Chizuka Ide

Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation

Bone marrow stromal cells (MSCs) have the capability under specific conditions of differentiating into various cell types such as osteocytes, chondrocytes, and adipocytes. Here we demonstrate a highly efficient and specific induction of cells with neuronal characteristics, without glial differentiation, from both rat and human MSCs using gene transfection with Notch intracellular domain (NICD) and subsequent treatment with bFGF, forskolin, and ciliary neurotrophic factor. MSCs expressed markers related to neural stem cells after transfection with NICD, and subsequent trophic factor administration induced neuronal cells. Some of them showed voltage-gated fast sodium and delayed rectifier potassium currents and action potentials compatible with characteristics of functional neurons. Further treatment of the induced neuronal cells with glial cell line-derived neurotrophic factor (GDNF) increased the proportion of tyrosine hydroxylase-positive and dopamine-producing cells. Transplantation of these GDNF-treated cells showed improvement in apomorphine-induced rotational behavior and adjusting step and paw-reaching tests following intrastriatal implantation in a 6-hydroxy dopamine rat model of Parkinson disease. This study shows that a population of neuronal cells can be specifically generated from MSCs and that induced cells may allow for a neuroreconstructive approach.

The Membrane-Anchored MMP Inhibitor RECK Is a Key Regulator of Extracellular Matrix Integrity and Angiogenesis

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

Peripheral nerve regeneration

Peripheral nerve regeneration comprises the formation of axonal sprouts, their outgrowth as regenerating axons and the reinnervation of original targets. This review focuses on the morphological features of axonal sprouts at the node of Ranvier and their subsequent outgrowth guided by Schwann cells or by Schwann cell basal laminae. Adhesion molecules such as N-CAM, L1 and N-cadherin are involved in the axon-to-axon and axon-to-Schwann cell attachment, and it is suggested that integrins such as alpha 1 beta 1 and alpha 6 beta 1 mediate the attachment between axons and Schwann cell basal laminae. The presence of synaptic vesicle-associated proteins such as synaptophysin, synaptotagmin and synapsin I in the growth cones of regenerating axons indicates the possibility that exocytotic fusion of vesicles with the surface axolemma supplies the membranous components for the extension of regenerating axons. Almost all the subtypes of protein kinase C have been localized in growth cones both in vivo and in vitro. Protein kinase C and GAP-43 are implicated to be involved in at least some part of the adhesion of growth cones to the substrate and their growth activity. The significance of tyrosine kinase in growth cones is emphasized. Tyrosine kinase plays an important role in intracellular signal transduction of the growth of regenerating axons mediated by both nerve trophic factors and adhesion molecules. Growth factors such as NGF, BDNF, CNTF and bFGF are also discussed mainly in terms of the influence of Schwann cells on regenerating axons.

Schwann cell basal lamina and nerve regeneration

Nerve segments approximately 7 mm long were excised from the predegenerated sciatic nerves of mice, and treated 5 times by repetitive freezing and thawing to kill the Schwann cells. Such treated nerve segments were grafted into the original places so as to be in contact with the proximal stumps. The animals were sacrificed 1, 2, 3, 5, 7 and 10 days after the grafting. The grafts were examined by electron microscopy in the middle part of the graft, i.e. 3-4 mm distal to the proximal end and/or near the proximal and distal ends of the graft. In other instances, the predegenerated nerve segments were minced with a razor blade after repetitive freezing and thawing. Such minced nerves were placed in contact with the proximal stumps of the same nerves. The animals were sacrificed 10 days after the grafting. Within 1-2 days after grafting, the dead Schwann cells had disintegrated into fragments. They were then gradually phagocytosed by macrophages. The basal laminae of Schwann cells, which were not attacked by macrophages, remained as empty tubes (basal lamina scaffolds). In the grafts we examined, no Schwann cells survived the freezing and thawing process. The regenerating axons always grew out through such basal lamina scaffolds, being in contact with the inner surface of the basal lamina (i.e. the side originally facing the Schwann cell plasma membrane). No axons were found outside of the scaffolds. One to two days after grafting, the regenerating axons were not associated with Schwann cells, but after 5-7 days they were accompanied by Schwann cells which were presumed to be migrating along axons from the proximal stumps. Ten days after grafting, proliferating Schwann cells observed in the middle part of the grafts had begun to sort out axons. In the grafts of minced nerves, the fragmented basal laminae of the Schwann cells re-arranged themselves into thicker strands or small aggregations of basal laminae. The regenerating axons, without exception, attached to one side of such modified basal laminae. Collagen fibrils were in contact with the other side, indicating that these modified basal laminae had the same polarity in terms of cell attachment as seen in the ordinary basal laminae of the scaffolds.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurogenesis in the ependymal layer of the adult rat 3rd ventricle

Neurogenesis has been described in limited regions of the adult mammalian brain. In this study, we showed that the ependymal layer of the 3rd ventricle is a neurogenic region in the adult rat brain. DiI labeling of the 3rd ventricle revealed that neural progenitor cells were derived from cells at the ependymal layer of the adult 3rd ventricle. The mitosis of these progenitor cells at the ependymal layer was promoted by bFGF administration. Combination of BrdU administration, nestin/GFAP immunohistochemistry, and labeling by GFP-recombinant adenoviral infection (vGFP) indicated that at least some tanycytes might be neural progenitor cells in the ependymal layer of the 3rd ventricle. Tracing by vGFP indicated that neural progenitor cells may have migrated from the 3rd ventricle to the hypothalamic parenchyma, where they were integrated into neural networks by forming synapses. In addition, some BrdU(+) neurons had immunoreactivity for orexin A in the hypothalamus. These results indicate that neural progenitor cells exist in the ependymal layer of the adult rat 3rd ventricle and that they may differentiate into neurons functioning in the hypothalamus.

Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation

The effects of bone marrow stromal cells (BMSCs) on the repair of injured spinal cord and on the behavioral improvement were studied in the rat. The spinal cord was injured by contusion using a weight-drop at the level of T8-9, and the BMSCs from the bone marrow of the same strain were infused into the cerebrospinal fluid (CSF) through the 4th ventricle. BMSCs were conveyed through the CSF to the spinal cord, where most BMSCs attached to the spinal surface although a few invaded the lesion. The BBB score was higher, and the cavity volume was smaller in the rats with transplantation than in the control rats. Transplanted cells gradually decreased in number and disappeared from the spinal cord 3 weeks after injection. The medium supplemented by CSF (250 microl in 3 ml medium) harvested from the rats in which BMSCs had been injected 2 days previously promoted the neurosphere cells to adhere to the culture dish and to spread into the periphery. These results suggest that BMSCs can exert effects by producing some trophic factors into the CSF or by contacting with host spinal tissues on the reduction of cavities and on the improvement of behavioral function in the rat. Considering that BMSCs can be used for autologous transplantation, and that the CSF infusion of transplants imposes a minimal burden on patients, the results of the present study are important and promising for the clinical use of BMSCs in spinal cord injury treatment.

Nectin an adhesion molecule involved in formation of synapses

The nectin–afadin system is a novel cell–cell adhesion system that organizes adherens junctions cooperatively with the cadherin–catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament–binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin–afadin system colocalizes with the cadherin–catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin–afadin system in the formation of synapses.

Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord

Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord‐derived neurosphere cells by either cell mixing or making monolayered‐feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered‐feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting.

Spinal cord injury treatment with intrathecal autologous bone marrow stromal cell transplantation: the first clinical trial case report.

Spinal cord injury often results in devastating dysfunction and disability. When a spinal cord is injured, various symptoms are presented depending on the segments of the damage and the degree. If cervical spinal damage is severe, tetraplegia results. If damage occurs at levels higher than C4, diaphragmatic movement will be impaired, and the patient has to live being connected with the ventilator on the bed. Patients will suffer from acute hyperesthesia or severe chronic pain, urinary and rectal dysfunction, and autonomic dystonia as well as motor and sensory deficits. In Japan, there are more than 100,000 victims suffering from spinal injury, and a new 5,000 to 6,000 patients are added every year. In the Unites States, about 250,000 to 400,000 people are living with spinal cord injury, and there are about 11,000 to 13,000 new injuries every year. The number of incidence is increasing. The majority of them result from motor vehicle or sports injuries, violence, or falls. An injured central nervous system never regenerates. This has long been thought as a medical common sense terms. Therefore, the principal object for the treatment of spinal injury was mainly purposed how to minimize the progression of secondary injuries and maintain the remnant function of the spine. For the purpose of preventing secondary spinal cord injury, spine stabilization for the fracture or dislocation and rehabilitation were the main strategy in the treatment. There has been no successful treatment for the severe spinal cord injury to recover the function satisfactorily. However, if spinal cord damage is functionally improved even at the minimum, it will affect not only the physical, mental, and economic status of patients and their families, but also the medical resources of society. Recently, regenerative treatments with stem cells are in the limelight. However, there are some serious problems such as ethical ones to be solved for the study with stem cells. We reported significant recovery of motor function in rats with experimental spinal cord injury treated by transplanting bone marrow stromal cells (BMSCs) in the cerebrospinal fluid (CSF). Based on that study, we aimed at the clinical application of this treatment, and actually planned a clinical trial of spinal cord injury treatment by transplanting patient’s autologous BMSCs into CSF in the acute phase after spinal cord injury, at Kansai Medical University Hospital. We have developed a detailed protocol for the clinical trial. The medical ethics committees of the institutions have approved the protocol officially. This clinical trial aims to treat a damaged spinal cord by a novel method of injecting BMSCs into CSF through the lumbar puncture, and assess the safety and efficacy of the procedure. Although we have experienced only a single case, a committee that monitors the data to assess the efficacy and safety of the trial with members independent of this study team has evaluated the safety of the trial in this case, approved to continue the study, and agreed to submit a report of the first case. In addition, Japan Spinal Cord Foundation strongly requested to disclose the course of the first case. Therefore, we would like to publish the report of the first case to enhance research work on the new strategy for the difficult treatment of spinal cord injury. Submitted for publication July 6, 2007. Accepted for publication September 18, 2007. Copyright

Alginate Enhances Elongation of Early Regenerating Axons in Spinal Cord of Young Rats

Freeze-dried alginate sponge cross-linked with covalent bonds has been demonstrated to enhance nerve regeneration in peripheral nerves and spinal cords. The present study examined, at early stages after surgery, the outgrowth of regenerating axons and reactions of astrocytes at the stump of transected spinal cord in young rats. Two segments (Th7-8) were resected, and alginate was implanted in the lesion. As controls, collagen gel was implanted in place of alginate or the lesion was left without implantation. Two and 4 weeks after surgery, nerve outgrowth and astrocyte reactions were examined. Many regenerating axons, some of which were accompanied by astrocytic processes, were found to extend from the stump into the alginate-implanted lesion. In the all nonimplanted animals, large cystic cavities were formed at both interfaces with no definite axonal outgrowth into the lesion. In collagen-implanted animals, cavity formation was found in some rats, and regenerating axons once formed at the stumps did not extend further into the lesion. Astrocytic processes extending into alginate-implanted lesion had no basal laminae, whereas those found in control experiments were covered by basal laminae. These findings suggest that alginate contributed to reducing the barrier composed of connective tissues and reactive astrocytic processes, and served as a scaffold for the outgrowth of regenerating axons and elongation of astrocytic processes.