RESUMO
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.
Assuntos
Neurônios/fisiologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Transplante de Células-Tronco/métodos , Células-Tronco/fisiologia , Análise de Variância , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Comportamento Animal , Encéfalo/irrigação sanguínea , Encéfalo/fisiopatologia , Bromodesoxiuridina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Contagem de Células , Modelos Animais de Doenças , Feminino , Lateralidade Funcional/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membro Posterior/inervação , Membro Posterior/fisiopatologia , Imuno-Histoquímica/métodos , Laminina/classificação , Laminina/metabolismo , Imageamento por Ressonância Magnética/métodos , Atividade Motora/fisiologia , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/metabolismo , Proteínas de Neurofilamentos/metabolismo , Oligopeptídeos/metabolismo , Oxigênio/sangue , Dor/etiologia , Dor/fisiopatologia , Medição da Dor , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores do Fator Natriurético Atrial/metabolismo , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Transplante de Células-Tronco/efeitos adversos , Fatores de Tempo , Transdução Genética/métodos , Tubulina (Proteína)/metabolismoRESUMO
The cochlear sensory epithelium and spiral ganglion neurons (SGNs) in the adult mammalian inner ear do not regenerate following severe injury. To replace the degenerated SGNs, neural stem cell (NSC) is an attractive alternative for substitution cell therapy. In this study, adult mouse NSCs were transplanted into normal and deafened inner ears of guinea pigs. To more efficiently drive the implanted cells into a neuronal fate, NSCs were also transduced with neurogenin 2 (ngn2) before transplantation. In deafened inner ears and in animals transplanted with ngn2-transduced NSCs, surviving cells expressed the neuronal marker neural class III beta-tubulin. Transplanted cells were found close to the sensory epithelium and adjacent to the SGNs and their peripheral processes. The results illustrate that adult NSCs can survive and differentiate in the injured inner ear. It also demonstrates the feasibility of gene transfer to generate specific progeny for cell replacement therapy in the inner ear.
Assuntos
Diferenciação Celular/fisiologia , Cóclea/fisiologia , Sobrevivência de Enxerto/fisiologia , Perda Auditiva Neurossensorial/terapia , Neurônios/transplante , Transplante de Células-Tronco/métodos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Biomarcadores , Linhagem da Célula/genética , Cóclea/citologia , Cóclea/cirurgia , Técnicas de Transferência de Genes , Cobaias , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/fisiologia , Camundongos , Camundongos Transgênicos , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/fisiologia , Transplante Heterólogo , Resultado do Tratamento , Tubulina (Proteína)/metabolismoRESUMO
Neural stem cells may present an ideal route for gene therapy as well as offer new possibilities for the replacement of neurons lost to injury or disease. However, it has proved difficult to express ectopic genes in stem cells. We report methods to introduce genes into adult neural stem cells using viral and nonviral vectors in vitro and in vivo. Adenoviral and VSV-G-pseudotyped retroviral vectors are more efficient than plasmid transfection or VSV-G lentiviral transduction in vitro. We further show that adult neural stem cells can be directed to a neuronal fate by ectopic expression of neurogenin 2 in vitro. Plasmids can be delivered in vivo when complexed with linear polyethyleneimine, and gene expression can be targeted specifically to neural stem or progenitor cells by the use of specific promoters. These techniques may be utilized both to study the function of various genes in the differentiation of neural stem cells to specific cell fates and, ultimately, for gene therapy or to generate specific differentiated progeny for cell transplantation.
