Title : Mesenchymal stem cell transplantation reduces glial cyst and improves functional outcome following spinal cord compression

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells that have a supportive role in regenerative therapies, especially in the central nervous system, where spontaneous regeneration is limited. MSCs can exert a paracrine activity and modulate the inflammatory response after a central nervous system injury. Spinal cord injury (SCI) leads to permanent neurologic deficits below the injury site, owing to neuronal and axonal damage. Among experimental treatments after SCI, cell transplantation has emerged as a promising approach. METHODS: Using a compression injury model in the mouse spinal cord, MSCs were acutely transplanted into the lesion cavity; injured mice without the graft served as controls. After 26 days, the survival of MSCs was investigated, and their effect on the formation of glial cyst and on injuryrelated inflammation was evaluated. RESULTS: Grafted MSCs remained permanently undifferentiated. The lesion volume was reduced by 31.6% compared with control mice despite the fact that astroglial and microglial activation was not altered by the graft. Sensory and motor tests showed that MSC cell therapy results in improvement on a battery of behavioral tests compared with control mice: MSC-treated mice versus control mice scored 0.00 versus 0.50 in the posture test, 0.00 versus 1.50 in the hindlimb flexion test, 3.00 versus 2.25 in the sensory test, and 7.50 mistakes versus 15.83 mistakes in the foot-fault test. CONCLUSIONS: These results underscore the therapeutic potential of MSCs, making them promising treatments for central nervous system pathologies. INTRODUCTION Mesenchymal stem cells (MSCs) are self-renewing, multipotent stem cells that can differentiate into several types of mesenchymal tissues (35). MSCs can also play an important supportive role in regenerative therapies, particularly in the CNS (where spontaneous regeneration is very limited), supporting axonal growth and the maintenance of synaptic connections, preventing neuronal death by reducing apoptosis and limiting free radical generation (17). MSCs also secrete cytokines and growth factors, thus exerting a paracrine influence on damaged tissues (11, 5). And finally, MSCs can interact with the host immune system, modulating the inflammatory response to insult (31, 15). Spinal cord injury (SCI) approximately concerns 6 million people worldwide, and profoundly affects the quality and expectancy of life of the victims, most of whom are relatively young (25, 39). Traumatic SCI leads to hemorrhage, ischemia, edema, a strong inflammatory response, reactive gliosis, the formation of glial scars and cystic cavities, and loss of neurons and glia; it is often accompanied by severe neurological dysfunction and disability (8, 25). Stem cells (SCs) hold great promise for treating SCI. In an earlier paper, we have demonstrated the benefits of using MSCs in a lesion model that involves spinal cord hemisection (3). Here we explore the potential benefits of using MSCs in a murine compression SCI model, focusing our analysis on how MSCs alter the inflammatory response and the formation of the glial cyst, and on how these changes influence functional recovery. To our knowledge, this is the first report to employ undifferentiated MSCs in an acute SCI model, and to document lesion volume reduction and behavioral improvement. MATERIALS AND METHODS Experimental animals Two month-old C57BL/6J male mice (Harlan-Italy, San Pietro al Natisone, Italy) were used to produce the SCI model. Animals had free access to food and water. All experimental procedures on live animals were performed according to the European Communities’ Council Directive of 24 November 1986 (86/609/EEC) and University of Torino’s institutional guidelines for animal welfare (DL 116/92); efforts were made to minimize the number of animals used and their suffering. BCF1 mice, which express Enhanced Green Fluorescent Protein (EGFP) under the betaactin promoter, were kindly provided by Dr. M. Okabe (Osaka University, Suita, Japan, 32), and were bred in our animal facility: EGFP mice were sacrificed and MSCs were harvested from bone marrow (see below). Isolation and culture of MSCs Murine MSCs were isolated and expanded in vitro for transplantation, as previously described (3). 15 seven-to-nine week-old EGFP mice were anesthetized and killed by cervical dislocation; their tibias and femurs were cleared of muscle and connective tissue, and bone marrow cells were aspirated using a 22-gauge needle; cells were washed twice (5 minutes each) by centrifugation at 1000 rpm in Eagle’s alpha minimum essential medium (a-MEM; Sigma) containing 2 mM Lglutamine (Invitrogen-Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen-Gibco). To culture MSCs, marrow cells were plated at a density of 700,000 cells/cm in 19.5 cm polystyrene dishes (BD Biosciences) that were pre-treated with a coating of fetal bovine serum (FBS; Sigma): they were grown in a-MEM supplemented with 10% FBS, in a humidified atmosphere of 95% air with 5% CO2 at 37°C. Medium was replaced on day 4 to remove free floating cells, and then replenished every 2-3 days. At 10 days in vitro, adherent cells were retrieved by trypsinisation (Trypsin, InvitrogenGibco) and immunodepleted of CD11b-positive granulocytic cells by magnetic cell sorting: cells were incubated with MicroBeads conjugated to monoclonal rat anti-mouse/human CD11b antibody (MiltenyiBiotec GmbH, BergischGladbach, Germany) and loaded onto a MACS column (MiltenyiBiotec). Cd11b-negative cells were harvested, washed and re-plated onto dishes as described above. When the cells were near confluence, they were incubated with trypsin for 5 min at 37°C. The cell suspension was diluted 1:2 at each passage. Before transplantation, MSCs were counted on a Burker chamber and re-suspended in saline solution at a final concentration of 50 x10 cells/μl. Surgery and Cell Injection Procedures Mice underwent surgical procedures to produce a spinal cord compression. Animals were divided into the following groups: (i) spinal cord injury (SCI) + saline solution group (vehicle treated – VT mice) n = 19; (ii) SCI + transplantation of mesenchymal stem cells (MSC mice) n = 14. Vehicle treated mice were the same as in (2). Spinal cord compression was induced according to Farooque (9). Briefly, adult C57BL/6J mice were deeply anaesthetized with 3% isoflurane vaporized in O2/N2O (50:50). A laminectomy was performed at the level of L2 neuromer. Compression was applied onto the spinal cord for 5 min, gently laying a round plate (diameter 1.5 mm), oriented longitudinally over the dorsal aspect of spinal cord, and weighing 10 g, in order to produce moderate injury. The plate was removed after 5 min. Immediately after injury, the suspension of MSCs (10 cells in 2 μl saline) was slowly (over about 60 seconds) injected at depth 1 mm via a glass micropipette (outer tip diameter 50 μm) assembled on a stereotaxic apparatus into the spinal cord, into the SCI lesion cavity. VT animals received vehicle alone.