Naoya Matsumoto

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.

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.

RECK modulates Notch signaling during cortical neurogenesis by regulating ADAM10 activity

We report that during cortical development in the mouse embryo, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) critically regulates Notch signaling by antagonizing the ectodomain shedding of Notch ligands, which is mediated by a disintegrin and metalloproteinase domain 10 (ADAM10). In the embryonic brain, RECK is specifically expressed in Nestin-positive neural precursor cells (NPCs). Reck-deficient NPCs undergo precocious differentiation that is associated with downregulated Nestin expression, impaired Notch signaling and defective self-renewal. These phenotypes were substantially rescued either by enhancing Notch signaling or by suppressing endogenous ADAM10 activity. Consequently, we found that RECK regulates the ectodomain shedding of Notch ligands by directly inhibiting the proteolytic activity of ADAM10. This mechanism appeared to be essential for Notch ligands to properly induce Notch signaling in neighboring cells. These findings indicate that RECK is a physiological inhibitor of ADAM10, an upstream regulator of Notch signaling and a critical modulator of brain development.

Grafting of choroid plexus ependymal cells promotes the growth of regenerating axons in the dorsal funiculus of rat spinal cord: a preliminary report.

Nerve regeneration in the central nervous system has been studied by grafting various tissues and cells. In the present study, we demonstrated that choroid plexus ependymal cells can promote nerve regeneration when grafted into spinal cord lesions. The choroid plexus was excised from the fourth ventricle of adult rats (Wistar), minced into small fragments, and grafted into the dorsal funiculus at the C2 level in adult rat spinal cord from the same strain. Electron microscopy and fluorescence histochemistry showed that ependymal cells of the grafted choroid plexus intimately interacted with growing axons, serving to support the massive growth of regenerating axons. CGRP-positive fibers closely interacted with grafted ependymal cells. HRP injection at the sciatic nerve showed that numerous HRP-labeled regenerating fibers from the fasciculus gracilis extended into the graft 7 days after grafting. This regenerating axons from the fasciculus gracilis was maintained for at least 10 months, with some axons elongating rostrally into the dorsal funiculus. Evoked potentials of long duration were recorded at a level ca. 5 mm rostral to the lesion in the rats 8 to 10 months after grafting. These findings indicate that choroid plexus ependymal cells have the ability to facilitate axonal growth in vivo, suggesting that they may be a promising candidate as graft for the promotion of nerve regeneration in the spinal cord.

Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.

We developed an in vivo electroporation method to introduce foreign genes into retinal ganglion cells (RGCs). After the intravitreous injection of the plasmid gene (20 μg), five electric pulses (6 V/cm, 100 ms duration) were each delivered twice with 5 min interval to the rat eye using a contact lens-type electrode (cathodal) attached to the cornea and a needle electrode (anodal) inserted to the middle of the forehead. The efficiency of the genetic introduction into RGCs and tissue damage to the eyeball was evaluated using a green fluorescent protein (GFP) gene, TUNEL and histological observation. DiI retrograde labeling revealed that 24.4±4.7% of all RGCs were electrointroduced with the GFP gene. TUNEL and histological analysis showed a few tissue damages in the cornea, lens and retina. To confirm whether this method can actually rescue damaged RGCs, glial cell line-derived neurotrophic factor (GDNF) was electrointroduced into RGCs after optic nerve transection. After the electrointroduction, a significant increase in the number of surviving RGCs was observed 2 and 4 weeks after the optic nerve transection, and the decrease of caspase 3 and 9 was detected by RT-PCR. These results suggest that this method may be useful for the delivery of genes into RGCs with simplicity and minimal tissue damage.

Interleukin-1β causes long-term potentiation deficiency in a mouse model of septic encephalopathy.

Sepsis induces multiple organ dysfunction syndrome including septic encephalopathy (SE), which results in cognitive impairment. However, an effective treatment for SE remains unknown. We determined the role of interleukin-1β (IL-1β) in long-term potentiation (LTP) deficiency after SE. At first, endotoxin level in the blood was increased at 24 h after cecum ligation and puncture (CLP) (i.e. SE model). Second, the expression of IL-1β and its receptor in the hippocampus was determined by immunohistochemistry and immunoblotting. The number of Iba1-positive cells and their expression of IL-1β were enhanced by CLP with disruption of the blood brain barrier. Also, Iba1, IL-1β, and occludin protein expressions were consistent with immunohistochemical results. Third, we used an electrophysiological technique and observed the LTP deficiency, a hallmark of learning and memory, in the slices of hippocampus after CLP. Since type 1 interleukin-1 receptors (IL-1R1s) on neuronal cells were increased in the hippocampus, we utilized IL-1R1 antagonist. Pre-incubation with IL-1R1 antagonist for 30 min before recording of field excitatory post-synaptic potentials (fEPSPs) in the hippocampus canceled LTP deficiency after CLP. These results suggest the novel importance of IL-1β in synaptic plasticity deficiency associated with sepsis-induced brain inflammation. In a mouse model of SE, IL-1R1 inhibition is important in protecting synaptic function of the hippocampus after induction of SE.

