Adult Olfactory Bulb Neural Precursor Cell Grafts Provide Temporary Protection From Motor Neuron Degeneration, Improve Motor Function, and Extend Survival in Amyotrophic Lateral Sclerosis Mice

J Neuropathol Exp Neurol Copyright Ó 2007 by the American Association of Neuropathologists, Inc. Vol. 66, No. 11 November 2007 pp. 1002Y1018 ORIGINAL ARTICLE Adult Olfactory Bulb Neural Precursor Cell Grafts Provide Temporary Protection From Motor Neuron Degeneration, Improve Motor Function, and Extend Survival in Amyotrophic Lateral Sclerosis Mice Lee J. Martin, PhD and Zhiping Liu, MD, PhD Abstract Amyotrophic lateral sclerosis is a fatal disease caused by degeneration of motor neurons (MNs). We transplanted multipotent neural precursor cell (NPC)-neurospheres from mouse olfactory bulb (OB) into the spinal cord of transgenic mice that develop MN degeneration because of human mutant superoxide dismutase-1 (mSOD1). Adult NPCs were isolated from the OB core of transgenic mice expressing green fluorescent protein, human wild-type SOD1, or human mSOD1. mSOD1 mice received lumbar spinal cord transplants of OB-NPC neurospheres at preclinical stages of disease (70 days old). Control mSOD1 mice received dead cells or recombinant green fluorescent protein. OB-NPCs attenuated the loss of motor function and wasting. They delayed disease onset to ~117 days, compared with control onset at ~90 days. The lifespan of NPC recipient mice was extended (~170 days) compared with the lifespan of controls (~140 days). Transplanted OB-NPCs differentiated into large spinal neurons positive for choline acetyltransferase, interneurons, and glial cells. Loss of endogenous MNs was attenuated in mSOD1 mice with transplants. New neurons formed myelinated axons and synapses. NPC-derived neurons issued axons that grew into peripheral nerve. OB-NPCs also differentiated into oligodendrocytes and astrocytes that contacted neuronal processes. We conclude that transplantation of adult OB-NPCs is therapeutic for mouse amyotrophic lateral sclerosis. Key Words: Adult stem cell, Motor neuron disease, Motor neuron replacement, Mutant superoxide dismutase-1 (SOD1), Spinal cord injury, Stem cell therapy. INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a fatal adultonset disease of motor neurons (MNs) in cerebral cortex, From the Department of Pathology (LJM, ZL), Division of Neuropathology and Department of Neuroscience (LJM), The Johns Hopkins University School of Medicine, Baltimore, Maryland. Send correspondence and reprint requests to: Lee J. Martin, PhD, Johns Hopkins University School of Medicine, Department of Pathology, 558 Ross Building, 720 Rutland Avenue, Baltimore, MD 21205-2196; E-mail: This work was supported by grants from the ALS Association and the National Institutes of Health National Institute of Neurological Diseases and Stroke (NS034100). 1002 brainstem, and spinal cord (1Y3). Patients with ALS become paralyzed and generally die within 5 years after clinical onset and diagnosis. No effective therapies are available for patients with ALS (1). Several neurotrophic factors have been tested in patients with ALS, but the success was insufficient to support their use clinically for disease treatment (3). Riluzole, a Na+ channel blocker with effects on glutamate release, is the only drug approved for the symptomatic treatment of ALS. In clinical trials the beneficial effects of riluzole in patients with ALS were marginal, and other drugs with glutamate antagonist actions are ineffective (1). Regenerative medicine through novel cell-based therapies (4) needs to be explored for treating ALS. Neural stem cells (NSCs) and other stem cell (SC) types could restore neurologic function in patients with ALS (5Y7). SCs can be derived from embryos and several adult tissues (4, 8). To date, the transplantation of allogenic or xenogenic embryonic SCs or adult SCs as a therapy to treat MN disorders is experimental, and more data needs to be collected on animal models to determine beneficial or harmful effects of this approach. Existing animal studies of traumatically injured or diseased spinal cord with grafts of embryonic mouse SCs (9), human embryonic germ cells (10), human fetal NSCs (11), human umbilical cord SCs (12, 13), adult mouse bone marrow SCs (14), or adult mouse multipotent neural precursor cells (NPCs) (15, 16) have demonstrated positive effects on behavioral or clinical outcome but very limited or no evidence for differentiation of grafted cells into cells with MN-like characteristics. In contrast, adult NPCs from the olfactory bulb (OB) core (17Y19) can adopt phenotypes of MNs in vitro and in an animal model of axotomy-induced MN degeneration (20). The OB of adult rat, mouse, and human contains multipotent (stem) NPCs (17Y21). The OB core is part of the anterior subventricular zone (SVZ)-rostral migratory stream system of NPCs (22). Because the OB core is the rostral extension of the SVZ, the NPCs within this structure are more accessible than those in the SVZ, lying deep within the forebrain, for potential experimental autologous transplantation. Thus, the OB could be of major importance to neuroregenerative medicine if proof of principle is further established that the OB core contains NPCs that are useful for transplantation in animal models of neurodegenerative disease. Studies evaluating the ability of J Neuropathol Exp Neurol  Volume 66, Number 11, November 2007 Copyright @ 2007 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol  Volume 66, Number 11, November 2007 adult OB-NPCs to alter the course of disease in genetic models of ALS have not been performed. This study, originally presented partly in abstract form (23), demonstrates the therapeutic benefits of transplanted adult OBNPCs in a human mutant superoxide dismutase-1 (mSOD1) transgenic mouse model of ALS. MATERIALS AND METHODS Animals Adult male transgenic (tg) mice expressing human mSOD1 were used to test the therapeutic benefit of transplanted adult OB-NPCs. The mSOD1 mice expressed the form of human SOD1 containing the Gly39YAla (G39A) substitution mutation (24, 25). A line (B6SJLTgN[SOD1-G93A]1Gur) with a high copy number of mutant allele and a rapid disease onset was used (The Jackson Laboratory, Bar Harbor, ME). Hemizygous tg mice develop symptoms at about 11 to 13 weeks of age and become profoundly paralyzed in all limbs and die at about 16 weeks of age and have severe degeneration of MNs in spinal cord (25). The sources of adult NPCs used for transplantation were adult heterozygotic tg male mice ubiquitously expressing green fluorescent protein (GFP) driven by a A-actin promoter (C57BL/6-TgN[ACTbEGFP]1Osb, The Jackson Laboratory) (20) and adult tg mice expressing human mutant G93A or the wild-type (wt) SOD1 gene driven by the human SOD1 promoter (21). Non-tg mice (C57BL/6), which contributed to the genetic background of the mSOD1 tg mice, were used as naive controls in motor function tests. For a positive control for the neuromuscular junction (NMJ) structural analysis, we used tg mice expressing yellow fluorescent pr (...truncated)