RESUMEN
Spinal cord injury presents a significant therapeutic challenge since the treatments available are mostly vain. The use of stem cells to treat this condition represents a promising new therapeutic strategy; therefore, a variety of stem cell treatments have been recently examined in animal models of CNS trauma. In this work, we analyzed the effects of third trimester amniotic fluid cells in a mouse model of spinal cord injury. Among the different cultures used for transplantation, some were able to induce a significant improvement in motor recovery (cultures #3.5, #3.6 and #7.30), evaluated by means of open field free locomotion. All effective cell cultures expressed the surface marker nerve/glial antigen 2, ortholog of the human chondroitin sulfate proteoglycan 4, which is present on several types of immature progenitor cells. The improved motor functional recovery was correlated with higher myelin preservation in the ventral horn white matter and an increased vascularization in the peri-lesion area. Real-Time PCR analysis showed higher expression levels of vascular endothelial growth factor and hypoxia-inducible factor-1α mRNA two days after cells transplantation compared to PBS-treated animals, indicating that an angiogenic pathway might have been activated by these cells, possibly through the production of hepatocyte growth factor. This cytokine appears to be produced mostly in filtering organs, such as the lung, of the transplanted animals and is likely released in the blood suggesting an endocrine role of hepatocyte growth factor in targeting the injury site.
Asunto(s)
Líquido Amniótico/citología , Células Madre Pluripotentes/trasplante , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Médula Espinal/patología , Trasplante de Células Madre/métodos , Proteínas Angiogénicas/genética , Animales , Antígenos/metabolismo , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Femenino , Factor de Crecimiento de Hepatocito/sangre , Factor de Crecimiento de Hepatocito/genética , Humanos , Pulmón/citología , Masculino , Ratones , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Embarazo , Tercer Trimestre del Embarazo , Proteoglicanos/metabolismo , Recuperación de la Función/fisiología , Médula Espinal/irrigación sanguínea , Resultado del TratamientoRESUMEN
PURPOSE: Our aim was the search for new sources of cells potentially useful for central nervous system regenerative medicine. Extra-embryonic tissues are promising sources of pluripotent stem cells. Among these, human second-trimester amniotic fluid (AF) contains cell populations exhibiting self-renewal capacity, multipotency and the expression of embryonic cell markers. METHODS: Here we report the properties of the easily available third-trimester AF cells (AFCs). Different cell types from 6 of 9 AF samples were separated, expanded, and characterized by assessing their morphological, proliferative, and differentiative properties. RESULTS: All isolated cultures presented CD105, CD90 and CD73 mesenchymal markers, whereas they differed among themselves in CD117, CD146, CD31, NG2 and CD133 expression. Their doubling time and telomere length were conserved throughout many passages. Importantly, immunofluorescence and Real-time PCR showed that, during their proliferative state and differentiation, several cultures expressed neuronal and glial markers such as nestin, GFAP, ß-tubulin III and neurofilament H indicating their potential attitude towards a neural fate. Indeed, these cells showed a rather poor capacity to differentiate in adipogenic and osteogenic lineages. CONCLUSIONS: In this work we report that cells with neural differentiation capability can be isolated from third-trimester AF, such properties could be useful for neuro-regenerative purposes.
Asunto(s)
Líquido Amniótico/citología , Diferenciación Celular/fisiología , Células Madre Pluripotentes/fisiología , Tercer Trimestre del Embarazo , Antígenos/genética , Antígenos/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Femenino , Feto/citología , Feto/fisiología , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Cariotipificación/métodos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Células Madre Pluripotentes/clasificación , Embarazo , Proteoglicanos/genética , Proteoglicanos/metabolismo , Factores de TiempoRESUMEN
The purpose of this study was to determine the fate and the effects of undifferentiated embryonic stem cells (ESCs) in mice after contusive lesion of the spinal cord (SCI). Reproducible traumatic lesion to the cord was performed at T8 level by means of the Infinite Horizon Device, and was followed by intravenous injection of one million of undifferentiated ESCs through the tail vein within 2 h from the lesion. The ESCs-treated animals showed a significant improvement of the recovery of motor function 28 days after lesion, with an average score of 4.61+/-0.13 points of the Basso Mouse Scale (n=14), when compared to the average score of vehicle treated mice, 3.58+/-0.23 (n=10). The number of identified ESCs found at the lesion site was 0.6% of the injected cells at 1 week after transplantation, and further reduced to 0.04% at 1 month. It is, thus, apparent that the promoted hind-limb recovery cannot be correlated to a substitution of the lost tissue performed by the exogenous ESC. The extensive evaluation of production of several neuroprotective and inflammatory cytokines did not reveal any effect by ESC-treatment, but unexpectedly the number of invading macrophages and neutrophils was greatly reduced. This may explain the improved preservation of lesion site ventral myelin, at both 1 week (29+/-11%) and 1 month (106+/-14%) after injury. No teratoma formation was observed, although an inappropriate colonization of the sacral cord by differentiated nestin- and beta-tubulin III-positive ESCs was detected.