Mammalian septin Sept2 modulates the activity of GLAST, a glutamate transporter in astrocytes

Sept2 is a member of the septin family of GTPases. Septins form filaments in a GTP‐form dependent manner, and are involved in cytokinesis from yeast to mammals; however, some mammalian septins, including Sept2, are expressed in the brain, a tissue in which almost all the cells are postmitotic. Recently, some functions of mammalian septin other than cytokinesis such as vesicle transport have been reported. However, mammalian septins physiological functions are still unclear. The present study revealed that Sept2 co‐localizes with the astrocyte glutamate transporter GLAST in the Bergmann glial processes facing axons and synapses. Biochemical analyses demonstrated that Sept2 bound directly to the carboxy‐terminal region of GLAST in a GDP‐form dependent manner. Expression of constitutive GDP‐form Sept2 mutant reduced the glutamate uptake activity of GLAST via internalization of GLAST from cell surface. Thus Sept2 may regulate GLAST‐mediated glutamate uptake by astrocytes, which is important for appropriate transmitter signalling in the cerebellum.

Choroid plexus ependymal cells host neural progenitor cells in the rat.

We previously demonstrated that choroid plexus epithelial (modified ependymal) cells (CPECs) differentiated into astrocytes after grafting into the spinal cord. In the present study, we examined whether CPECs from rats at postnatal 1 day (P1), 7 day (P7), and 8 weeks (P8W) can function as neural progenitor cells that give rise to neurons and glial cells. Cell spheres were produced in cultures of whole tissue of the choroid plexus from the fourth ventricle of rats at each postnatal period. β‐tubulin class III (Tuj‐1), glial fibrillary acid protein (GFAP)‐, and O4‐positive cells differentiated from cell spheres in the differentiation medium. We produced a monoclonal antibody 3E6 specifically labeling microvilli of CPECs. Using this monoclonal antibody, CPECs were isolated from the choroid plexus of P8W rats by cell sorter (FACS). Immunocytochemistry confirmed that there was no contamination from fibroblasts, endothelial cells, macrophages, or Schwann cells in the FACS‐isolated 3E6‐labeled cells. Cell spheres formed in the cultures of these 3E6‐labeled CPECs. After expansion, these cell spheres gave rise to Tuj‐1‐ (5%), GFAP‐ (45%), and O4‐positive cells (0.16%). The remaining cells (45%) were unlabeled neural or glial markers. Some CPECs of the P8W rat were immunohistochemically stained with lineage‐associated markers of Musashi‐1 and epidermal growth factor‐receptor (EGF‐R). In addition, infusion of EGF or fibroblast growth factor‐2 (FGF2) into the ventricle increased the number of bromodeoxyuridine (BrdU)‐positive cells among CPECs from 0.03% (untreated) to 1.14% (38‐fold, EGF) and 1.03% (35‐fold, FGF2), respectively. These findings indicate that neural progenitor cells exist among CPECs in the rat.

Differentiation of choroid plexus ependymal cells into astrocytes after grafting into the pre-lesioned spinal cord in mice.

Choroid plexus epithelial cells represent a continuation of, and have the same origin as, ventricular ependymal cells, and are regarded as modified ependymal cells. To extend previous studies of the use of choroid plexus ependymal cell (CPEC) grafting for nerve regeneration in the spinal cord, we investigated the capacity of cultured choroid plexus ependymal cells to differentiate into other types of glial cells in the spinal cord tissue. The choroid plexuses were excised from the fourth ventricle of green fluorescent protein (GFP)‐transgenic mice and the cells were dissociated and cultured for 4–6 weeks. CPECs were harvested from the monolayer cultures and injected into the pre‐lesioned spinal cords of wild‐type mice of the same strain using a Hamilton syringe. One week after injection, some GFP‐positive transplanted cells became immunohistochemically positive for glial fibrillary acidic protein (GFAP) but negative for neurofilament and myelin basic protein. All the GFAP‐positive transplanted cells were negative for vimentin. Two weeks after grafting, immunoelectron microscopy showed that the GFP‐positive transplanted cells that had gained GFAP immunoreactivity contained numerous bundles of intermediate filaments, a morphological characteristic similar to that of astrocytes, and were in close contact with adjacent host tissue. These results indicate that, when grafted into the spinal cord, at least some cultured choroid plexus ependymal cells have the capacity to differentiate into astrocytes. GLIA 36:364–374, 2001.