346 VOLUME 8 | NUMBER 3 | MARCH 2005 NATURE NEUROSCIENCE ARTICLES Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome Christoph P Hofstetter 1 , Niklas A V Holmström 2 , Johan A Lilja 1 , Petra Schweinhardt 1,4 , Jinxia Hao 3 , Christian Spenger 1 , Zsuzsanna Wiesenfeld-Hallin 3 , Shekar N Kurpad 5 , Jonas Frisén 2 & Lars Olson 1 Several studies have reported functional improvement after transplantation of neural stem cells into injured spinal cord. We now provide evidence that grafting of adult neural stem cells into a rat thoracic spinal cord weight-drop injury improves motor recovery but also causes aberrant axonal sprouting associated with allodynia-like hypersensitivity of forepaws. Transduction of neural stem cells with neurogenin-2 before transplantation suppressed astrocytic differentiation of engrafted cells and prevented graft-induced sprouting and allodynia. Transduction with neurogenin-2 also improved the positive effects of engrafted stem cells, including increased amounts of myelin in the injured area, recovery of hindlimb locomotor function and hindlimb sensory responses, as determined by functional magnetic resonance imaging. These findings show that stem cell transplantation into injured spinal cord can cause severe side effects and call for caution in the consideration of clinical trials. Spontaneous recovery after spinal cord injury is hindered by the limited ability of the mammalian central nervous system to re-establish functional neural connections, remyelinate spared nerve fibers and replace lost cells 1 . Complete spinal cord injury leads to total and permanent sensorimotor loss and disruption of autonomic nervous system control caudal to the level of injury. Most spinal cord injury victims also develop chronic pain conditions 2 that severely reduce quality of life 3 . Although most forms of spinal cord injuries in humans are incomplete and are characterized by variable degrees of tissue sparing across the lesion 4 , surviving axons are often compromised with respect to the propagation of electrical impulses because of loss of myelin 5 . Grafting of embryonic stem cells 6 and marrow stromal cells 7 , as well as neural stem cells (NSCs) 8 , into the injured spinal cord improves functio- nal recovery. Transplanted neural stem cells give rise almost exclusively to astrocytes and to only relatively few oligodendrocytes and occasional neurons 8,9 , suggesting that the beneficial effects are mediated either by trophic support provided by astrocytes 10 or by remyelination of spared axons by graft-derived oligodendrocytes 11 . Neurotrophic factors have also been reported to cause severe side effects. Studies in rodents using several different trophic factors 12,13 as well as a clinical trial using nerve growth factor for Alzheimer disease 14 have reported neuropathic pain such as allodynia: that is, pain from stimuli that normally are not noxious. To further explore the potential function of graft-derived astrocytes and oligodendrocytes in the treatment effects, we transplanted naive NSCs and NSCs in which astrocytic differentiation was suppressed by ectopic expression of neurogenin-2. Neurogenin-2, a member of the basic helix-loop-helix family of transcription factors, is involved in the determination and differentiation of multiple neural lineages during development 15 . Neurogenins inhibit gliogenesis by sequestering CBP-Smad1 away from astrocyte differentiation genes and by blocking activation of STAT1/3 (ref. 16). Here we report that although transplantation of naive NSCs improved motor function, it also caused aberrant host fiber sprouting associated with allodynia-like hypersensitivity of non-affected forepaws. Suppression of astroglial differentiation effectively reduced both sprouting and allo- dynia and allowed for further sensory and motor recovery. RESULTS Neurogenin-2 modulates the fate of engrafted NSCs We studied the differentiation of adult spinal cord–derived NSCs grafted to the site of a 1-week-old low-thoracic spinal cord weight-drop injury. The fate of naive NSCs was compared with that of NSCs that had been transduced to express neurogenin-2 (Ngn2-NSCs). Two weeks after transplantation, NSC-derived cells were present mainly in the area of injury, close to the injection site. Engrafted cells had round cell bodies and some extended processes (Fig. 1a). Ngn2-NSCs were also located mainly near the injection site at this time. However, many cells had migrated beyond the astroglial barrier into the white matter, where they extended processes oriented along spared nerve fibers (Fig. 1a). The total number of engrafted 5-bromodeoxyuridine (BrdU)- immunoreactive cells at all four depositions was determined using stereological cell counting. A total of 79,496 ± 3,350 NSC-derived cells 1 Department of Neuroscience, 2 Department of Cell and Molecular Biology and 3 Division of Clinical Neurophysiology, Karolinska Institutet, 17177 Stockholm, Sweden. 4 Department of Human Anatomy, and Genetics, University of Oxford, OX1 1QX Oxford, UK. 5 Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. Correspondence should be addressed to (christoph.hofstetter@neuro.ki.se). Published online 13 February 2005; doi:10.1038/nn1405 © 2005 Nature Publishing Group http://www.nature.com/natureneuroscience