of spatial learning and memory function. Dysfunctional GABAergic interneurons within the hippocampal formation have been shown to cause hyperactivity of neural circuits within the limbic system. Transplants of GABAergic interneurons into the hippocampus of rodent models of AD demonstrate normalization of this hyperactivity and restoration of learning and memory. In AD subjects with the APOE4 genotype, astrocytes derived from patients? iPSCs exhibit abnormal morphology. Transplants of astrocytes in other rodent models of neurodegenerative diseases restore neurological function. The translation of cell replacement therapy to the clinic for treating AD requires a source of authentic human progenitor cells. Recent studies utilizing blastocyst complementation in gene- edited animals have resulted in the generation of authentic cells and organs such as islet cells and pancreas, renal cells and kidney, and pulmonary cells and lung. We propose to use this approach to generate authentic hippocampal GABAergic interneurons and astrocytes in mice for cell therapy in the APOE4 knock-in (KI) mouse model of AD. A single Specific Aim is designed to characterize GABAergic neurons and astrocytes in the brains of HHEX KO mouse fetuses following complementation with pluripotent murine stem cells. Two Sub-Aims will (i) compare fetal GABAergic hippocampal interneurons and hippocampal astrocytes derived from intra-species murine chimeras vs. wild-type fetuses; and (ii) transplant exogenic hippocampal GABAergic interneurons and astrocytes derived from intra-species HHEX KO mouse chimeras into the brains of APOE4 KI mice to determine efficacy of using exogenic neural cells as a source for transplantation. The results from these studies will provide proof-of-principle that blastocyst complementation in gene edited animals may serve as a platform for generating exogenic neural cells for transplantation in AD, and as a potential future human therapeutic modality.